Non-cytotoxic fusion proteins comprising EGF muteins

The present invention relates to non-cytotoxic fusion proteins, and to the use thereof as therapeutics for suppressing conditions such as mucus hypersecretion, inflammation, neuroendocrine disorders, and neuroendocrine tumours.

Non-cytotoxic proteases are a discrete group of proteases, which act on target cells by incapacitating cellular function. Importantly, non-cytotoxic proteases do not kill the target cells upon which they act. Some of the best known examples of non-cytotoxic proteases include clostridial neurotoxins and IgA proteases.

Non-cytotoxic proteases act by proteolytically-cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)--see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle formation, and thus to secretion of molecules via vesicle transport from a cell. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell.

In view of the ubiquitous nature of SNARE proteins, non-cytotoxic proteases have been successfully employed in a wide range of therapies, such as: the treatment of pain (see WO96/33274); the treatment of mucus hypersecretion conditions such as COPD, asthma (see WO00/10598); the treatment of non-neuronal conditions such as endocrine conditions, exocrine conditions, immunological conditions, cardiovascular conditions, bone conditions (see WO01/21213); the treatment of neurological disorders such as Parkinson's disease (see U.S. Pat. No. 6,620,415, U.S. Pat. No. 6,306,403); the treatment of neuropsychiatric disorders (see US2004/0180061, US2003/0211121); the treatment of endocrine disorders (see U.S. Pat. No. 6,827,931); the treatment of thyroid disorders (see U.S. Pat. No. 6,740,321); the treatment of diabetes (see U.S. Pat. No. 6,337,075, U.S. Pat. No. 6,416,765); and the treatment of pancreatic disorders (see U.S. Pat. No. 6,261,572, U.S. Pat. No. 6,143,306). Each of the above publications is herein incorporated in its entirety by reference thereto.

Generally, administration of a non-cytotoxic protease is well tolerated. However, administration in some applications can be challenging because of the larger doses required to achieve a beneficial effect. Larger doses can increase the likelihood of undesirable antigenic responses. Similarly, larger doses are associated with increased manufacture costs.

In common with any other drug substances, a therapeutic dosing range exists which identifies the lower and upper limits of safe, effective therapy. Often, the upper limit is determined by the increasing significance of off-target effects that lead to undesirable (e.g. potentially harmful) side-effects of drug treatment. In the case of non-cytotoxic proteases, this could lead to the paralysis of cellular secretion in off-target cells, which, in turn, may be fatal.

The use of non-cytotoxic protease molecules in therapeutic treatments of humans and other mammals is attracting increasing interest. In this regard, one focused area of interest lies in the use of epidermal growth factor (EGF) re-targeted non-cytotoxic proteases. These therapeutics have been shown to be useful in reducing mucus secretion (see WO00/10598), and inflammation (see U.S. Ser. No. 11/806,648). The present inventors have also found that EGF re-targeted non-cytotoxic proteases are useful in suppressing neuroendocrine disorders, and neuroendocrine tumours.

However, one problem associated with the therapeutic application of EGF-based non-cytotoxic proteases is that efficacy may require the use of relatively high dosage levels. As mentioned above, this is undesirable for want of increased manufacture costs and/or potential immunogenicity problems.

Thus, there is a need in the art to develop means for reducing dosage sizes and/or for reducing undesirable antigenic responses, whilst maintaining the potency of the non-cytotoxic protease. This need is exacerbated by the growing use of non-cytotoxic proteases, which places an ever-increasing need on the part of the pharmaceutical industry to develop alternative and/or improved therapeutic molecules.

The present invention addresses the above need(s), and solves one or more of the above-mentioned problems. In more detail, the present invention provides alternative and/or improved EGF re-targeted non-cytotoxic proteases, which are useful for various clinical and therapeutic applications, in particular for treating inflammation, mucus secretion-related disorders such as asthma and COPD, as well as neuroendocrine disorders, and neuroendocrine tumours.

In more detail, a first aspect of the present invention provides a polypeptide, comprising: a a non-cytotoxic protease that is capable of cleaving a SNARE protein; b. a translocation peptide that is capable of translocating said non-cytotoxic protease from within an endosome of a mammalian cell, across the endosomal membrane thereof and into the cytosol of the mammalian cell; and c. an epidermal growth factor (EGF) mutein, wherein (i) said EGF mutein comprises an amino acid sequence having at least 65% sequence identity to the amino acid sequence of naturally-occurring human EGF (SEQ ID NO: 1), and wherein said EGF mutein amino acid sequence differs from the amino acid sequence of SEQ ID NO: 1 by at least one amino acid insertion, deletion or substitution at a position selected from amino acid positions 15-17 or 48-51 of SEQ ID NO: 1; and (ii) said EGF mutein amino acid sequence has an amino acid sequence backbone that retains all 6 cysteine amino acid residues as they appear at positions 6, 14, 20, 31, 33 and 42 in SEQ ID NO: 1.

Without wishing to be bound by any theory, the present inventors believe that EGF binds to its natural receptor (ie. the EGF receptor; also known as ErbB.sub.1) via a binding reaction, which involves two distinct binding interfaces present on the EGF molecule, namely a first binding interface (ie. Binding Interface 1) provided by the sequence of amino acid residues at positions 31-40 of SEQ ID NO: 1, and a second binding interface (ie. Binding Interface 2) provided the sequence of amino acid residues at positions 41-45 of SEQ ID NO: 1. These two Binding Interfaces are believed to bind to either side of a cleft, which is present in the EGF receptor, and into which a small portion (referred to as the Leading Edge) of the EGF molecule is then inserted. The Leading Edge is provided by the sequence of amino acid residues at positions 48-51 of SEQ ID NO: 1 and includes a scaffold support sequence provided by positions 15-17 of SEQ ID NO: 1. This binding arrangement is illustrated in FIG. 2, in which the U-shaped cleft of the receptor is positioned at the top and left-hand side of FIG. 2 and the `open` face of the cleft points towards the centre of the FIG. 2.

In more detail, the present inventors believe that `bulky` amino acid residues present within the Leading Edge (positions 15-17 and 48-51 of SEQ ID NO: 1) of EGF reduce the EGF receptor activation ability of EGF-based non-cytotoxic fusion molecules. Thus, by reducing the overall size of amino acid residues present within the Leading Edge, the present inventors have provided a new EGF mutein, which confers improved receptor activation properties on the EGF non-cytotoxic fusion molecules of the present invention when compared with corresponding wild-type EGF fusions.

In one embodiment, the EGF mutein is modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more `bulky` amino acid residues present in the Leading Edge, wherein said `bulky` amino acid residues are selected from phenylalanine (F), tryptophan (W), or tyrosine (Y). By way of example, suitable substitutions may be selected from the group consisting of: leucine (L), isoleucine (I), valine (V), alanine (A), glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), and methionine (M). In a preferred embodiment, said substitution or deletion is at positions 48-51, preferably at position 49 and/or position 50 (compared with SEQ ID NO: 1). In this regard, position 49 is preferably substituted (compared with SEQ ID NO: 1) to leucine (L), isoleucine (I) or valine (V), preferably to leucine (L); and/or position 50 is preferably substituted (compared with SEQ ID NO: 1) to alanine (A), glycine (G), serine (S), threonine (T) or methionine (M), preferably to alanine (A).

In one embodiment, the EGF mutein may be separately or further modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more amino acid residues present at positions 15-17 of the Leading Edge. Modifications within this region are believed to increase the stability of the Leading Edge, for example by introduction of one or more additional inter- or intra-molecular hydrogen bonds. By way of example, suitable substitutions may be selected from the group consisting of: asparagine (N), glutamine (Q), aspartate (D), cysteine (C), glycine (G), leucine (L), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y). In this regard, position 16 is preferably substituted (compared with SEQ ID NO: 1) to asparagine (N) or glutamine (Q).

In addition to the above-described Leading Edge modifications, the EGF mutein may be further modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more amino acid residues present at positions 23-29. These positions are close to the Binding Interface 1 (discussed above), and help provide additional stability to the EGF mutein. By way of example, suitable substitutions may be selected from the group consisting of: glycine (G), alanine (A), serine (S), threonine (T), methionine (M), arginine (R), lysine (K), and histidine (H). In this regard, preferred substitutions are introduced at one or more of positions 24-28. For example, position 24 is preferably substituted (compared with SEQ ID NO: 1) to glycine (G), alanine (A), serine (S), threonine (T) or methionine (M), preferably to glycine (G); and/or position 25 is preferably substituted (compared with SEQ ID NO: 1) to threonine (T), glycine (G), alanine (A), serine (S), or methionine (M), preferably to threonine (T); and/or position 28 is preferably substituted (compared with SEQ ID NO: 1) to arginine (R), lysine (K) or histidine (H), preferably to arginine (R).

In addition to the above-described Leading Edge modifications, and optionally in addition to the above-described modification at position 23-29, the EGF mutein may be further modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more amino acid residues present at positions 3-5. These positions are close to where the EGF mutein is typically fused to the larger body of the fusion protein, and help provide additional stability to the EGF mutein. By way of example, suitable substitutions may be selected from the group consisting of: proline (P), arginine (R), lysine (K), and histidine (H). In this regard, position 4 is preferably substituted (compared with SEQ ID NO: 1) to proline (P), and/or position 5 is preferably substituted (compared with SEQ ID NO: 1) to arginine (R), lysine (K) or histidine (H), preferably lysine (K).

In addition to the above-described Leading Edge modifications, and optionally in addition to the above-described modification at position 23-29 and/or positions 3-5, the EGF mutein may be further modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more amino acid residues present at positions 10-12. These positions are close to where the EGF mutein is typically fused to the larger body of the fusion protein, and help provide additional stability to the EGF mutein. By way of example, suitable substitutions may be selected from the group consisting of: glutamic acid (E) and aspartic acid (D). In this regard, position 11 is preferably substituted (compared with SEQ ID NO: 1) to glutamic acid (E) or aspartic acid (D), preferably to glutamic acid.

In addition to the above-described Leading Edge modifications, and optionally in addition to the above-described modification at position 23-29 and/or positions 3-5 and/or positions 10-12, the EGF mutein may be further modified (compared with SEQ ID NO: 1) by substitution or deletion of one or more amino acid residues present at positions 37-39. These positions are close to both the Binding Interface 1 and Binding Interface 2 (discussed above), and help provide additional stability to the EGF mutein. By way of example, suitable substitutions may be selected from the group consisting of: valine (V), leucine (L), and isoleucine (I). In this regard, position 38 is preferably substituted (compared with SEQ ID NO: 1) to valine (V), leucine (L) or isoleucine (I), preferably to valine (V).

The present inventors have noted that the activation ability of an EGF molecule (for its natural EGF receptor) is significantly reduced when the EGF molecule is present as part of a much larger fusion protein, as is the case when such a molecule is used as a Targeting Moiety in a non-cytotoxic fusion protein. This problem is addressed by the present invention by the introduction of one or more mutations, which increase the activation ability of said EGF molecule when it is present as part of a larger non-cytotoxic fusion protein. This, in turn, improves the cell targeting efficiency of the polypeptides of the present invention, and means that lower dosage regimens may be employed. The latter reduces manufacturing costs, and minimises undesirable, patient-related antigenic effects against the polypeptides of the invention.

It is routine to confirm that an EGF mutein of the present invention has improved activation ability for an EGF (eg. ErbB) receptor--by way of example, we refer to Examples 4 & 5.

In one embodiment, the EGF fusions of the present invention demonstrate a binding affinity to an EGF receptor (e.g. ErbB.sub.1) that is greater than 4 or 2 nM, or greater than 0.4 or 0.2 nM, or greater than 0.04 or 0.02 nM.

In another embodiment, the EGF fusions of the present invention demonstrate a binding activation of an EGF receptor (e.g. ErbB.sub.1) that is greater than 6 pEC.sub.50, or greater than 7 pEC.sub.50, or greater than 8 pEC.sub.50. Examples of suitable assays are provided in Examples 9 & 10.

The EGF mutein comprises at least one amino acid deletion, substitution or insertion vis-6-vis naturally-occurring human EGF (SEQ ID NO: 1), though with the proviso that none of the 6 cysteine amino acid residues of naturally-occurring human EGF is so altered. In a preferred embodiment, the EGF mutein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 such deletions, substitutions or insertions. Of said modifications, substitutions are the most preferred, as they have less effect on secondary or tertiary structure. In the case of deletions or insertions, the EGF mutein preferably has at most 4 or 5, more preferably at most 2 or 3, and particularly preferably at most 1 such deletion and/or insertion.

In one embodiment, the EGF mutein comprises no more than 15, 14 or 13, preferably no more than 12, 11 or 10 amino acid deletions, substitutions or insertions vis-a-vis naturally-occurring human EGF (SEQ ID NO: 1). Of said modifications, substitutions are the most preferred, as they have less effect on secondary or tertiary structure. In the case of deletions or insertions, the EGF mutein preferably has at most 4 or 5, more preferably at most 2 or 3, and particularly preferably at most 1 such deletion and/or insertion.

In one embodiment, the EGF mutein comprises (or consists of) at least 37 amino acid residues. For example, an EGF mutein comprising this length has a primary amino acid sequence that closely mimics the 6 cysteine consensus sequence backbone of naturally-occurring hEGF (eg. SEQ ID NO:1). Such a 37 amino acid sequence is characterised by cysteine residues at positions (in an N-terminal to C-terminal direction) 1, 9, 15, 26, 28 and 37. In another embodiment (preferably including the above-defined backbone sequence), the EGF mutein comprises at least 39 or 41, preferably at least 43 or 45, more preferably at least 47 or 49 contiguous amino acid residues. In another embodiment, the EGF mutein comprises at least 51, 52 or 53 amino acid residues.

In one embodiment, the EGF mutein differs from naturally-occurring human EGF in that it comprises at least one (or more, as detailed above) deletion, substitution or insertion at any of the positions: D.sub.3S.sub.4E.sub.5, P.sub.7L.sub.8S.sub.9, G.sub.12Y.sub.13, L.sub.15H.sub.16, M.sub.21Y.sub.22I.sub.23E.sub.24A.sub.25, I.sub.38G.sub.39E.sub.40R.sub.41, Q.sub.43Y.sub.44R.sub.45D.sub.46L.sub.47K.sub.48W.sub.49W.sub.50E.sub.51L- .sub.52 (positions and letters refer to the one-letter amino acid code of naturally-occurring human EGF--SEQ ID NO:1).

For example: D.sub.3 can be substituted with G, N, Y, A or F; S.sub.4 can be substituted with T, P, F, Q or R; E.sub.5 can be substituted with G, K, or Q, P.sub.7 can be substituted with S, L.sub.8 can be substituted with P, S, R or Q; S.sub.9 can be substituted by P; G.sub.12 can be substituted with E, D, or Q; Y.sub.13 can be substituted with H or W; L.sub.15 can be substituted by A, I, M, F or V; H.sub.16 can be substituted with Q, N, A, E, D or Y, M.sub.21 can be substituted with V, R, or K; Y.sub.22 can be substituted with H, I.sub.23 can be substituted with V or L; E.sub.24 can be substituted with K, G or V; A.sub.25 can be substituted with S, T, or Q; I.sub.38 can be substituted with T, S, A, N, L, or V; G.sub.39 can be substituted with E, Q, K, D, I, L, or F; E.sub.40 can be substituted with D; R.sub.41 can be substituted by D; Q.sub.43 can be substituted with E; Y.sub.44 can be substituted with H or T; R.sub.45 can be substituted by G, Q, or P; R.sub.46 can be substituted by G; L.sub.47 can be substituted by G, D, or R; K.sub.48 can be substituted with R, T, or D; W.sub.49 can be substituted by R; W.sub.50 can be substituted by L; E.sub.51 can be substituted by G, A, W, K, or Y; and/or L.sub.52 can be substituted by P, R, or T.

In addition or separately, Q.sub.18 can be substituted with E, Q, K, F or L; and/or V.sub.35 can be substituted with E; D17 can be substituted by G; V19 can be substituted by A.

In addition or separately, N.sub.1 can be substituted by S, K, Y, T or H; S.sub.2 can be substituted by G or R; E.sub.5 can be substituted by G or K; H.sub.10 can be substituted by Y; D.sub.11 can be substituted by N, S, or E; L.sub.26 can be substituted by V; K.sub.28 can be substituted by R, S, or T; A.sub.30 can be substituted by V; N.sub.32 can be substituted by S; V.sub.34 can be substituted by A; V.sub.35 can be substituted by A.

In one embodiment, the EGF mutein differs from naturally-occurring human EGF in that it comprises at least one (or more, as detailed above) deletion, substitution or insertion at any of the positions G.sub.12Y.sub.13, H.sub.16 (positions and letters refer to the one-letter amino acid code of naturally-occurring human EGF--SEQ ID NO:1). By way of example, G.sub.12 may be substituted by an amino acid residue such as glutamine (Q) or asparagine (N). Similarly, Y.sub.13 may be substituted by a residue such as tryptophan (W) or phenylalanine (F), and H.sub.16 may be substituted by a residue such as aspartic acid (D), glutamic acid (E), glycine (G), alanine (A), serine (S), or threonine (T).

In one embodiment, the EGF mutein comprises an amino acid sequence as set forth in any of SEQ ID NOs: 6-32, 34, 36, 38, 40, 42, 44, 46, 49, 50, 52, 54, 56, 58, and 60. This embodiment embraces variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity thereto, though with the proviso that said variants always retain the specific amino acid substitution(s) illustrated in said SEQ ID NOs when compared with wild-type human EGF (ie. SEQ ID NO: 1). This embodiment also embraces variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity thereto, though with the proviso that said variants always retain a conservative amino acid substitution of the specific amino acid substitution(s) illustrated in said SEQ ID NOs when compared with wild-type human EGF (ie. SEQ ID NO: 1).

The biologically active component of the polypeptides of the present invention is a non-cytotoxic protease. Non-cytotoxic proteases are produced by a variety of plants, and by a variety of microorganisms such as clostridial sp. and neisserial sp. (e.g. N. gonorrhoeae).

In a preferred embodiment, the non-cytotoxic protease of the present invention is a clostridial neurotoxin protease or a neisserial IgA protease.

Turning now to the translocation peptide (also referred to as the translocation domain) of the present invention, this component serves to translocate the non-cytotoxic protease across the endosomal membrane and into the cytosol of a target cell, where the protease component may then exert its proteolytic effect on SNARE proteins. Translocation peptides are well known in the art, and are produced by a variety of plants and microorganisms.

In a preferred embodiment, the translocation peptide of the present invention is a clostridial neurotoxin translocation peptide (also known as a clostridial translocation domain, or H.sub.N).

The polypeptide of the present invention comprises a modified EGF molecule, which acts as a Targeting Moiety (TM) to direct the polypeptide to a selected target cell(s), for example by binding to an EGF receptor on a mucus-secreting cell. In a preferred embodiment, the EGF receptor is an ErbB receptor, preferably an ErbB.sub.1 receptor.

According to a second aspect of the present invention, there is provided a non-cytotoxic polypeptide (as defined above), for use in treating a range of medical conditions and diseases.

In one embodiment, the present invention provides use of said non-cytotoxic polypeptides and corresponding methods for the suppression of mucus hypersecretion, in particular conditions or diseases in which mucus hypersecretion is a causative element, such as (chronic) bronchitis, chronic obstructive pulmonary disease (COPD), and asthma.

In one embodiment, the present invention provides use and corresponding methods for the suppression of inflammation.

In another embodiment, the present invention provides use and corresponding methods for the suppression of endocrine neoplasia such as MEN, thyrotoxicosis and neuroendocrine disorders such as Cushing's disease, acromegaly, hyperandrogenism, chronic anovulation, polycystic ovarian syndrome, carcinoid syndrome, hypoglycaemic syndrome, necrolytic migratory erythema, Zollinger-Ellison syndrome, and Verner-Morrison syndrome.

In one embodiment, the present invention provides use and corresponding methods for the suppression of neuroendocrine tumours such as non-carcinoid gastroenteropancreatic neuroendocrine tumours, carcinoid tumours, pituitary tumours and phaeochromocytomas, and for suppressing cancers such as colorectal cancer, prostate cancer, breast cancer, and lung cancer.

In use, a polypeptide of the invention binds to an EGF (eg. ErbB) receptor (the Binding Site), which is present on and preferably characteristic of a target cell. Thus, in the context of mucus applications, the EGF TM binds to mucus-secreting cells (e.g. epithelial goblet cells, or submucosal gland mucus-secreting cells). In the context of anti-inflammatory applications, the EGF TM binds to inflammatory leukocyte cells (e.g. neutrophils). In the context of neuroendocrine conditions, the EGF TM may bind to a tumour cell itself, or to a growth hormone-secreting cell (eg. a pituitary cell). Following binding, the polypeptide of the invention (at least the non-cytotoxic protease component thereof) becomes endocytosed into a vesicle, and the translocation component then directs transport of the non-cytotoxic protease across the endosomal membrane and into the cytosol of the target cell. Once inside the target cell, the non-cytotoxic protease component inhibits the cellular exocytic fusion process, and thereby inhibits release/secretion from the target cell.

Polypeptide Preparation

The polypeptides of the present invention comprise 3 principal components: a `warhead` (ie. a non-cytotoxic protease); an EGF mutein TM; and a translocation domain. The general technology associated with the preparation of such fusion proteins is often referred to as re-targeted toxin technology. By way of exemplification, we refer to: WO94/21300; WO96/33273; WO98/07864; WO00/10598; WO01/21213; WO06/059093; WO00/62814; WO00/04926; WO93/15766; WO00/61192; and WO99/58571. All of these publications are herein incorporated by reference thereto.

In more detail, the TM component of the present invention may be fused to either the protease component or the translocation component of the present invention. Said fusion is preferably by way of a covalent bond, for example either a direct covalent bond or via a spacer/linker molecule. The protease component and the translocation component are preferably linked together via a covalent bond, for example either a direct covalent bond or via a spacer/linker molecule. Suitable spacer/linked molecules are well known in the art, and typically comprise an amino acid-based sequence of between 5 and 40, preferably between 10 and 30 amino acid residues in length.

In use, the polypeptides have a di-chain conformation, wherein the protease component and the translocation component are linked together, preferably via a disulphide bond.

The polypeptides of the present invention may be prepared by conventional chemical conjugation techniques, which are well known to a skilled person. By way of example, reference is made to Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press.

Alternatively, the polypeptides may be prepared by recombinant preparation of a single polypeptide fusion protein (see, for example, WO98/07864). This technique is based on the in vivo bacterial mechanism by which native clostridial neurotoxin (ie. holotoxin) is prepared, and results in a fusion protein having the following `simplified` structural arrangement: NH.sub.2-[protease component]-[translocation component]-[EGF TM]-COOH

According to WO98/07864, the TM is placed towards the C-terminal end of the fusion protein. The fusion protein is then activated by treatment with a protease, which cleaves at a site between the protease component and the translocation component. A di-chain protein is thus produced, comprising the protease component as a single polypeptide chain covalently attached (via a disulphide bridge) to another single polypeptide chain containing the translocation component plus TM.

Alternatively, the fusion proteins of the present invention may be prepared according to WO06/05093 such that the TM has an N-terminal domain that is `free` for interaction with a Binding Site on a target cell. In this system, the TM component of the fusion protein is located towards the middle of the linear fusion protein sequence, between the protease cleavage site and the translocation component. Subsequent cleavage at the protease cleavage site exposes the N-terminal portion of the TM, and provides the di-chain polypeptide fusion protein.

The above-mentioned protease cleavage sequence(s) may be introduced (and/or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed (ie. clostridial, or non-clostridial), the following are preferred:

TABLE-US-00001 Enterokinase (DDDDK.dwnarw.) (SEQ I D NO: 74) Factor Xa (IEGR.dwnarw./IDGR.dwnarw.) (SEQ I D NO: 75/76) TEV(Tobacco Etch virus) (ENLYFQ.dwnarw.G) (SEQ I D NO: 77) Thrombin (LVPR.dwnarw.GS) (SEQ I D NO: 78) PreScission (LEVLFQ.dwnarw.GP) (SEQ I D NO: 79) CleanCut (WELQ.dwnarw.X) (SEQ I D NO: 80) (X indicates any amino acid excluding proline)

Also embraced by the term protease cleavage site is an intein, which is a self-cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present. The above-mentioned `activation` cleavage sites may also be employed as a `destructive` cleavage site (discussed below) should one be incorporated into a polypeptide of the present invention.

In a preferred embodiment, the fusion protein of the present invention may comprise one or more N-terminal and/or C-terminal located purification tags. Whilst any purification tag may be employed, the following are preferred:

His-tag (e.g. 6.times. histidine), preferably as a C-terminal and/or N-terminal tag

MBP-tag (maltose binding protein), preferably as an N-terminal tag

GST-tag (glutathione-S-transferase), preferably as an N-terminal tag

His-MBP-tag, preferably as an N-terminal tag

GST-MBP-tag, preferably as an N-terminal tag

Thioredoxin-tag, preferably as an N-terminal tag

CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.

One or more peptide spacer/linker molecules may be included in the fusion protein. For example, a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule.

Thus, a third aspect of the present invention provides a nucleic acid (e.g. DNA) sequence encoding a polypeptide as described above.

Said nucleic acid may be included in the form of a vector, such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid integration site, a promoter, a terminator, and a ribosome binding site.

The present invention also includes a method for expressing the above-described nucleic acid sequence (i.e. the third aspect of the present invention) in a host cell, in particular in E. coli.

The present invention also includes a method for activating a polypeptide of the present invention, said method comprising contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the non-cytotoxic protease component and the translocation component, thereby converting the polypeptide into a di-chain polypeptide wherein the non-cytotoxic protease and translocation components are joined together by a disulphide bond. In a preferred embodiment, the recognition site is not native to a naturally-occurring clostridial neurotoxin and/or to a naturally-occurring IgA protease.

The polypeptides of the present invention may be further modified to reduce or prevent unwanted side-effects associated with dispersal into non-targeted areas. According to this embodiment, the polypeptide comprises a destructive cleavage site. The destructive cleavage site is distinct from the `activation` site (i.e. di-chain formation), and is cleavable by a second protease and not by the non-cytotoxic protease. Moreover, when so cleaved at the destructive cleavage site by the second protease, the polypeptide has reduced potency (e.g. reduced binding ability to the intended target cell, reduced translocation activity and/or reduced non-cytotoxic protease activity). For completeness, any of the `destructive` cleavage sites of the present invention may be separately employed as an `activation` site in a polypeptide of the present invention.

Thus, according to this embodiment, the present invention provides a polypeptide that can be controllably inactivated and/or destroyed at an off-site location.

In a preferred embodiment, the destructive cleavage site is recognised and cleaved by a second protease (i.e. a destructive protease) selected from a circulating protease (e.g. an extracellular protease, such as a serum protease or a protease of the blood clotting cascade), a tissue-associated protease (e.g. a matrix metalloprotease (MMP), such as an MMP of muscle), and an intracellular protease (preferably a protease that is absent from the target cell.

Thus, in use, should a polypeptide of the present invention become dispersed away from its intended target cell and/or be taken up by a non-target cell, the polypeptide will become inactivated by cleavage of the destructive cleavage site (by the second protease).

In one embodiment, the destructive cleavage site is recognised and cleaved by a second protease that is present within an off-site cell-type. In this embodiment, the off-site cell and the target cell are preferably different cell types. Alternatively (or in addition), the destructive cleavage site is recognised and cleaved by a second protease that is present at an off-site location (e.g. distal to the target cell). Accordingly, when destructive cleavage occurs extracellularly, the target cell and the off-site cell may be either the same or different cell-types. In this regard, the target cell and the off-site cell may each possess a receptor to which the same polypeptide of the invention binds.

The destructive cleavage site of the present invention provides for inactivation/destruction of the polypeptide when the polypeptide is in or at an off-site location. In this regard, cleavage at the destructive cleavage site minimises the potency of the polypeptide (when compared with an identical polypeptide lacking the same destructive cleavage site, or possessing the same destructive site but in an uncleaved form). By way of example, reduced potency includes: reduced binding (to a mammalian cell receptor) and/or reduced translocation (across the endosomal membrane of a mammalian cell in the direction of the cytosol), and/or reduced SNARE protein cleavage.

When selecting destructive cleavage site(s) in the context of the present invention, it is preferred that the destructive cleavage site(s) are not substrates for any proteases that may be separately used for post-translational modification of the polypeptide of the present invention as part of its manufacturing process. In this regard, the non-cytotoxic proteases of the present invention typically employ a protease activation event (via a separate `activation` protease cleavage site, which is structurally distinct from the destructive cleavage site of the present invention). The purpose of the activation cleavage site is to cleave a peptide bond between the non-cytotoxic protease and the translocation or the binding components of the polypeptide of the present invention, thereby providing an `activated` di-chain polypeptide wherein said two components are linked together via a di-sulfide bond.

Thus, to help ensure that the destructive cleavage site(s) of the polypeptides of the present invention do not adversely affect the `activation` cleavage site and subsequent di-sulfide bond formation, the former are preferably introduced into polypeptide of the present invention at a position of at least 20, at least 30, at least 40, at least 50, and more preferably at least 60, at least 70, at least 80 (contiguous) amino acid residues away from the `activation` cleavage site.

The destructive cleavage site(s) and the activation cleavage site are preferably exogenous (i.e. engineered/artificial) with regard to the native components of the polypeptide. In other words, said cleavage sites are preferably not inherent to the corresponding native components of the polypeptide. By way of example, a protease or translocation component based on BoNT/A L-chain or H-chain (respectively) may be engineered according to the present invention to include a cleavage site. Said cleavage site would not, however, be present in the corresponding BoNT native L-chain or H-chain. Similarly, when the Targeting Moiety component of the polypeptide is engineered to include a protease cleavage site, said cleavage site would not be present in the corresponding native sequence of the corresponding Targeting Moiety.

In a preferred embodiment of the present invention, the destructive cleavage site(s) and the `activation` cleavage site are not cleaved by the same protease. In one embodiment, the two cleavage sites differ from one another in that at least one, more preferably at least two, particularly preferably at least three, and most preferably at least four of the tolerated amino acids within the respective recognition sequences is/are different.

By way of example, in the case of a polypeptide chimaera containing a Factor Xa `activation` site between clostridial L-chain and H.sub.N components, it is preferred to employ a destructive cleavage site that is a site other than a Factor Xa site, which may be inserted elsewhere in the L-chain and/or H.sub.N and/or TM component(s). In this scenario, the polypeptide may be modified to accommodate an alternative `activation` site between the L-chain and H.sub.N components (for example, an enterokinase cleavage site), in which case a separate Factor Xa cleavage site may be incorporated elsewhere into the polypeptide as the destructive cleavage site. Alternatively, the existing Factor Xa `activation` site between the L-chain and H.sub.N components may be retained, and an alternative cleavage site such as a thrombin cleavage site incorporated as the destructive cleavage site.

When identifying suitable sites within the primary sequence of any of the components of the present invention for inclusion of cleavage site(s), it is preferable to select a primary sequence that closely matches with the proposed cleavage site that is to be inserted. By doing so, minimal structural changes are introduced into the polypeptide. By way of example, cleavage sites typically comprise at least 3 contiguous amino acid residues. Thus, in a preferred embodiment, a cleavage site is selected that already possesses (in the correct position(s)) at least one, preferably at least two of the amino acid residues that are required in order to introduce the new cleavage site. By way of example, in one embodiment, the Caspase 3 cleavage site (DMQD) may be introduced. In this regard, a preferred insertion position is identified that already includes a primary sequence selected from, for example, Dxxx, xMxx, xxQx, xxxD, DMxx, DxQx, DxxD, xMQx, xMxD, xxQD, DMQx, xMQD, DxQD, and DMxD.

Similarly, it is preferred to introduce the cleavage sites into surface exposed regions. Within surface exposed regions, existing loop regions are preferred.

In a preferred embodiment of the present invention, the destructive cleavage site(s) are introduced at one or more of the following position(s), which are based on the primary amino acid sequence of BoNT/A. Whilst the insertion positions are identified (for convenience) by reference to BoNT/A, the primary amino acid sequences of alternative protease domains and/or translocation domains may be readily aligned with said BoNT/A positions.

For the protease component, one or more of the following positions is preferred: 27-31, 56-63, 73-75, 78-81, 99-105, 120-124, 137-144, 161-165, 169-173, 187-194, 202-214, 237-241, 243-250, 300-304, 323-335, 375-382, 391-400, and 413-423. The above numbering preferably starts from the N-terminus of the protease component of the present invention.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 8 amino acid residues, preferably greater than 10 amino acid residues, more preferably greater than 25 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the protease component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 20 amino acid residues, preferably greater than 30 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the protease component.

For the translocation component, one or more of the following positions is preferred: 474-479, 483-495, 507-543, 557-567, 576-580, 618-631, 643-650, 669-677, 751-767, 823-834, 845-859. The above numbering preferably acknowledges a starting position of 449 for the N-terminus of the translocation domain component of the present invention, and an ending position of 871 for the C-terminus of the translocation domain component.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the translocation component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the translocation component.

In a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the N-terminus of the TM component. Similarly, in a preferred embodiment, the destructive cleavage site(s) are located at a position greater than 10 amino acid residues, preferably greater than 25 amino acid residues, more preferably greater than 40 amino acid residues, particularly preferably greater than 50 amino acid residues from the C-terminus of the TM component.

The polypeptide of the present invention may include one or more (e.g. two, three, four, five or more) destructive protease cleavage sites. Where more than one destructive cleavage site is included, each cleavage site may be the same or different. In this regard, use of more than one destructive cleavage site provides improved off-site inactivation. Similarly, use of two or more different destructive cleavage sites provides additional design flexibility.

The destructive cleavage site(s) may be engineered into any of the following component(s) of the polypeptide: the non-cytotoxic protease component; the translocation component; the Targeting Moiety; or the spacer peptide (if present). In this regard, the destructive cleavage site(s) are chosen to ensure minimal adverse effect on the potency of the polypeptide (for example by having minimal effect on the targeting/binding regions and/or translocation domain, and/or on the non-cytotoxic protease domain) whilst ensuring that the polypeptide is labile away from its target site/target cell.

Preferred destructive cleavage sites (plus the corresponding second proteases) are listed in the Table immediately below. The listed cleavage sites are purely illustrative and are not intended to be limiting to the present invention.

TABLE-US-00002 Destructive cleavage site Tolerated recognition sequence variance Second recognition P4-P3-P2-P1--P1'-P2'-P3' protease sequence P4 P3 P2 P1 P1' P2' P3' Thrombin LVPRGS A, F, G, I, A, F, G, P R Not D Not -- (SEQ ID NO: 78) L, T, V I, L, T, or E D or E or M V, W or A Thrombin GRG G R G Factor Xa IEGR A, F, G, I, D or E G R -- -- -- (SEQ ID NO: 75) L, T, V or M ADAM17 PLAQAVRSSS (SEQ ID NO: 81) Human SKGRSLIGRV airway (SEQ ID NO: 82) trypsin-like protease (HAT) ACE -- -- -- -- Not P Not N/A (peptidyl- D or E dipeptidase A) Elastase MEAVTY M, R E A, H V, T V, T, H Y -- (leukocyte) (SEQ ID NO: 83) Furin RXR/KR R X R R (SEQ ID NO: 84) or K Granzyme IEPD I E P D -- -- -- Caspase 1 (SEQ ID NO: 85) F, W, Y, L -- H, D Not -- -- A, T P, E.D. Q.K or R Caspase 2 DVAD D V A D Not -- -- (SEQ ID NO: 86) P, E.D. Q.K or R Caspase 3 DMQD D M Q D Not -- -- (SEQ ID NO: 87) P, E.D. Q.K or R Caspase 4 LEVD L E V D Not -- -- (SEQ ID NO: 88) P, E.D. Q.K or R Caspase 5 L or W E H D -- -- -- Caspase 6 V E H D Not -- -- or I P, E.D. Q.K or R Caspase 7 DEVD D E V D Not -- -- (SEQ ID NO: 89) P, E.D. Q.K or R Caspase 8 I or L E T D Not -- -- P, E.D. Q.K or R Caspase 9 LEHD L E H D -- -- -- (SEQ ID NO: 90) Caspase IEHD I E H D -- -- -- 10 (SEQ ID NO: 91)

Matrix metalloproteases (MMPs) are a preferred group of destructive proteases in the context of the present invention. Within this group, ADAM17 (EC 3.4.24.86, also known as TACE), is preferred and cleaves a variety of membrane-anchored, cell-surface proteins to "shed" the extracellular domains. Additional, preferred MMPs include adamalysins, serralysins, and astacins.

Another group of preferred destructive proteases is a mammalian blood protease, such as Thrombin, Coagulation Factor Vila, Coagulation Factor IXa, Coagulation Factor Xa, Coagulation Factor Xla, Coagulation Factor Xlla, Kallikrein, Protein C, and MBP-associated serine protease.

In one embodiment of the present invention, said destructive cleavage site comprises a recognition sequence having at least 3 or 4, preferably 5 or 6, more preferably 6 or 7, and particularly preferably at least 8 contiguous amino acid residues. In this regard, the longer (in terms of contiguous amino acid residues) the recognition sequence, the less likely non-specific cleavage of the destructive site will occur via an unintended second protease.

It is preferred that the destructive cleavage site of the present invention is introduced into the protease component and/or the Targeting Moiety and/or into the translocation component and/or into the spacer peptide. Of these four components, the protease component is preferred. Accordingly, the polypeptide may be rapidly inactivated by direct destruction of the non-cytotoxic protease and/or binding and/or translocation components.

Polypeptide Delivery

In use, the present invention employs a pharmaceutical composition, comprising a polypeptide, together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The polypeptides of the present invention may be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.

In the case of a polypeptide that is to be delivered locally, the polypeptide may be formulated as a cream (eg. for topical application), or for sub-dermal injection.

Local delivery means may include an aerosol, or other spray (eg. a nebuliser). In this regard, an aerosol formulation of a polypeptide enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.

One route of administration is via laproscopic and/or localised injection. Alternatively (or in addition), delivery may be systemic such as via intravenous administration.

In the case of formulations for injection, it is optional to include a pharmaceutically active substance to assist retention at or reduce removal of the polypeptide from the site of administration. One example of such a pharmaceutically active substance is a vasoconstrictor such as adrenaline. Such a formulation confers the advantage of increasing the residence time of polypeptide following administration and thus increasing and/or enhancing its effect.

Polypeptides of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.

The dosage ranges for administration of the polypeptides of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the polypeptide or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

Suitable daily dosages (per kg weight of patient) are in the range 0.0001-1 ng/kg, preferably 0.0001-0.5 ng/kg, more preferably 0.002-0.5 ng/kg, and particularly preferably 0.004-0.5 ng/kg. The unit dosage can vary from less that 1 picogram to 30 ng, but typically will be in the region of 0.01 to 1 ng per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.

A particularly preferred dosing regimen is based on 2.5 ng of polypeptide as the 1.times. dose. In this regard, preferred dosages are in the range 1.times.-100.times. (i.e. 2.5-250 ng).

Fluid dosage forms are typically prepared utilising the polypeptide and a pyrogen-free sterile vehicle. The polypeptide, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the polypeptide can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.

Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.

DEFINITIONS SECTION

Targeting Moiety (TM) means any chemical structure that functionally interacts with a Binding Site to cause a physical association between the polypeptide of the invention and the surface of a target cell (typically a mammalian cell, especially a human cell). The term TM embraces any molecule (ie. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a Binding Site on the target cell, which Binding Site is capable of internalisation (eg. endosome formation)--also referred to as receptor-mediated endocytosis. The TM may possess an endosomal membrane translocation function, in which case separate TM and Translocation Domain components need not be present in an agent of the present invention. Throughout this specification, specific TMs have been described, for example by reference to SEQ ID NOs. Reference to said TMs is merely exemplary, and the present invention embraces all variants and derivatives thereof, which retain the basic binding (i.e. targeting) ability of the exemplified TMs.

The TM of the present invention binds (preferably specifically binds) to the target cell in question. The term "specifically binds" preferably means that a given TM (e.g. with the additional fusion protein components such as the translocation component and/or the endopeptidase component) binds to the target cell with a binding affinity (Ka) of 10.sup.8M.sup.-1 or greater, preferably 10.sup.9M.sup.-1 or greater, more preferably 10.sup.10 M.sup.-1 orgreater, and most preferably, 10.sup.11 M.sup.-1 or greater.

Reference to TM in the present specification embraces fragments and variants thereof, which retain an ability to bind to the target cell and/or EGF (eg. ErbB) receptor in question. By way of example, a variant may have at least 70% or 75%, or at least 80% or 85%, or at least 90% or 95% amino acid sequence homology with a reference TM (eg. SEQ ID NO: 1, and/or the 37 amino acid sequence thereof that comprises the 6-cysteine backbone thereof). A variant may include one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage. Also, by way of example, the term fragment, when used in relation to a TM, means a peptide having at least thirty seven, or at least forty, or at least forty five, or at least fifty amino acid residues of the reference TM. The term fragment also relates to the above-mentioned variants. Thus, by way of example, a fragment of the present invention may comprise a peptide sequence having at least 37, 40, 45 or 50 amino acids, wherein the peptide sequence has at least 80% sequence homology over a corresponding peptide sequence (of contiguous) amino acids of the reference peptide. The EGF TM of the present invention has 99% or less, 97% or less, 95% or less, 93% or less, 91% or less, 89% or less, 87% or less, 85% or less, 83% or less, 81% or less, 79% or less, 77% or less, 75% or less, 73% or less, 71% or less sequence identity to naturally-occurring human EGF (SEQ ID NO: 1, and/or the 37 amino acid sequence thereof that comprises the 6-cysteine backbone thereof).

It is routine to confirm that a TM binds to the selected target cell. For example, a simple radioactive displacement experiment may be employed in which tissue or cells representative of a target cell (e.g. a mucus-secreting cell, or an inflammatory cell) are exposed to labelled (eg. tritiated) TM in the presence of an excess of unlabelled TM. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the TM binds to the target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further comprise observing a loss of TM binding. Examples of this type of experiment can be found in Hulme, E. C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, In Receptor biochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford University Press.

The polypeptides of the present invention may lack a functional H.sub.C domain of a clostridial neurotoxin. Accordingly, said polypeptides are not able to bind rat synaptosomal membranes (via a clostridial H.sub.C component) in binding assays as described in Shone et al. (1985) Eur. J. Biochem. 151, 75-82. In a preferred embodiment, the polypeptides preferably lack the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. In another embodiment, the polypeptides preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. Alternatively, the Hc binding activity may be negated/reduced by mutagenesis--by way of example, referring to BoNT/A for convenience, modification of one or two amino acid residue mutations (W1266 to L and Y1267 to F) in the ganglioside binding pocket causes the H.sub.C region to lose its receptor binding function. Analogous mutations may be made to non-serotype A clostridial peptide components, e.g. a construct based on botulinum B with mutations (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F). Other mutations to the active site achieve the same ablation of H.sub.C receptor binding activity, e.g. Y1267S in botulinum type A toxin and the corresponding highly conserved residue in the other clostridial neurotoxins. Details of this and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which is hereby incorporated by reference thereto.

The H.sub.C peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25 kDa each, namely the N-terminal region (commonly referred to as the H.sub.CN peptide or domain) and the C-terminal region (commonly referred to as the H.sub.CC peptide or domain). This fact is confirmed by the following publications, each of which is herein incorporated in its entirety by reference thereto: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Moreover, it has been well documented that the C-terminal region (H.sub.CC), which constitutes the C-terminal 160-200 amino acid residues, is responsible for binding of a clostridial neurotoxin to its natural cell receptors, namely to nerve terminals at the neuromuscular junction--this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy-chain lacking a functional heavy chain H.sub.C peptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional H.sub.CC peptide. In other words, the H.sub.CC peptide region is either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to inactivate its native binding ability for nerve terminals at the neuromuscular junction.

Thus, in one embodiment, a clostridial H.sub.N peptide of the present invention lacks part of a C-terminal peptide portion (H.sub.CC) of a clostridial neurotoxin and thus lacks the H.sub.C binding function of native clostridial neurotoxin. By way of example, in one embodiment, the C-terminally extended clostridial H.sub.N peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of a clostridial neurotoxin heavy-chain. In another embodiment, the clostridial H.sub.N peptide of the present invention lacks the entire C-terminal peptide portion (H.sub.CC) of a clostridial neurotoxin and thus lacks the H.sub.C binding function of native clostridial neurotoxin. By way of example, in one embodiment, the clostridial H.sub.N peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of a clostridial neurotoxin heavy-chain. By way of further example, the clostridial H.sub.N peptide of the present invention lacks a clostridial H.sub.CC reference sequence selected from the group consisting of: Botulinum type A neurotoxin--amino acid residues (Y1111-L1296) Botulinum type B neurotoxin--amino acid residues (Y1098-E1291) Botulinum type C neurotoxin--amino acid residues (Y1112-E1291) Botulinum type D neurotoxin--amino acid residues (Y1099-E1276) Botulinum type E neurotoxin--amino acid residues (Y1086-K1252) Botulinum type F neurotoxin--amino acid residues (Y1106-E1274) Botulinum type G neurotoxin--amino acid residues (Y1106-E1297) Tetanus neurotoxin--amino acid residues (Y1128-D1315).

The above-identified reference sequences should be considered a guide as slight variations may occur according to sub-serotypes.

The protease of the present invention embraces all non-cytotoxic proteases that are capable of cleaving one or more SNARE proteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease (or fragment thereof). More preferably the bacterial protease is selected from the genera Clostridium (e.g. a clostridial L-chain). The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity).

The present invention also embraces variant non-cytotoxic proteases (ie. variants of naturally-occurring protease molecules), so long as the variant proteases still demonstrate the requisite protease activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95 or at least 98% amino acid sequence homology with a reference protease sequence. Thus, the term variant includes non-cytotoxic proteases having enhanced (or decreased) endopeptidase activity--particular mention here is made to the increased K.sub.cat/K.sub.m of BoNT/A mutants Q161A, E54A, and K165L see Ahmed, S. A. (2008) Protein J. DOI 10.1007/s10930-007-9118-8, which is incorporated by reference thereto. The term fragment, when used in relation to a protease, typically means a peptide having at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 amino acid residues of the reference protease. As with the TM `fragment` component (discussed above), protease `fragments` of the present invention embrace fragments of variant proteases based on a reference sequence.

Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.

Exemplary neurotoxins are produced by clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum. The above-mentioned abbreviations are used throughout the present specification. For example, the nomenclature BoNT/A denotes the source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes.

BoNTs are the most potent toxins known, with median lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg depending on the serotype. BoNTs are adsorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine.

BoNTs share a common structure, being di-chain proteins of .about.150 kDa, consisting of a heavy chain (H-chain) of .about.100 kDa covalently joined by a single disulphide bond to a light chain (L-chain) of .about.50 kDa. The H-chain consists of two domains, each of .about.50 kDa. The C-terminal domain (H.sub.C) is required for the high-affinity neuronal binding, whereas the N-terminal domain (H.sub.N) is proposed to be involved in membrane translocation. The L-chain is a zinc-dependent metalloprotease responsible for the cleavage of the substrate SNARE protein.

The term L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically cleaving a non-neuronal SNARE protein.

Examples of suitable protease (reference) sequences include: Botulinum type A neurotoxin--amino acid residues (1-448) Botulinum type B neurotoxin--amino acid residues (1-440) Botulinum type C neurotoxin--amino acid residues (1-441) Botulinum type D neurotoxin--amino acid residues (1-445) Botulinum type E neurotoxin--amino acid residues (1-422) Botulinum type F neurotoxin--amino acid residues (1-439) Botulinum type G neurotoxin--amino acid residues (1-441) Tetanus neurotoxin--amino acid residues (1-457)

The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences: Botulinum type A neurotoxin--amino acid residues (M1-K448) Botulinum type B neurotoxin--amino acid residues (M1-K441) Botulinum type C neurotoxin--amino acid residues (M1-K449) Botulinum type D neurotoxin--amino acid residues (M1-R445) Botulinum type E neurotoxin--amino acid residues (M1-R422) Botulinum type F neurotoxin--amino acid residues (M1-K439) Botulinum type G neurotoxin--amino acid residues (M1-K446) Tetanus neurotoxin--amino acid residues (M1-A457)

A variety of clostridial toxin fragments comprising the light chain can be useful in aspects of the present invention with the proviso that these light chain fragments can specifically target the core components of the neurotransmitter release apparatus and thus participate in executing the overall cellular mechanism whereby a clostridial toxin proteolytically cleaves a substrate. The light chains of clostridial toxins are approximately 420-460 amino acids in length and comprise an enzymatic domain. Research has shown that the entire length of a clostridial toxin light chain is not necessary for the enzymatic activity of the enzymatic domain. As a non-limiting example, the first eight amino acids of the BoNT/A light chain are not required for enzymatic activity. As another non-limiting example, the first eight amino acids of the TeNT light chain are not required for enzymatic activity. Likewise, the carboxyl-terminus of the light chain is not necessary for activity. As a non-limiting example, the last 32 amino acids of the BoNT/A light chain (residues 417-448) are not required for enzymatic activity. As another non-limiting example, the last 31 amino acids of the TeNT light chain (residues 427-457) are not required for enzymatic activity. Thus, aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids and at least 450 amino acids. Other aspects of this embodiment can include clostridial toxin light chains comprising an enzymatic domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino acids and at most 450 amino acids.

The non-cytotoxic protease component of the present invention preferably comprises a BoNT/A, C or D serotype L-chain (or fragment or variant thereof).

The polypeptides of the present invention, especially the protease component thereof, may be PEGylated--this may help to increase stability, for example duration of action of the protease component. PEGylation is particularly preferred when the protease comprises a BoNT/E protease. PEGylation preferably includes the addition of PEG to the N-terminus of the protease component. By way of example, the N-terminus of a protease may be extended with one or more amino acid (e.g. cysteine) residues, which may be the same or different. One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto. An example of this technology is described in WO2007/104567, which is incorporated in its entirety by reference thereto.

The polypeptides of the present invention may include mutations and/deletions at one or more "secondary modification sites"--these sites are targeted and acted upon by enzymes (such as intracellular enzymes), which alter the biological persistence of the polypeptides of the invention. Such mutations/deltions may comprise the mutation or deletion of part or all of said one or more secondary modification sites. Such an increase in biological persistence is particular desired when longevity of action of the non-cytotoxic protease component of the present invention is desired. Alternatively, the polypeptides of the invention may include the addition of one or more secondary modification site.

The mutation or deletion of part or all of a secondary modification site found in a polypeptide of the present invention, or the addition of a secondary modification site to a polypeptide of the present invention, may lead to an enhanced biological persistence of the polypeptide. In general terms, the biological persistence of the polypeptide may be about 20% to about 300% greater than in the absence of any structural modification to a secondary modification site. Thus, inhibition of secretion from target cells is increased by about 20% to about 300%.

The enhanced biological persistence may take the form of an increased biological half-life of the polypeptide. The biological half-life of the polypeptide is preferably increased by about 10%; more preferably, the biological half-life of the polypeptide is increased by about 100%.

By way of example, botulinum neurotoxin A and botulinum neurotoxin E have the following potential secondary modification sites, as shown in Tables A and B, respectively. These sites may be targeted for mutation or deletion of all or part of the site.

TABLE-US-00003 TABLE A N-glycosylation: 173-NLTR; 382-NYTI; 411-NFTK; 417-NFTG Casein kinase II (CK-2) phosphorylation sites: 51-TNPE; 70-SYYD; 79-TDNE; 120-STID; 253-SGLE; 258-SFEE; 275-SLQE; 384-TIYD N-terminal myristylation sites: 15-GVDIAY; 141-GSYRSE; 254-GLEVSF Protein kinase C (PKC) phosphorylation sites: 142-SYR; 327-SGK; 435-TSK Tyrosine phosphorylation sites: 92-KLFERIY; 334-KLKFDKLY N-glycosylation: 97-NLSG; 138-NGSG; 161-NSSN; 164-NISL; 365-NDSI; 370-NISE

TABLE-US-00004 TABLE B Casein kinase II (CK-2) phosphorylation sites: 51-TPQD; 67-SYYD; 76-SDEE; 130-SAVE; 198-SMNE; 247-TNIE; 333-SFTE; 335-TEFD N-terminal myristylation sites: 220-GLYGAK; 257-GTDLNI; 386-GQNANL Protein kinase C (PKC) phosphorylation sites: 60-SLK; 166-SLR; 191-SFR; 228-TTK; 234-TQK; 400-TGR; 417-SVK Tyrosine kinase phosphorylation sites: 62-KNGDSSY (SEQ ID NO: 92); 300-KDVFEAKY (SEQ ID NO: 93)

A further example is the addition of a casein kinase II phosphorylation site, such as TDNE to a polypeptide of the present invention.

Further details are described in WO 2002/40506, which is herein incorporated in its entirety by reference thereto.

The polypeptides of the present invention may comprise one or more tyrosine phosphorylation sites in addition to any naturally existing tyrosine phosphorylation sites already present. The one or more additional tyrosine phosphorylation sites may be present in the non-cytotoxic protease component of the polypeptide, in the translocating domain of the polypeptide, or in the targeting domain of the polypeptide. Such sites may be added to the polypeptide as an addition to the polypeptide sequence, or may be substituted into the polypeptide sequence. By way of example, the tyrosine phosphorylation site may substitute about 1-8, or about 1-4, consecutive amino acids of the non-cytotoxic protease component.

The additional presence of such sites may increase the biological persistence of the polypeptides. In this regard, increased biological persistence may result in an increased duration of action of the polypeptide, or an increased half-life of the polypeptide, or both.

Any tyrosine phosphorylation site is suitable for use in the polypeptides of the present invention. By way of example, suitable tyrosine phosphorylation sites include KLFERIY (SEQ ID NO: 94) and KLKFDKLY (SEQ ID NO: 95).

Further details are provided in U.S. Pat. No. 7,223,577, which is herein incorporated in its entirety by reference thereto.

The polypeptides of the present invention may also have their biological persistence enhanced by the presence within the polypeptide of leucine-based motifs. The leucine-based motifs may be added in addition to the polypeptide sequence or included as a substitution.

A leucine-based motif may comprise seven contiguous amino acids. These may be further described as consisting of a group of five amino acids (a quintet) and a group immediately adjacent of two amino acids (a duplet). The duplet of amino acids may be located at either the N-terminal or C-terminal end of the leucine-based motif.

The quintet of amino acids may include at least one acidic amino acid selected from the group consisting of glutamic acid and aspartic acid.

The duplet of amino acids includes at least one hydrophobic acid; examples of such hydrophobic amino acids include leucine, isoleucine, methionine, alanine, phenylalanine, tryptophan, valine and tyrosine. The duplet of amino acids is preferably a leucine-leucine, a leucine-isoleucine, an isoleucine-leucine, an isoleucine-isoleucine or a leucine-methionine. The duplet of amino acids is even more preferably a leucine-leucine.

The quintet of amino acids may comprise at least one amino acid containing a hydroxyl group; examples of such amino acids include serine, threonine and tyrosine. The hydroxyl-containing amino acid is preferably phosphorylated. Even more preferably, the hydroxyl-containing amino acid is a serine which can be phosphorylated to allow for the binding of adapter proteins.

The above-described leucine-based motifs may also comprise modified amino acids. By way of example, a leucine-based motif may include a halogenated leucine, preferably a fluorinated leucine.

Examples of suitable leucine-based motifs include (where x can be any amino acid) xDxxxLL (SEQ ID NO: 96), xExxxLL (SEQ ID NO: 97), xDxxxLI (SEQ ID NO: 98), xDxxxLM (SEQ ID NO: 99), xExxxLI (SEQ ID NO: 100), xExxxIL (SEQ ID NO: 101), xExxxLM (SEQ ID NO: 102). A further example of a suitable leucine-based motif is phenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine.

Additional examples of leucine-based motifs (derived from various species) which are suitable for use in the polypeptides of the present invention are found in the table below.

TABLE-US-00005 Species Sequence Botulinum type A FEFYKLL (SEQ ID NO: 103) Rat VMAT1 EEKRAIL (SEQ ID NO: 104) Rat VMAT2 EEKMAIL (SEQ ID NO: 105) Rat VAChT SERDVLL (SEQ ID NO: 106) Rat .delta. VDTQVLL (SEQ ID NO: 107) Mouse .delta. AEVQALL (SEQ ID NO: 108) Frog .gamma./.delta. SDKQNLL (SEQ ID NO: 109) Chicken .gamma./.delta. SDRQNLI (SEQ ID NO: 110) Sheep .delta. ADTQVLM (SEQ ID NO: 111) Human CD3.gamma. SDKQTLL (SEQ ID NO: 112) Human CD4 SQIKRLL (SEQ ID NO: 113) Human .delta. ADTQALL (SEQ ID NO: 114) S. cerevisiae Vam3p NEQSPLL (SEQ ID NO: 115) VMAT: vesicular monoamine transporter. VAChT: vesicular acetylcholine transporter. S. cerevisiae Vam3p: a yeast homologue of synaptobrevin. Underlined serine residues are potential sites of phosphorylation.

In addition to the use of leucine-based motifs as described above, the polypeptides of the present invention may also comprise tyrosine-based motifs. The presence of a tyrosine-based motif may act to increase the biological persistence of the polypeptide. Tyrosine-based motifs suitable for use in the present invention comprise the sequence Y-X-X-Hy (SEQ ID NO: 116), where Y is tyrosine, X is any amino acid, and Hy is a hydrophobic amino acid. An example of such a tyrosine-based motif described in U.S. Pat. No. 7,223,577 is YKLL (SEQ ID NO: 117).

Further details are provided in WO 2005068494, which is herein incorporated in its entirety by reference thereto.

A Translocation Domain is a molecule that enables translocation of a protease into a target cell such that a functional expression of protease activity occurs within the cytosol of the target cell. Whether any molecule (e.g. a protein or peptide) possesses the requisite translocation function of the present invention may be confirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K.sup.+ and/or labelled NAD, which may be readily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120].

Additional methodology to enable assessment of membrane fusion and thus identification of Translocation Domains suitable for use in the present invention are provided by Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The present invention also embraces variant translocation domains, so long as the variant domains still demonstrate the requisite translocation activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with a reference translocation domain. The term fragment, when used in relation to a translocation domain, means a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference translocation domain. In the case of a clostridial translocation domain, the fragment preferably has at least 100, preferably at least 150, more preferably at least 200, and most preferably at least 250 amino acid residues of the reference translocation domain (eg. H.sub.N domain). As with the TM `fragment` component (discussed above), translocation `fragments` of the present invention embrace fragments of variant translocation domains based on the reference sequences.

The Translocation Domain is preferably capable of formation of ion-permeable pores in lipid membranes under conditions of low pH. Preferably it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Hence, in one embodiment, the Translocation Domain is a translocating domain of an enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be employed in the present invention.

The Translocation Domain may be of a clostridial origin, such as the H.sub.N domain (or a functional component thereof). H.sub.N means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. The H-chain lacks the natural binding function of the H.sub.C component of the H-chain. In this regard, the H.sub.C function may be removed by deletion of the H.sub.C amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the H.sub.C function may be inactivated by chemical or biological treatment. Thus, the H-chain is incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.

Examples of suitable (reference) Translocation Domains include: Botulinum type A neurotoxin--amino acid residues (449-871) Botulinum type B neurotoxin--amino acid residues (441-858) Botulinum type C neurotoxin--amino acid residues (442-866) Botulinum type D neurotoxin--amino acid residues (446-862) Botulinum type E neurotoxin--amino acid residues (423-845) Botulinum type F neurotoxin--amino acid residues (440-864) Botulinum type G neurotoxin--amino acid residues (442-863) Tetanus neurotoxin--amino acid residues (458-879)

The above-identified reference sequence should be considered a guide as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference thereto) cites slightly different clostridial sequences: Botulinum type A neurotoxin--amino acid residues (A449-K871) Botulinum type B neurotoxin--amino acid residues (A442-S858) Botulinum type C neurotoxin--amino acid residues (T450-N866) Botulinum type D neurotoxin--amino acid residues (D446-N862) Botulinum type E neurotoxin--amino acid residues (K423-K845) Botulinum type F neurotoxin--amino acid residues (A440-K864) Botulinum type G neurotoxin--amino acid residues (S447-S863) Tetanus neurotoxin--amino acid residues (S458-V879)

In the context of the present invention, a variety of Clostridial toxin H.sub.N regions comprising a translocation domain can be useful in aspects of the present invention with the proviso that these active fragments can facilitate the release of a non-cytotoxic protease (e.g. a clostridial L-chain) from intracellular vesicles into the cytoplasm of the target cell and thus participate in executing the overall cellular mechanism whereby a clostridial toxin proteolytically cleaves a substrate. The H.sub.N regions from the heavy chains of Clostridial toxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a H.sub.N region from a Clostridial toxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial toxin H.sub.N regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial toxin H.sub.N regions comprising translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.

For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H.sub.N embraces naturally-occurring neurotoxin H.sub.N portions, and modified H.sub.N portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified H.sub.N portions still demonstrate the above-mentioned translocation function.

Alternatively, the Translocation Domain may be of a non-clostridial origin. Examples of non-clostridial (reference) Translocation Domain origins include, but not be restricted to, the translocation domain of diphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], the translocation domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-3559], the translocation domains of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides of translocating function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirror the Translocation Domain present in a naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain.

Particular examples of viral (reference) Translocation Domains suitable for use in the present invention include certain translocating domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e. membrane fusion and vesiculation) function of a number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus haemagglutinin. Other virally expressed membrane fusion proteins known to have the desired translocating activity are a translocating domain of a fusogenic peptide of Semliki Forest Virus (SFV), a translocating domain of vesicular stomatitis virus (VSV) glycoprotein G, a translocating domain of SER virus F protein and a translocating domain of Foamy virus envelope glycoprotein. Virally encoded Aspike proteins have particular application in the context of the present invention, for example, the E1 protein of SFV and the G protein of the G protein of VSV.

Use of the (reference) Translocation Domains listed in Table (below) includes use of sequence variants thereof. A variant may comprise one or more conservative nucleic acid substitutions and/or nucleic acid deletions or insertions, with the proviso that the variant possesses the requisite translocating function. A variant may also comprise one or more amino acid substitutions and/or amino acid deletions or insertions, so long as the variant possesses the requisite translocating function.

TABLE-US-00006 Translocation Amino acid Domain source residues References Diphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of 405-613 Prior et al., 1992, pseudomonas Biochemistry exotoxin 31, 3555-3559 Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus GLFGAIAGFIENGWE Plank et al., 1994, haemagglutinin GMIDGWYG, (SEQ J. Biol. Chem. ID NO: 118), and 269, 12918-12924 Variants thereof Wagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 Semliki Forest virus Translocation domain Kielian et al., 1996, fusogenic protein J Cell Biol. 134(4), 863-872 Vesicular Stomatitis 118-139 Yao et al., 2003, virus glycoprotein G Virology 310(2), 319-332 SER virus F protein Translocation domain Seth et al., 2003, J Virol 77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et envelope al., 2003, J Virol. glycoprotein 77(8), 4722-4730

The polypeptides of the present invention may further comprise a translocation facilitating domain. Said domain facilitates delivery of the non-cytotoxic protease into the cytosol of the target cell and are described, for example, in WO 08/008,803 and WO 08/008,805, each of which is herein incorporated by reference thereto.

By way of example, suitable translocation facilitating domains include an enveloped virus fusogenic peptide domain, for example, suitable fusogenic peptide domains include influenzavirus fusogenic peptide domain (eg. influenza A virus fusogenic peptide domain of 23 amino acids), alphavirus fusogenic peptide domain (eg. Semliki Forest virus fusogenic peptide domain of 26 amino acids), vesiculovirus fusogenic peptide domain (eg. vesicular stomatitis virus fusogenic peptide domain of 21 amino acids), respirovirus fusogenic peptide domain (eg. Sendai virus fusogenic peptide domain of 25 amino acids), morbiliivirus fusogenic peptide domain (eg. Canine distemper virus fusogenic peptide domain of 25 amino acids), avulavirus fusogenic peptide domain (eg. Newcastle disease virus fusogenic peptide domain of 25 amino acids), henipavirus fusogenic peptide domain (eg. Hendra virus fusogenic peptide domain of 25 amino acids), metapneumovirus fusogenic peptide domain (eg. Human metapneumovirus fusogenic peptide domain of 25 amino acids) or spumavirus fusogenic peptide domain such as simian foamy virus fusogenic peptide domain; or fragments or variants thereof.

By way of further example, a translocation facilitating domain may comprise a Clostridial toxin H.sub.CN domain or a fragment or variant thereof. In more detail, a Clostridial toxin H.sub.CN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a Clostridial toxin H.sub.CN translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include: Botulinum type A neurotoxin--amino acid residues (872-1110) Botulinum type B neurotoxin--amino acid residues (859-1097) Botulinum type C neurotoxin--amino acid residues (867-1111) Botulinum type D neurotoxin--amino acid residues (863-1098) Botulinum type E neurotoxin--amino acid residues (846-1085) Botulinum type F neurotoxin--amino acid residues (865-1105) Botulinum type G neurotoxin--amino acid residues (864-1105) Tetanus neurotoxin--amino acid residues (880-1127)

The above sequence positions may vary a little according to serotype/sub-type, and further examples of suitable (reference) Clostridial toxin H.sub.CN domains include: Botulinum type A neurotoxin--amino acid residues (874-1110) Botulinum type B neurotoxin--amino acid residues (861-1097) Botulinum type C neurotoxin--amino acid residues (869-1111) Botulinum type D neurotoxin--amino acid residues (865-1098) Botulinum type E neurotoxin--amino acid residues (848-1085) Botulinum type F neurotoxin--amino acid residues (867-1105) Botulinum type G neurotoxin--amino acid residues (866-1105) Tetanus neurotoxin--amino acid residues (882-1127)

Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with non-clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a Clostridial toxin H.sub.CN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a Clostridial toxin H.sub.CN facilitating domain may be combined or with a clostridial translocation domain peptide, examples of which include: Botulinum type A neurotoxin--amino acid residues (449-1110) Botulinum type B neurotoxin--amino acid residues (442-1097) Botulinum type C neurotoxin--amino acid residues (450-1111) Botulinum type D neurotoxin--amino acid residues (446-1098) Botulinum type E neurotoxin--amino acid residues (423-1085) Botulinum type F neurotoxin--amino acid residues (440-1105) Botulinum type G neurotoxin--amino acid residues (447-1105) Tetanus neurotoxin--amino acid residues (458-1127) Sequence Homology:

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals; A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M--A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

Alignment Scores for Determining Sequence Identity

TABLE-US-00007 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

The percent identity is then calculated as:

.times..times..times..times..times..times..times..times..times..times..ti- mes..times..times..times..times..times..times..times..times..times..times.- .times..times..times..times..times..times..times..times..times..times..tim- es..times..times..times..times..times..times..times..times..times..times..- times..times..times..times..times..times..times..times..times. ##EQU00001##

Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

Conservative Amino Acid Substitutions

Basic: arginine

lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and .alpha.-methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention. Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

SUMMARY OF EXAMPLES

Example 1 Preparation of a Lha Backbone Construct Example 2 Construction of LH.sub.N/A-EGFv1 fusion protein Example 3 Expression and purification of a LH.sub.N/A-CT-EGF v1 fusion protein Example 4 EGF binding affinity assay Example 5 EGF binding affinity assay Example 6 Treatment of a patient suffering from chronic bronchitis Example 7 Method for treating acromegalic patients resistant to somatostatin analogues Example 8 Method for suppressing neuroendocrine tumour cells Example 9 Comparison of EGF and EGF fusion proteins in an EGF receptor activation assay Example 10 Comparison of EGF, EGF mutein and EGF mutein fusion proteins in an EGF receptor activation assay Example 11 Treatment of a patient suffering from rhinitis Example 12 Treatment of a patient suffering from asthma Example 13 Treatment of a patient suffering from COPD Example 14 Treatment of a patient suffering from asthma Example 15 Treatment of a patient suffering from colon cancer Example 16 Treatment of a patient suffering from psoriasis Example 17 Treatment of a patient suffering from eczema Example 18 Treatment of a patient suffering from colorectal cancer Example 19 Treatment of a patient suffering from prostate cancer Example 20 Treatment of a patient suffering from nsc lung cancer Example 21 Treatment of a patient suffering from breast cancer

SUMMARY OF SEQ ID NOS

SEQ ID 1 Amino acid sequence for naturally-occurring, human epidermal growth factor (EGF)

SEQ ID 2 DNA sequence of LHA

SEQ ID 3 DNA sequence of LHB

SEQ ID 4 DNA sequence of LHC

SEQ ID 5 DNA sequence of LHD

SEQ ID 6 Protein sequence of EGF variant H16N

SEQ ID 7 Protein sequence of EGF variant v1

SEQ ID 8 Protein sequence of EGF variant H16Q

SEQ ID 9 Protein sequence of EGF variant v2

SEQ ID 10 Protein sequence of EGF variant W49L

SEQ ID 11 Protein sequence of EGF variant v3

SEQ ID 12 Protein sequence of EGF variant W491

SEQ ID 13 Protein sequence of EGF variant v4

SEQ ID 14 Protein sequence of EGF variant W49V

SEQ ID 15 Protein sequence of EGF variant v5

SEQ ID 16 Protein sequence of EGF variant W49A

SEQ ID 17 Protein sequence of EGF variant v6 (G12Q)

SEQ ID 18 Protein sequence of EGF variant W49G

SEQ ID 19 Protein sequence of EGF variant v7 (H16D)

SEQ ID 20 Protein sequence of EGF variant W49S

SEQ ID 21 Protein sequence of EGF variant v8 (Y13W)

SEQ ID 22 Protein sequence of EGF variant W49T

SEQ ID 23 Protein sequence of EGF variant v9 (Q43A)

SEQ ID 24 Protein sequence of EGF variant W49N

SEQ ID 25 Protein sequence of EGF variant v10 (H16A)

SEQ ID 26 Protein sequence of EGF variant W49Q

SEQ ID 27 Protein sequence of EGF variant v11 (L15A)

SEQ ID 28 Protein sequence of EGF variant H.sub.16N_W49L

SEQ ID 29 Protein sequence of EGF variant v12 (V19E)

SEQ ID 30 Protein sequence of EGF variant H16Q_W49L

SEQ ID 31 Protein sequence of EGF variant v13 (V34D)

SEQ ID 32 Protein sequence of EGF variant H.sub.16N_W491

SEQ ID 33 Protein sequence of LHA-EGFv1 (Xa activation)

SEQ ID 34 Protein sequence of EGF variant H16Q_W491

SEQ ID 35 Protein sequence of LHA-EGFv2 (Xa activation)

SEQ ID 36 Protein sequence of EGF variant H.sub.16N_W50A

SEQ ID 37 Protein sequence of LHA-EGFv3

SEQ ID 38 Protein sequence of EGF variant H16Q_W50A

SEQ ID 39 Protein sequence of LHA-EGFv4

SEQ ID 40 Protein sequence of EGF variant H.sub.16N_W49L_W50A

SEQ ID 41 Protein sequence of LHA-EGFv5

SEQ ID 42 Protein sequence of EGF variant H16Q_W49L_W50A

SEQ ID 43 Protein sequence of LHA-EGFv6

SEQ ID 44 Protein sequence of EGF variant H.sub.16N_W49I_W50A

SEQ ID 45 Protein sequence of LHC-EGFv7

SEQ ID 46 Protein sequence of EGF variant H16Q_W49I_W50A

SEQ ID 47 Protein sequence of LHC-EGFv8

SEQ ID 48 Protein sequence of EGF variant H.sub.16N_W49L_W50A_E24G

SEQ ID 49 Protein sequence of LHC-EGFv9

SEQ ID 50 Protein sequence of EGF variant H.sub.16N_W49L_W50A_E24G_A25T

SEQ ID 51 Protein sequence of LHC-EGFv10

SEQ ID 52 Protein sequence of EGF variant H.sub.16N_W49L_W50A_E24G_A25S

SEQ ID 53 Protein sequence of LHC-EGFv11

SEQ ID 54 Protein sequence of EGF variant

H.sub.16N_W49L_W50A_E24G_A25T_K28R

SEQ ID 55 Protein sequence of LHC-EGFv12

SEQ ID 56 Protein sequence of EGF variant H.sub.16N_W49L_W50A_E24G_A25T_K28R_S4P

SEQ ID 57 Protein sequence of LHC-EGFv13

SEQ ID 58 Protein sequence of EGF variant H.sub.16N_W49L_W50A_E24G_A25T_K28R_S4P_E5K

SEQ ID 59 Protein sequence of LHB-EGFv1

SEQ ID 60 Protein sequence of EGF variant v3

SEQ ID 61 Protein sequence of LHB-EGFv5

SEQ ID 62 Protein sequence of Tetanus LHN-EGFv1

SEQ ID 63 Protein sequence of LHD-EGFv6 (protease sensitivity site)

SEQ ID 64 Protein sequence of LHD-EGFv3

SEQ ID 65 Protein sequence of LH D-EGFv11

SEQ ID 66 Protein sequence of M26-IgA1-HC-EGFv3

SEQ ID 67 Protein sequence of M26-IgA1-HC-EGFv11

SEQ ID 68 Protein sequence of Tetanus LHN-EGFv3

SEQ ID 69 Protein sequence of LHA-CP-EGFv2

SEQ ID 70 Protein sequence of LHD-EGFv2

SEQ ID 71 Protein sequence of LHC-CP-EGFv2

SEQ ID 72 Protein sequence of LHC-EGFv3

SEQ ID 73 DNA sequence of a EGF variant v3

FIG. 1 illustrates the data generated in Example 10 in which EGF, EGF mutein, or EGF mutein fusion protein is tested for its ability (at increasing concentrations) to activate an EGF receptor. A low pEC.sub.50 value indicates relatively poor receptor activation.

FIG. 2 illustrates a 3D model of EGF-EGF.sup.R binding, in which Binding Interface 1 (residues 31-40 of EGF), Binding Interface 2 (residues 41-45 of EGF), and the Leading Edge (residues 48-51 in combination with 15-17 of EGF) are identified.

EXAMPLE 1

Preparation of a LHA Backbone Construct

The following procedure creates a clone for use as an expression backbone for multidomain protein expression. This example is based on preparation of a serotype A based clone (SEQ ID 2), though the procedures and methods are equally applicable to other LH.sub.N serotypes such as serotype B (SEQ ID 3), serotype C (SEQ ID 4) and serotype D (SEQ ID 5)

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector chosen due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pET (Novagen) expression vector which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (NdeI-BamHI-SalI-PstI-SpeI-XbaI-HindIII). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options.

Preparation of Lca

The DNA sequence is designed by back translation of the LC/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) using one of a variety of reverse translation software tools (for example Backtranslation tool v2.0 (Entelechon)). BamHI/SalI recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as SeqBuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence containing the LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

Preparation of H.sub.N/A Insert

The DNA sequence is designed by back translation of the H.sub.N/A amino acid sequence (obtained from freely available database sources such as GenBank (accession number P10845) using one of a variety of reverse translation software tools (for example Back translation tool v2.0 (Entelechon)). A PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII to the C-terminus ensuring the correct reading frame in maintained. The DNA sequence is screened (using software such as SeqBuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

Preparation of the Interdomain (LC-H.sub.N Linker)

The LC-H.sub.N linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and H.sub.N) has the sequence VRGIIPFKTKSLDEGYNKALNDL (SEQ ID NO: 119). This sequence information is freely available from available database sources such as GenBank (accession number P10845). An alternative linker can be used for example composed of a Glycine-Serine linker (GGGGS.sub.3) (SEQ ID NO: 120). For generation of a specific protease cleavage site, the native recognition sequence for Factor Xa (IEGR (SEQ ID NO: 75) or IDGR (SEQ ID NO: 76)) can be used for example in the modified sequence VDGIITSKTKSLIDGR (SEQ ID NO: 121) or GGGGSGGGGSGGGGSIEGRGGGGSGGGGSGGGGS (SEQ ID NO: 122) or GGGGSGGGGSGGGGSIEGR (SEQ ID NO: 123) or the recognition sequence for the Light Chain of Enterokinase (DDDDK (SEQ ID NO: 74)) can be inserted, for example into the activation loop to generate the sequence VDGIITSKTKSLDDDDK (SEQ ID NO: 124) or GGGGSGGGGSGGGGSDDDDKGGGGSGGGGSGGGGS (SEQ ID NO: 125).

Using one of a variety of reverse translation software tools (for example Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the linker region is determined. BamHI/SalI and PstI/XbaI/stop codon/HindIII restriction enzyme sequences are incorporated at either end, in the correct reading frames. The DNA sequence is screened (using software such as Seqbuilder, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed by the Backtranslation tool from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. If it is desired to clone the linker out of pCR 4 vector, the vector is cleaved with either BamHI+SalI or PstI+XbaI combination restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of either the LC DNA (cleaved with BamHI/SalI) or H.sub.N DNA (cleaved with PstI/XbaI) from (SEQ ID 2). Once the LC or the H.sub.N encoding DNA is inserted upstream or downstream of the linker DNA, the entire LC-linker or linker-H.sub.N DNA fragment can the be isolated and transferred to the backbone clone.

Assembly and Confirmation of the Backbone Clone

The LC-linker or the linker-H.sub.N is cut out from the pCR 4 cloning vector using BamHI/PstI or SalI/XbaI restriction enzymes or XhoI/NotI digests respectively. The pET expression vector containing LH.sub.N/A (SEQ ID 2) is digested with the same enzymes but is also treated with antarctic phosphatase as an extra precaution to prevent re-circularisation. The LC-linker or linker-H.sub.N region and the pET vector backbone are gel purified. The purified insert and vector backbone are ligated together using T4 DNA ligase and the product is transformed with TOP10 cells which are then screened for LC-linker or linker-H.sub.N insertion using BamHI/SalI or BamHI/PstI or XhoI/NotI restriction enzymes to ensure the final backbone is correct. The integrity of the ORF DNA is checked by sequencing.

EXAMPLE 2

Construction of LH.sub.N/A-EGFv3 Expression Vector

The following procedure creates a clone for use as an expression construct for multidomain fusion expression. This example is based on preparation of an EGFv3 fusion protein (eg. SEQ ID 37), though the procedures and methods are equally applicable to all EGF variants of the present invention, as well as to other LH.sub.N serotypes such as serotype B (SEQ ID 3), serotype C (SEQ ID 4) and serotype D (SEQ ID 5) fusion proteins of the present invention.

Preparation of Spacer-EGFv3 Insert

Activation Spacer

For presentation of EGF variant sequence at the C-terminus of the H.sub.N domain, a DNA sequence is designed to flank the spacer and targeting moiety (TM) regions allowing incorporation into the backbone clone (SEQ ID 1). The DNA sequence can be arranged as BamHI-SalI-PstI-XbaI-spacer-EGFv3-stop codon-HindIII. The DNA sequence can be designed using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)). Once the TM DNA is designed, the additional DNA required to encode the preferred spacer is created in silico. It is important to ensure the correct reading frame is maintained for the spacer, EGFv3 and restriction sequences and that the XbaI sequence is not preceded by the bases, TC which would result in DAM methylation. The DNA sequence is screened for restriction sequence incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the % GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

Insertion of Spacer-EGFv3 into Backbone

In order to create a LC-linker-H.sub.N-spacer-EGFv3 construct using the backbone construct (SEQ ID 1) and the newly synthesised pCR 4-spacer-TM vector encoding the EGFv3 .TM. (SEQ ID 73), the following two-step method is employed. Firstly, the H.sub.N domain is excised from the backbone clone using restriction enzymes PstI and XbaI and ligated into similarly digested pCR 4-spacer-EGFv3 vector. This creates an H.sub.N-spacer-EGFv3 ORF in pCR 4 that can be excised from the vector using restriction enzymes PstI and HindIII for subsequent ligation into similarly cleaved backbone or expression construct. The final construct contains the LC-linker-H.sub.N-spacer-EGFv3 ORF for transfer into expression vectors for expression to result in a fusion protein of the sequence (eg. SEQ ID 37).

Screening with restriction enzymes is sufficient to ensure the final backbone is correct as all components are already sequenced confirmed, either during synthesis or following PCR amplification. However, during the sub-cloning of some components into the backbone, where similar size fragments are being removed and inserted, sequencing of a small region to confirm correct insertion is required.

EXAMPLE 3

Expression and Purification of a LH.sub.N/A-CT-EGF v3 Fusion Protein

This example is based on preparation of a CT-EGF-A fusion activated by Factor Xa, though the procedures and methods are equally applicable to any other fusion protein of the present invention.

Expression of LH.sub.N/A-CT-EGF v3 Fusion Protein

Expression of the CT-EGF v3-A fusion activated by Factor Xa fusion protein is achieved using the following protocol. Inoculate 100 ml of modified TB containing 0.2% glucosamine and 30 .mu.g/ml kanamycin in a 250 ml flask with a single colony from the CT-EGF v3-A fusion. Grow the culture at 37.degree. C., 225 rpm for 16 hours. Inoculate 1 L of modified TB containing 0.2% glucosamine and 30 .mu.g/ml kanamycin in a 2 L flask with 10 ml of overnight culture. Grow cultures at 37.degree. C. until an approximate OD.sub.600nm of 0.5 is reached at which point reduce the temperature to 16.degree. C. After 1 hour induce the cultures with 1 mM IPTG and grow at 16.degree. C. for a further 16 hours.

Purification of CT-EGF v3-A Fusion

Defrost falcon tube containing 35 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 (DE3) cell paste. Sonicate the cell paste on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4.degree. C. for 30 minutes. Load the supernatant onto a 0.1 M NiSO.sub.4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Using a step gradient of 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4.degree. C. overnight and measure the OD of the dialysed fusion protein. Add 10 mg of Factor Xa per 1 mg fusion protein and incubate at 25.degree. C. static overnight. Load onto a 0.1 M NiSO.sub.4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 40 and 100 mM imidazole, wash away the non-specific bound protein and elute the fusion protein with 200 mM imidazole. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2 150 mM NaCl at 4.degree. C. overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20.degree. C. Test purified protein using OD, BCA and purity analysis.

EXAMPLE 4

EGF Binding Affinity Assay

To compare binding affinity of EGF variants to wtEGF, a cell (NR6, a murine 3T3-derived fibroblast cell line; WT-EGF) that lacks endogenous EGF receptor (ErbB:-EGFR) is stably transfected with wild-type human EGFR to generate WT+EGF. Prior to performing binding assays, confluent WT-EGF cells were dislodged from tissue culture plates with Versene. EGF competition binding was measured in two ways to insure that equilibrium had been reached: 5.times.10.sup.4 NR6WT cells were incubated with Alexa-488 labelled EGF wild-type for 30 min at 4.degree. C. Increasing concentrations of unlabeled EGF wild-type or mutants were added, and samples were incubated for an additional 6 h at 4.degree. C., with constant mixing. Alternatively, increasing concentrations of unlabeled EGF wild-type and mutants were first added to the cells for 30 min at 4.degree. C., and Alexa-488 EGF wild-type was added for an additional 6 h at 4.degree. C. Fluorescence intensity of cell surface Alexa-488 EGF wild-type labeling was measured by flow cytometry. Binding assays were performed in PBS supplemented with 1 mg/ml BSA (pH 7.4), under conditions where ligand depletion was negligible. Competition binding curves were fit using a four-point binding equation. Standard deviation represents replicate binding experiments performed in at least triplicate on different days using two different protein preparations.

EXAMPLE 5

EGF Binding Affinity Assay

To compare binding affinity of EGF variants for the EGF receptor the affinity was determined by measuring its competition with .sup.125I-rEGF (recombinant EGF) for binding to paraformaldehyde-fixed A431 cells, a system wherein ligand and receptor internalisation processes are thus inhibited. Pure rEGF was used as a standard. A431 cells were grown to confluence in 96-well plates and then fixed on the day of the assay with 3% paraformaldehyde in PBS. The EGF variants and rEGF were serially diluted with 1.0 nM .sup.125I-rEGF in PBS containing 0.1% BSA. Incubations were then carried out at 37.degree. C. for 2 hours before each well was washed 3 times with PBS containing 0.15 mg/ml BSA. Finally wells were snapped apart and counted directly in a gamma counter.

EXAMPLE 6

Treatment of a Patient Suffering from Chronic Bronchitis

A 62 year old male suffering from chronic bronchitis (FEV.sub.1 reduced to 80% of normal predicted value; daily sputum volume of 30 ml) presents at his GP. Despite treatment with inhaled steroids, the patient presents with difficulty in performing everyday tasks due continued shortness of breath. The GP prescribes a 6-month course of an EGF mutein fusion protein according to the present invention in nebuliser form, 80 .mu.g to be taken monthly. Following discussion with the physician, the patient selects the most appropriate nebuliser for their personal situation from a range of suitable devices. After a single dose of EGF-based fusion protein experiences reduced sputum volume (to 15 ml) and an improvement in FEV1 (to 90%).

EXAMPLE 7

Method for Treating Acromegalic Patients Resistant to Somatostatin Analogues

After six years' successful control of circulating GH and IGF-1 by somatostatin analogues (SSA), a 60-year-old acromegalic fairground tarot reader reports increasingly obvious oily skin and also prominent body odour as a result of hyperhydrosis. She is found to be glucose-intolerant and to have elevated circulating IGF-1 levels and raising the SSA dosage does not control these. She is treated by localised injection of an EGF mutein fusion protein according to the present invention. Within 14 days the patient reports a significant reduction in sweating. Over the following month her oily skin returns to normal and at this time her GH and IGF-1 levels are both within the normal range. This situation remains over the next five years.

EXAMPLE 8

Method for Suppressing Neuroendocrine Tumour Cells

A 35 year old male member of a regional badminton team undergoes a spinal X-ray for lower back pain. The consultant notices abnormal bone growth and, on questioning, the man reports increasing incidents of sleep apnoea and also increasingly oily skin. The physician recommends measurement of circulating IGF-1 and these are found to be elevated. Subsequent tests also show above-normal circulating GH levels so a cranial MRI scan is carried out. This shows a pituitary tumour of 9 mm diameter. The patient is treated with an EGF mutein fusion protein of the present invention by injection. At intervals of 1 week circulating IGF-1 levels are measured and are seen to be lower at the first measurement and to reduce steadily to 15% above normal over the following six weeks. The level of circulating GH is found to be normal at this time. A further dose of the medication with two-weekly IGF-1 measurements shows this hormone to have stabilised at the upper end of normal. At six weeks after the second treatment a cranial MRI scan reveals shrinkage of the tumour to 6 mm. The therapy is continued at a reduced dosage at two-monthly intervals with IGF-1 and GH levels measured on the seventh week. These are both stable in the normal range and the sleep apnoea and oily skin are now absent. A spinal X-ray at one year following the first treatment shows no increased bone size from the original observation.

EXAMPLE 9

Comparison of EGF and EGF fusion proteins in an EGF receptor activation assay

A431 cells (1.times.10.sup.5) were incubated with increasing concentrations of EGF, SXN100516 (LH.sub.N/A-EGF), SXN100988 (LH.sub.N/B-EGF) or SXN100501 (LH.sub.N/C-EGF) for 20 min at 37.degree. C.

Cells were washed with ice-cold PBS and lysed. Lysates were diluted 1:10 before the level of phosphorylated ErbB1 receptor was measured using a sandwich immunoassay and MSD platform.

TABLE-US-00008 pEC.sub.50 estimates of Syntaxin molecules at the ErbB1 receptor in A431 cells Agonist/SXN Chimaera pEC.sub.50 .+-. s.e.mean EGF per se 9.13 .+-. 0.13 SXN100501 fusion protein 7.15 .+-. 0.11 SXN100516 fusion protein 7.47 .+-. 0.16 SXN100988 fusion protein 7.61 .+-. 0.19

EXAMPLE 10

Comparison of EGF, EGF Mutein and EGF Mutein Fusion Proteins in an EGF Receptor Activation Assay

A431 cells (1.times.10.sup.5) were incubated with increasing concentrations of Syntaxin EGF mutein fusion or EGF for 20 min at 37.degree. C. in triplicate eppendorf tubes. After washing, the cells were lysed and the lysates added to MSD 96 well plates coated with a capture antibody specific for phosphoY1068 of the EGF receptor.

Following incubation, the plates were washed and incubated with an anti-phospho EGF receptor antibody labeled with an electrochemiluminescent MSD SULFO-TAG. MSD Read buffer was added to the plate and the light emitted from each well of the plate (RLU) measured on the MSD sector imager 6000.

Molecules and Syntaxin Chimaeras Tested

TABLE-US-00009 SXN Number/ligand Batch Construct SXN101181 LC080805 pK7-Hx-EGFv3 SXN101784 OW090711 pK7-LcA-XA-HnA-EGFv3-10HT SXN101886 JW090728B pK7-6HT-Xa-LC-Xa-HC-GS20- EGFv3N16Y SXN101887 JW090729 pK7-6HT-Xa-LC-Xa-HC-GS20- EGFv3L49W

Results (See FIG. 1)

TABLE-US-00010 Ligand/Chimaera pEC.sub.50 EGF 8.74 .+-. 0.04 EGFv3 7.61 SXN101181 8.47 .+-. 0.08 SXN101784 8.13 SXN101886 7.04 SXN101887 7.69

There is no significant difference in the pEC.sub.50 of SXN101181 compared to EGF (p>0.05, t-test).

A log-unit difference in potency (pEC.sub.50) was observed between EGF (8.74.+-.0.04) and EGFv3 (7.61).

EXAMPLE 11

Treatment of a Patient Suffering from Rhinitis

A 24 years old female who presents annually with typical seasonal allergic rhinitis symptoms is treated by local administration (by nasal spray) with an EGF mutein fusion protein of the present invention. Within 2 to 7 days the patient reports a subsidence of symptoms. The effect is sustained for the remainder of the allergy season.

EXAMPLE 12

Treatment of a Patient Suffering from Asthma

A 43 years old female with a reduced quality of life due to chronic asthma is treated with by systemic injection of an EGF mutein fusion protein of the present invention. Within 3 to 7 days the symptoms of the patient have cleared and the patient can breathe more freely. The effect is sustained for 2 to 3 months, whereupon the treatment is repeated and the improvement to the quality of the patient's life is sustained.

EXAMPLE 13

Treatment of a Patient Suffering from COPD

A 64 years old male with chronic obstructive pulmonary disease who has a reduced quality of life due to inability to breath effectively is treated by local administration (by aerosol) of an EGF mutein fusion protein of the present invention. Within 2 to 5 days the patient has cleared most of the excess airway mucus and can breathe more freely. The effect is sustained for 2 to 3 months, whereupon the treatment is repeated and the improvement to the quality of the patient's life is sustained.

EXAMPLE 14

Treatment of a Patient Suffering from Asthma

A 25 years old male with severe exacerbation of his asthmatic symptoms due to a rhinovirus infection is treated by local administration (inhalation by aerosol) with an EGF mutein fusion protein of the present invention. Within 2 to 7 days the patient reports reduced airway mucus and the exacerbation subsides.

EXAMPLE 15

Treatment of a Patient Suffering from Colon Cancer

A 32 years old male patient with jaundice is diagnosed with advanced hepatocellular carcinoma which has spread to the colon. The patient is treated with a systemic injection of an EGF mutein fusion protein of the present invention. Within two to three weeks the growth of the metastasized tumours has been arrested. Two months after the treatment the tumours have decreased in size and the jaundice has gone. A second application of treatment continues the decrease in tumour size and maintains the alleviation of symptoms.

EXAMPLE 16

Treatment of a Patient Suffering from Psoriasis

A 32 years old male patient diagnosed with psoriasis and who is experiencing significant physical discomfort from itching and scratching the affected areas of his back and arms is treated by topical administration with an EGF mutein fusion protein of the present invention. Within 2 to 7 days the symptoms are relieved and the effect lasts for 1 to 2 months. A second application of treatment maintains the alleviation of symptoms.

EXAMPLE 17

Treatment of a Patient Suffering from Eczema

A 45 years old male patient diagnosed with eczema and who is experiencing significant physical discomfort from itching and scratching the affected areas of his buttocks and legs is treated by topical administration with an EGF mutein fusion protein of the present invention. Within 2 to 7 days the symptoms are relieved and the effect lasts for 1 to 2 months. A second application of treatment maintains the alleviation of symptoms.

EXAMPLE 18

Treatment of a Patient Suffering from Colorectal Cancer

A 70 years old male patient is diagnosed with (stage IV) colorectal cancer. Surgery is not recommended. After treatment with a systemic injection of an EGF mutein fusion protein of the present invention the metastasised tumours have stopped increasing in size. Within one month the tumours have decreased in size and the patient reports feeling better. A second application of treatment further reduces the tumour size and maintains the alleviation of symptoms.

EXAMPLE 19

Treatment of a Patient Suffering from Prostate Cancer

A 46 years old male patient is diagnosed with prostate cancer which has metastasized to the vertebrae. He complains of pains in the spine and pelvis. As surgery is inappropriate the patient is treated with a systemic injection of an EGF mutein fusion protein of the present invention. Within two weeks the tumours have decreased in size and the patient reports less pain. A second application of treatment continues the decrease in tumour size and maintains the alleviation of symptoms.

EXAMPLE 20

Treatment of a Patient Suffering from nsc Lung Cancer

A 46 years old female patient is diagnosed with (stage IV) non-small cell lung carcinoma with a prognosis of 2% chance of living two years. The patient is treated with a systemic injection of an EGF mutein fusion protein of the present invention. Within two weeks the growth rate of the metastasized tumours has been arrested. Two months after the treatment the tumours have decreased in size and the patient feels better. A second application of treatment continues to decrease the tumour size and maintains the alleviation of symptoms. The patient increases her chances of survival beyond two years.

EXAMPLE 21

Treatment of a Patient Suffering from Breast Cancer

A 36 years old female patient with jaundice is diagnosed with advanced breast cancer which has spread to the liver. The patient is treated with a systemic injection of an EGF mutein fusion protein of the present invention. Within two weeks the growth rate of the metastasized tumour has been arrested. Two months after the treatment the tumours have decreased in size and the jaundice has gone. A second application of treatment continues the decrease in tumour size and maintains the alleviation of symptoms.

TABLE-US-00011 SEQ ID NOS SEQ ID 1 Amino acid sequence for naturally-occurring, human epidermal growth factor (EGF) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 2 DNA sequence of LHA ATGGAGTTCGTTAACAAACAGTTCAACTATAAAGACCCAGTTAACGGTGTTGACATTGCTT ACATCAAAATCCCGAACGCTGGCCAGATGCAGCCGGTAAAGGCATTCAAAATCCACAACA AAATCTGGGTTATCCCGGAACGTGATACCTTTACTAACCCGGAAGAAGGTGACCTGAACC CGCCACCGGAAGCGAAACAGGTGCCGGTATCTTACTATGACTCCACCTACCTGTCTACC GATAACGAAAAGGACAACTACCTGAAAGGTGTTACTAAACTGTTCGAGCGTATTTACTCC ACCGACCTGGGCCGTATGCTGCTGACTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGG TTCTACCATCGATACCGAACTGAAAGTAATCGACACTAACTGCATCAACGTTATTCAGCCG GACGGTTCCTATCGTTCCGAAGAACTGAACCTGGTGATCATCGGCCCGTCTGCTGATATC ATCCAGTTCGAGTGTCTGAGCTTTGGTCACGAAGTTCTGAACCTCACCCGTAACGGCTAC GGTTCCACTCAGTACATCCGTTTCTCTCCGGACTTCACCTTCGGTTTTGAAGAATCCCTG GAAGTAGACACGAACCCACTGCTGGGCGCTGGTAAATTCGCAACTGATCCTGCGGTTAC CCTGGCTCACGAACTGATTCATGCAGGCCACCGCCTGTACGGTATCGCCATCAATCCGA ACCGTGTCTTCAAAGTTAACACCAACGCGTATTACGAGATGTCCGGTCTGGAAGTTAGCT TCGAAGAACTGCGTACTTTTGGCGGTCACGACGCTAAATTCATCGACTCTCTGCAAGAAA ACGAGTTCCGTCTGTACTACTATAACAAGTTCAAAGATATCGCATCCACCCTGAACAAAGC GAAATCCATCGTGGGTACCACTGCTTCTCTCCAGTACATGAAGAACGTTTTTAAAGAAAAA TACCTGCTCAGCGAAGACACCTCCGGCAAATTCTCTGTAGACAAGTTGAAATTCGATAAA CTTTACAAAATGCTGACTGAAATTTACACCGAAGACAACTTCGTTAAGTTCTTTAAAGTTCT GAACCGCAAAACCTATCTGAACTTCGACAAGGCAGTATTCAAAATCAACATCGTGCCGAA AGTTAACTACACTATCTACGATGGTTTCAACCTGCGTAACACCAACCTGGCTGCTAATTTT AACGGCCAGAACACGGAAATCAACAACATGAACTTCACAAAACTGAAAAACTTCACTGGT CTGTTCGAGTTTTACAAGCTGCTGTGCGTCGACGGCATCATTACCTCCAAAACTAAATCT GACGATGACGATAAAAACAAAGCGCTGAACCTGCAGTGTATCAAGGTTAACAACTGGGAT TTATTCTTCAGCCCGAGTGAAGACAACTTCACCAACGACCTGAACAAAGGTGAAGAAATC ACCTCAGATACTAACATCGAAGCAGCCGAAGAAAACATCTCGCTGGACCTGATCCAGCAG TACTACCTGACCTTTAATTTCGACAACGAGCCGGAAAACATTTCTATCGAAAACCTGAGCT CTGATATCATCGGCCAGCTGGAACTGATGCCGAACATCGAACGTTTCCCAAACGGTAAAA AGTACGAGCTGGACAAATATACCATGTTCCACTACCTGCGCGCGCAGGAATTTGAACACG GCAAATCCCGTATCGCACTGACTAACTCCGTTAACGAAGCTCTGCTCAACCCGTCCCGTG TATACACCTTCTTCTCTAGCGACTACGTGAAAAAGGTCAACAAAGCGACTGAAGCTGCAA TGTTCTTGGGTTGGGTTGAACAGCTTGTTTATGATTTTACCGACGAGACGTCCGAAGTAT CTACTACCGACAAAATTGCGGATATCACTATCATCATCCCGTACATCGGTCCGGCTCTGA ACATTGGCAACATGCTGTACAAAGACGACTTCGTTGGCGCACTGATCTTCTCCGGTGCG GTGATCCTGCTGGAGTTCATCCCGGAAATCGCCATCCCGGTACTGGGCACCTTTGCTCT GGTTTCTTACATTGCAAACAAGGTTCTGACTGTACAAACCATCGACAACGCGCTGAGCAA ACGTAACGAAAAATGGGATGAAGTTTACAAATATATCGTGACCAACTGGCTGGCTAAGGT TAATACTCAGATCGACCTCATCCGCAAAAAAATGAAAGAAGCACTGGAAAACCAGGCGGA AGCTACCAAGGCAATCATTAACTACCAGTACAACCAGTACACCGAGGAAGAAAAAAACAA CATCAACTTCAACATCGACGATCTGTCCTCTAAACTGAACGAATCCATCAACAAAGCTATG ATCAACATCAACAAGTTCCTGAACCAGTGCTCTGTAAGCTATCTGATGAACTCCATGATCC CGTACGGTGTTAAACGTCTGGAGGACTTCGATGCGTCTCTGAAAGACGCCCTGCTGAAA TACATTTACGACAACCGTGGCACTCTGATCGGTCAGGTTGATCGTCTGAAGGACAAAGTG AACAATACCTTATCGACCGACATCCCTTTTCAGCTCAGTAAATATGTCGATAACCAACGCC TTTTGTCCACT SEQ ID 3 DNA sequence of LHB ATGCCGGTTACCATCAACAACTTCAACTACAACGACCCGATCGACAACAACAACATCATTA TGATGGAACCGCCGTTCGCACGTGGTACCGGACGTTACTACAAGGCTTTTAAGATCACC GACCGTATCTGGATCATCCCGGAACGTTACACCTTCGGTTACAAACCTGAGGACTTCAAC AAGAGTAGCGGGATTTTCAATCGTGACGTCTGCGAGTACTATGATCCAGATTATCTGAAT ACCAACGATAAGAAGAACATATTCCTTCAGACTATGATTAAACTCTTCAACCGTATCAAAA GCAAACCGCTCGGTGAAAAACTCCTCGAAATGATTATCAACGGTATCCCGTACCTCGGTG ACCGTCGTGTCCCGCTTGAAGAGTTCAACACCAACATCGCAAGCGTCACCGTCAACAAAC TCATCAGCAACCCAGGTGAAGTCGAACGTAAAAAAGGTATCTTCGCAAACCTCATCATCT TCGGTCCGGGTCCGGTCCTCAACGAAAACGAAACCATCGACATCGGTATCCAGAACCAC TTCGCAAGCCGTGAAGGTTTCGGTGGTATCATGCAGATGAAATTCTGCCCGGAATACGTC AGTGTCTTCAACAACGTCCAGGAAAACAAAGGTGCAAGCATCTTCAACCGTCGTGGTTAC TTCAGCGACCCGGCACTCATCCTCATGCATGAACTCATCCACGTCCTCCACGGTCTCTAC GGTATCAAAGTTGACGACCTCCCGATCGTCCCGAACGAGAAGAAATTCTTCATGCAGAGC ACCGACGCAATCCAGGCTGAGGAACTCTACACCTTCGGTGGCCAAGACCCAAGTATCAT AACCCCGTCCACCGACAAAAGCATCTACGACAAAGTCCTCCAGAACTTCAGGGGTATCGT GGACAGACTCAACAAAGTCCTCGTCTGCATCAGCGACCCGAACATCAATATCAACATATA CAAGAACAAGTTCAAAGACAAGTACAAATTCGTCGAGGACAGCGAAGGCAAATACAGCAT CGACGTAGAAAGTTTCGACAAGCTCTACAAAAGCCTCATGTTCGGTTTCACCGAAACCAA CATCGCCGAGAACTACAAGATCAAGACAAGGGCAAGTTACTTCAGCGACAGCCTCCCGC CTGTCAAAATCAAGAACCTCTTAGACAACGAGATTTACACAATTGAAGAGGGCTTCAACAT CAGTGACAAAGACATGGAGAAGGAATACAGAGGTCAGAACAAGGCTATCAACAAACAGG CATACGAGGAGATCAGCAAAGAACACCTCGCAGTCTACAAGATCCAGATGTGCGTCGAC GGCATCATTACCTCCAAAACTAAATCTGACGATGACGATAAAAACAAAGCGCTGAACCTG CAGTGCATCGACGTTGACAACGAAGACCTGTTCTTCATCGCTGACAAAAACAGCTTCAGT GACGACCTGAGCAAAAACGAACGTATCGAATACAACACCCAGAGCAACTACATCGAAAAC GACTTCCCGATCAACGAACTGATCCTGGACACCGACCTGATAAGTAAAATCGAACTGCCG AGCGAAAACACCGAAAGTCTGACCGACTTCAACGTTGACGTTCCGGTTTACGAAAAACAG CCGGCTATCAAGAAAATCTTCACCGACGAAAACACCATCTTCCAGTACCTGTACAGCCAG ACCTTCCCGCTGGACATCCGTGACATCAGTCTGACCAGCAGTTTCGACGACGCTCTGCT GTTCAGCAACAAAGTTTACAGTTTCTTCAGCATGGACTACATCAAAACCGCTAACAAAGTT GTTGAAGCAGGGCTGTTCGCTGGTTGGGTTAAACAGATCGTTAACGACTTCGTTATCGAA GCTAACAAAAGCAACACTATGGACAAAATCGCTGACATCAGTCTGATCGTTCCGTACATC GGTCTGGCTCTGAACGTTGGTAACGAAACCGCTAAAGGTAACTTTGAAAACGCTTTCGAG ATCGCTGGTGCAAGCATCCTGCTGGAGTTCATCCCGGAACTGCTGATCCCGGTTGTTGG TGCTTTCCTGCTGGAAAGTTACATCGACAACAAAAACAAGATCATCAAAACCATCGACAAC GCTCTGACCAAACGTAACGAAAAATGGAGTGATATGTACGGTCTGATCGTTGCTCAGTGG CTGAGCACCGTCAACACCCAGTTCTACACCATCAAAGAAGGTATGTACAAAGCTCTGAAC TACCAGGCTCAGGCTCTGGAAGAGATCATCAAATACCGTTACAACATCTACAGTGAGAAG GAAAAGAGTAACATCAACATCGACTTCAACGACATCAACAGCAAACTGAACGAAGGTATC AACCAGGCTATCGACAACATCAACAACTTCATCAACGGTTGCAGTGTTAGCTACCTGATG AAGAAGATGATCCCGCTGGCTGTTGAAAAACTGCTGGACTTCGACAACACCCTGAAAAAG AACCTGCTGAACTACATCGACGAAAACAAGCTGTACCTGATCGGTAGTGCTGAATACGAA AAAAGTAAAGTGAACAAATACCTGAAGACCATCATGCCGTTCGACCTGAGTATCTACACC AACGACACCATCCTGATCGAAATGTTCAACAAATACAACTCT SEQ ID 4 DNA sequence of LHC ATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTGT ACCTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACTCA AAACATTTGGGTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAACCT CCGCGTCCGACGTCCAAATATCAGAGCTATTACGATCCCTCATATCTCAGTACGGACGAA CAAAAAGATACTTTCCTTAAAGGTATCATTAAACTGTTTAAGCGTATTAATGAGCGCGATA TCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGTTCCCCGTTCATGGGCGATAGCTCTA CCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATCGCGGTAGAGAAGTTTG AGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTTGGTCCGC TGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGA GTTTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTT TTTCAGATGTCACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGG ACCCGGTTATTGCACTGATGCACGAACTGACGCACTCTCTGCATCAACTGTATGGGATCA ACATCCCCAGTGACAAACGTATTCGTCCCCAGGTGTCTGAAGGATTTTTCTCACAGGATG GGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGAGGCCTGGACGTAGAGATCATT CCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTATAAGGATATTGC AAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGATAAA TATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTTGTGGT TAACATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTG TATAGTTCCCAATACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGG TTTTCGCGAACATCCTTGACGATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAA CAAGGGCTTCAATATTGAAAATTCAGGCCAGAACATTGAACGCAACCCGGCCTTGCAGAA ACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAAGTCTGCGTCGACAAAAGCGAAGA GAAGCTGTACGATGACGATGACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGT GAAAAACAATCGGCTGCCTTATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGA AAATAAAATTATCACTGACGAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACG AAAGCATCTTAGATGGCCAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGA ACGTGAATATGGAACCGTTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTA CGAAATATGTGGACTACCTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAA CGTGGAAAACATTACTCTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGAT TTATACCTTCCTCCCGTCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTT CCTCAACTGGGCGAATGAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATAC CCTGGATAAAATCTCCGACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATC GGTAATAGTGCGCTGCGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTT CCTCCTGGAGGGCTTTCCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCT TCCATCCAGGAGCGTGAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGT GAAACGCTGGAAAGATTCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGAC CCAATTCAACCATATTAATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATT AAAGCCAAAATTGATCTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAA GCCAGGTGGAGAACCTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACA TTAACAAATTCATTCGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGT TATTGATGAACTGAATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGAC TCCCACAACATTATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAG CTTTGAAAATACGATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATA TCATCAATGAATATTTCAAT SEQ ID 5 DNA sequence of LHD ATGACGTGGCCAGTTAAGGATTTCAACTACTCAGATCCTGTAAATGACAACGATATTCTGT ACCTTCGCATTCCACAAAATAAACTGATCACCACACCAGTCAAAGCATTCATGATTACTCA AAACATTTGGGTCATTCCAGAACGCTTTTCTAGTGACACAAATCCGAGTTTATCTAAACCT CCGCGTCCGACGTCCAAATATCAGAGCTATTACGATCCCTCATATCTCAGTACGGACGAA CAAAAAGATACTTTCCTTAAAGGTATCATTAAACTGTTTAAGCGTATTAATGAGCGCGATA TCGGGAAAAAGTTGATTAATTATCTTGTTGTGGGTTCCCCGTTCATGGGCGATAGCTCTA CCCCCGAAGACACTTTTGATTTTACCCGTCATACGACAAACATCGCGGTAGAGAAGTTTG AGAACGGATCGTGGAAAGTCACAAACATCATTACACCTAGCGTCTTAATTTTTGGTCCGC TGCCAAACATCTTAGATTATACAGCCAGCCTGACTTTGCAGGGGCAACAGTCGAATCCGA GTTTCGAAGGTTTTGGTACCCTGAGCATTCTGAAAGTTGCCCCGGAATTTCTGCTCACTT TTTCAGATGTCACCAGCAACCAGAGCTCAGCAGTATTAGGAAAGTCAATTTTTTGCATGG ACCCGGTTATTGCACTGATGCACGAACTGACGCACTCTCTGCATCAACTGTATGGGATCA ACATCCCCAGTGACAAACGTATTCGTCCCCAGGTGTCTGAAGGATTTTTCTCACAGGATG GGCCGAACGTCCAGTTCGAAGAGTTGTATACTTTCGGAGGCCTGGACGTAGAGATCATT CCCCAGATTGAGCGCAGTCAGCTGCGTGAGAAGGCATTGGGCCATTATAAGGATATTGC AAAACGCCTGAATAACATTAACAAAACGATTCCATCTTCGTGGATCTCGAATATTGATAAA TATAAGAAAATTTTTAGCGAGAAATATAATTTTGATAAAGATAATACAGGTAACTTTGTGGT TAACATTGACAAATTCAACTCCCTTTACAGTGATTTGACGAATGTAATGAGCGAAGTTGTG TATAGTTCCCAATACAACGTTAAGAATCGTACCCATTACTTCTCTCGTCACTACCTGCCGG TTTTCGCGAACATCCTTGACGATAATATTTACACTATTCGTGACGGCTTTAACTTGACCAA CAAGGGCTTCAATATTGAAAATTCAGGCCAGAACATTGAACGCAACCCGGCCTTGCAGAA ACTGTCGAGTGAATCCGTGGTTGACCTGTTTACCAAAGTCTGCGTCGACAAAAGCGAAGA GAAGCTGTACGATGACGATGACAAAGATCGTTGGGGATCGTCCCTGCAGTGTATTAAAGT GAAAAACAATCGGCTGCCTTATGTAGCAGATAAAGATAGCATTAGTCAGGAGATTTTCGA AAATAAAATTATCACTGACGAAACCAATGTTCAGAATTATTCAGATAAATTTTCACTGGACG AAAGCATCTTAGATGGCCAAGTTCCGATTAACCCGGAAATTGTTGATCCGTTACTGCCGA ACGTGAATATGGAACCGTTAAACCTCCCTGGCGAAGAGATCGTATTTTATGATGACATTA CGAAATATGTGGACTACCTTAATTCTTATTACTATTTGGAAAGCCAGAAACTGTCCAATAA CGTGGAAAACATTACTCTGACCACAAGCGTGGAAGAGGCTTTAGGCTACTCAAATAAGAT TTATACCTTCCTCCCGTCGCTGGCGGAAAAAGTAAATAAAGGTGTGCAGGCTGGTCTGTT CCTCAACTGGGCGAATGAAGTTGTCGAAGACTTTACCACGAATATTATGAAAAAGGATAC CCTGGATAAAATCTCCGACGTCTCGGTTATTATCCCATATATTGGCCCTGCGTTAAATATC GGTAATAGTGCGCTGCGGGGGAATTTTAACCAGGCCTTTGCTACCGCGGGCGTCGCGTT CCTCCTGGAGGGCTTTCCTGAATTTACTATCCCGGCGCTCGGTGTTTTTACATTTTACTCT TCCATCCAGGAGCGTGAGAAAATTATCAAAACCATCGAAAACTGCCTGGAGCAGCGGGT GAAACGCTGGAAAGATTCTTATCAATGGATGGTGTCAAACTGGTTATCTCGCATCACGAC CCAATTCAACCATATTAATTACCAGATGTATGATAGTCTGTCGTACCAAGCTGACGCCATT AAAGCCAAAATTGATCTGGAATATAAAAAGTACTCTGGTAGCGATAAGGAGAACATCAAAA GCCAGGTGGAGAACCTTAAGAATAGTCTGGATGTGAAAATCTCTGAAGCTATGAATAACA TTAACAAATTCATTCGTGAATGTTCGGTGACGTACCTGTTCAAGAATATGCTGCCAAAAGT TATTGATGAACTGAATAAATTTGATCTGCGTACCAAAACCGAACTTATCAACCTCATCGAC TCCCACAACATTATCCTTGTGGGCGAAGTGGATCGTCTGAAGGCCAAAGTAAACGAGAG CTTTGAAAATACGATGCCGTTTAATATTTTTTCATATACCAATAACTCCTTGCTGAAAGATA TCATCAATGAATATTTCAAT SEQ ID6 EGFH16N NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID7 Protein sequence of a EGF variant targeting moiety v1 NSDSECPLSHDGYCLHGGVCMYIKAVDRYACNCVVGYIGERCQYRDLTWWGPR SEQ ID8 EGFH16Q NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID9 Protein sequence of a EGF variant targeting moiety v2 SRGSKCPPSHDGYCLQGGVCMYIEALDRYACNCVVGYAGERCQYRDLTWWGRR SEQ ID10 EGFW49L NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKLWELR SEQ ID11 Protein sequence of a EGF variant targeting moiety v3 NSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID12 EGFW49I NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKIWELR SEQ ID13 Protein sequence of a EGF variant targeting moiety v4 NSYSECPPSYDGYCLHDGVCRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR SEQ ID14 EGFW49V NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKVWELR SEQ ID15 Protein sequence of a EGF variant targeting moiety v5 NSDSGCPSFHDGYCLNGGVCMYIEALDKYACNCVIGYNGDRCQTRDLKWWELR SEQ ID16 EGFW49A NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKAWELR SEQ ID17 Protein sequence of a EGF variant targeting moiety v6 (G12Q) NSDSECPLSHDQYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID18 EGFW49G NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKGWELR SEQ ID19 Protein sequence of a EGF variant targeting moiety v7 (H16D) NSDSECPLSHDGYCLDDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID20 EGFW49S NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKSWELR SEQ ID21 Protein sequence of a EGF variant targeting moiety v8 (Y13W) NSDSECPLSHDGWCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID22 EGFW49T NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKTWELR SEQ ID23 Protein sequence of a EGF variant targeting moiety v9 (Q43A) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCAYRDLKWWELR SEQ ID24 EGFW49N NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKNWELR SEQ ID25 Protein sequence of a EGF variant targeting moiety v10 (H16A)

NSDSECPLSHDGYCLADGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID26 EGF_W49Q NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKQWELR SEQ ID27 Protein sequence of a EGF variant targeting moiety v11 (L15A) NSDSECPLSHDGYCAHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID28 EGF_H16N_W49L NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKLWELR SEQ ID29 Protein sequence of a EGF variant targeting moiety v12 (V19E) NSDSECPLSHDGYCLHDGECMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID30 EGF_H16Q_W49L NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKLWELR SEQ ID31 Protein sequence of a EGF variant targeting moiety v13 (V34D) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCDVGYIGERCQYRDLKWWELR SEQ ID 32 EGF_H16N_W49I NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKIWELR SEQ ID 33 Protein sequence of LHA-EGFv1 (Xa activation) MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSIDGRNKALNL QCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDSECPLSHDQYCLHDGV CMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 34 EGF_H16Q_W49I NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKIWELR SEQ ID 35 Protein sequence of LHA-EGFv2 (Xa activation) MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSIDGRNKALNL QCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDSRGSKCPPSHDGYCLQGG VCMYIEALDRYACNCVVGYAGERCQYRDLTWWGRR SEQ ID 36 EGF_H16N_W50A NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWAELR SEQ ID 37 Protein sequence of LHA-EGFv3 (enhanced mutation MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECLSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALN LQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGV CMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID 38 EGF_H16Q_W50A NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWAELR SEQ ID 39 Protein sequence of LHA-EGFv4 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALN LQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSYSECPPSYDGYCLHDGV CRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR SEQ ID 40 EGF_H16N_W49L_W50A NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKLAELR SEQ ID 41 Protein sequence of LHA-EGFv5 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALN LQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDSGCPSFHDGYCLNGG VCMYIEALDKYACNCVIGYNGDRCQTRDLKWWELR SEQ ID 42 EGF_H16Q_W49L_W50A NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKLAELR SEQ ID 43 Protein sequence of LHA-EGFv6 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKNKALN LQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISI ENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNML YKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINK AMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNN TLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDSECPLSHDQYCLHDGV CMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 44 EGF_H16N_W49I_W50A NSDSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYIGERCQYRDLKIAELR SEQ ID 45 Protein sequence of LHC-EGFv7 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLDDGVC MYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 46 EGF_H16Q_W49I_W50A NSDSECPLSHDGYCLQDGVCMYIEALDKYACNCVVGYIGERCQYRDLKIAELR SEQ ID 47 Protein sequence of LHC-EGFv8 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLDDGVC MYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 48 EGF_H16N_W49L_W50A_E24G NSDSECPLSHDGYCLNDGVCMYIGALDKYACNCVVGYIGERCQYRDLKLAELR SEQ ID 49 Protein sequence of LHC-EGFv9 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVC MYIEALDKYACNCVVGYIGERCAYRDLKWWELR SEQ ID 50 EGF_H16N_W49L_W50A_E24G_A25T NSDSECPLSHDGYCLNDGVCMYIGTLDKYACNCVVGYIGERCQYRDLKLAELR SEQ ID 51 Protein sequence of LHC-EGFv10 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLADGVC MYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 52 EGF_H16N_W49L_W50A_E24G_A25S NSDSECPLSHDGYCLNDGVCMYIGSLDKYACNCVVGYIGERCQYRDLKLAELR SEQ ID 53 Protein sequence of LHC-EGFv11 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCAHDGVC MYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 54 EGF_H16N_W49L_W50A_E24G_A25T_K28R NSDSECPLSHDGYCLNDGVCMYIGTLDRYACNCVVGYIGERCQYRDLKLAELR SEQ ID 55 Protein sequence of LHC-EGFv12 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR

ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGEC MYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 56 EGF_H16N_W49L_W50A_E24G_A25T_K28R_S4P NSDPECPLSHDGYCLNDGVCMYIGTLDRYACNCVVGYIGERCQYRDLKLAELR SEQ ID 57 Protein sequence of LHC-EGFv13 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKLYNKTLQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHDGYCLHDGVC MYIEALDKYACNCDVGYIGERCQYRDLKWWELR SEQ ID 58 EGF_H16N_W49L_W50A_E24G_A25T_K28R_S4P_E5K NSDPKCPLSHDGYCLNDGVCMYIGTLDRYACNCVVGYIGERCQYRDLKLAELR SEQ ID 59 Protein sequence of LHB-EGFv1 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGI FNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFN TNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKF CPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQ STDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKD KYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEI YTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGS SLQCIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTE SLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSM DYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIGLALNVGNETAKGNFE NAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLST VNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNF INGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALDSRGSKCPPSHDGYCLQGGVCMYI EALDRYACNCVVGYAGERCQYRDLTWWGRR SEQ ID 60 EGFv3 NSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID 61 Protein sequence of LHB-EGFv5 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGI FNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFN TNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKF CPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQ STDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKD KYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEI YTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCVDEEKLYDDDDKDRWGS SLQCIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTE SLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSM DYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDAIADISLIVPYIGLALNVGNETAKGNFE NAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLST VNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNF INGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNDTILIEMFNKYNSLEGGGGSGGGGSGGGGSALDNSDSGCPSFHDGYCLNGGVCMYI EALDKYACNCVIGYNGDRCQTRDLKWWELR SEQ ID 62 Protein sequence of Tetanus LHN-EGFv1 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLI EGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDT NSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSI MQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQE IYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQ IYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLL DDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCVDGIITSKTKSDDD DKNKALNLQCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITL PNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINST KIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQG YEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKA KWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANK AMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKV FSTPIPFSYSKNLDCWVDNEEDIDVGLEGGGGSGGGGSGGGGSALDNSDSECPLSHDQYCL HDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 63 Protein sequence of LHD-EGFv6 (protease sensitivity site) MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRP TSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDF TRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKV APEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFS LDGRNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKI FSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANIL DDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKD RWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEI VDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYS NKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNS ALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSY QWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSL DVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAK VNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHD QYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 64 Protein sequence of LHD-EGFv3 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRP TSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDF TRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKV APEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFS LDGRNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKI FSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANIL DDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKD RWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEI VDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYS NKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNS ALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSY QWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSL DVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAK VNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDPKCPLSHE GYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID 65 Protein sequence of LHD-EGFv11 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRP TSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDF TRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKV APEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFS QDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKI FSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANIL DDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKSEEKLYDDDDKD RWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEI VDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYS NKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNS ALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSY QWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSL DVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAK VNESFENTMPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSECPLSHD GYCLDDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 66 Protein sequence of M26-IgA1-HC-EGFv3 MESNQPEKNGTATKPENSGNTTSENGQTEPEKKLELRNVSDIELYSQTNGTYRQHVSLDGIP ENTDTYFVKVKSSAFKDVYIPVASITEEKRNGQSVYKITAKAEKLQQELENKYVDNFTFYLDKK AKEENTNFTSFSNLVKAINQNPSGTYHLAASLNANEVELGPDERSYIKDTFTGRLIGEKDGKN YAIYNLKKPLFENLSGATVEKLSLKNVAISGKNDIGSLANEATNGTKIKQVHVDGCVDEEKLYD DDDKDRWGSSLQCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKN TSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIE EALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIG PALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKR WKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVE NLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDPKC PLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID 67 Protein sequence of M26-IgA1-HC-EGFv11 MESNQPEKNGTATKPENSGNTTSENGQTEPEKKLELRNVSDIELYSQTNGTYRQHVSLDGIP ENTDTYFVKVKSSAFKDVYIPVASITEEKRNGQSVYKITAKAEKLQQELENKYVDNFTFYLDKK AKEENTNFTSFSNLVKAINQNPSGTYHLAASLNANEVELGPDERSYIKDTFTGRLIGEKDGKN YAIYNLKKPLFENLSGATVEKLSLKNVAISGKNDIGSLANEATNGTKIKQVHVDGCVDEEKLYD DDDKDRWGSSLQCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKN TSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIE EALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIG PALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKR WKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVE NLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEV DKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNLEGGGGSGGGGSGGGGSALDNSDSEC PLSHDGYCAHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR SEQ ID 68 Protein sequence of Tetanus LHN-EGFv3 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLI EGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDT NSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSI MQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQE IYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQ IYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLL DDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCVDGIITSKTKSDDD DKNKALNLQCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITL PNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINST KIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQG YEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKA KWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANK AMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKV FSTPIPFSYSKNLDCWVDNEEDIDVGLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCL NDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELR SEQ ID 69 Protein sequence of LHA-CP-EGFv2 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGGGGSGGGGSGGGGSA DDDDKSRGSKCPPSHDGYCLQGGVCMYIEALDRYACNCVVGYAGERCQYRDLTWWGRRP LAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEEN ISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEF EHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEV STTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYT EEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALL KYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEALASG SEQ ID 70 Protein sequence of LHD-EGFv2 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPP EAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEL KVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFT FGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLE VSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGGGGSADDDDKSRGSK CPPSHDGYCLQGGVCMYIEALDRYACNCVVGYAGERCQYRDLTWWGRRALAGGGGSGGG GSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLT FNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTN SVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIP YIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRN EKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNID DLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIG QVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLS SEQ ID 71 Protein sequence of LHC-CP-EGFv2 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKSRGSKCPP SHDGYCLQGGVCMYIEALDRYACNCVVGYAGERCQYRDLTWWGRRAALAGGGGSALALQ CRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPS IDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYF PTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGN FTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGT WLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAM NNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQN TIPFNIFSYTNNSLLKDIINEYF SEQ ID 72 Protein sequence of LHC-EGFv3 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVT SPKSGYYDPNYLSTDSDKDTFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDV DFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISP RFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQ YNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKL IRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDN VYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCVDADDDDKNSDPKCPL SHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRAALAGGGGSALALQCR ELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSID SESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFP TLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFT EAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTW LSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMN NINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTI PFNIFSYTNNSLLKDIINEYF SEQ ID 73 DNA sequence of a EGF variant targeting moiety v3

AATAGTGACCCAAAGTGTCCATTAAGCCATGAAGGATATTGTCTAAACGATGGTGTTTGTA TGTACATAGGGACATTGGATAGGTATGCTTGCAATTGCGTAGTGGGATACGTAGGTGAAC GATGCCAATATAGAGACTTAAAACTGGCAGAGCTTAGA

SEQUENCE LISTINGS

1

73153PRTHomo sapiens 1Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 5022595DNAArtificial SequenceDNA sequence of LHA 2atggagttcg ttaacaaaca gttcaactat aaagacccag ttaacggtgt tgacattgct 60tacatcaaaa tcccgaacgc tggccagatg cagccggtaa aggcattcaa aatccacaac 120aaaatctggg ttatcccgga acgtgatacc tttactaacc cggaagaagg tgacctgaac 180ccgccaccgg aagcgaaaca ggtgccggta tcttactatg actccaccta cctgtctacc 240gataacgaaa aggacaacta cctgaaaggt gttactaaac tgttcgagcg tatttactcc 300accgacctgg gccgtatgct gctgactagc atcgttcgcg gtatcccgtt ctggggcggt 360tctaccatcg ataccgaact gaaagtaatc gacactaact gcatcaacgt tattcagccg 420gacggttcct atcgttccga agaactgaac ctggtgatca tcggcccgtc tgctgatatc 480atccagttcg agtgtctgag ctttggtcac gaagttctga acctcacccg taacggctac 540ggttccactc agtacatccg tttctctccg gacttcacct tcggttttga agaatccctg 600gaagtagaca cgaacccact gctgggcgct ggtaaattcg caactgatcc tgcggttacc 660ctggctcacg aactgattca tgcaggccac cgcctgtacg gtatcgccat caatccgaac 720cgtgtcttca aagttaacac caacgcgtat tacgagatgt ccggtctgga agttagcttc 780gaagaactgc gtacttttgg cggtcacgac gctaaattca tcgactctct gcaagaaaac 840gagttccgtc tgtactacta taacaagttc aaagatatcg catccaccct gaacaaagcg 900aaatccatcg tgggtaccac tgcttctctc cagtacatga agaacgtttt taaagaaaaa 960tacctgctca gcgaagacac ctccggcaaa ttctctgtag acaagttgaa attcgataaa 1020ctttacaaaa tgctgactga aatttacacc gaagacaact tcgttaagtt ctttaaagtt 1080ctgaaccgca aaacctatct gaacttcgac aaggcagtat tcaaaatcaa catcgtgccg 1140aaagttaact acactatcta cgatggtttc aacctgcgta acaccaacct ggctgctaat 1200tttaacggcc agaacacgga aatcaacaac atgaacttca caaaactgaa aaacttcact 1260ggtctgttcg agttttacaa gctgctgtgc gtcgacggca tcattacctc caaaactaaa 1320tctgacgatg acgataaaaa caaagcgctg aacctgcagt gtatcaaggt taacaactgg 1380gatttattct tcagcccgag tgaagacaac ttcaccaacg acctgaacaa aggtgaagaa 1440atcacctcag atactaacat cgaagcagcc gaagaaaaca tctcgctgga cctgatccag 1500cagtactacc tgacctttaa tttcgacaac gagccggaaa acatttctat cgaaaacctg 1560agctctgata tcatcggcca gctggaactg atgccgaaca tcgaacgttt cccaaacggt 1620aaaaagtacg agctggacaa atataccatg ttccactacc tgcgcgcgca ggaatttgaa 1680cacggcaaat cccgtatcgc actgactaac tccgttaacg aagctctgct caacccgtcc 1740cgtgtataca ccttcttctc tagcgactac gtgaaaaagg tcaacaaagc gactgaagct 1800gcaatgttct tgggttgggt tgaacagctt gtttatgatt ttaccgacga gacgtccgaa 1860gtatctacta ccgacaaaat tgcggatatc actatcatca tcccgtacat cggtccggct 1920ctgaacattg gcaacatgct gtacaaagac gacttcgttg gcgcactgat cttctccggt 1980gcggtgatcc tgctggagtt catcccggaa atcgccatcc cggtactggg cacctttgct 2040ctggtttctt acattgcaaa caaggttctg actgtacaaa ccatcgacaa cgcgctgagc 2100aaacgtaacg aaaaatggga tgaagtttac aaatatatcg tgaccaactg gctggctaag 2160gttaatactc agatcgacct catccgcaaa aaaatgaaag aagcactgga aaaccaggcg 2220gaagctacca aggcaatcat taactaccag tacaaccagt acaccgagga agaaaaaaac 2280aacatcaact tcaacatcga cgatctgtcc tctaaactga acgaatccat caacaaagct 2340atgatcaaca tcaacaagtt cctgaaccag tgctctgtaa gctatctgat gaactccatg 2400atcccgtacg gtgttaaacg tctggaggac ttcgatgcgt ctctgaaaga cgccctgctg 2460aaatacattt acgacaaccg tggcactctg atcggtcagg ttgatcgtct gaaggacaaa 2520gtgaacaata ccttatcgac cgacatccct tttcagctca gtaaatatgt cgataaccaa 2580cgccttttgt ccact 259532619DNAArtificial SequenceDNA sequence of LHB 3atgccggtta ccatcaacaa cttcaactac aacgacccga tcgacaacaa caacatcatt 60atgatggaac cgccgttcgc acgtggtacc ggacgttact acaaggcttt taagatcacc 120gaccgtatct ggatcatccc ggaacgttac accttcggtt acaaacctga ggacttcaac 180aagagtagcg ggattttcaa tcgtgacgtc tgcgagtact atgatccaga ttatctgaat 240accaacgata agaagaacat attccttcag actatgatta aactcttcaa ccgtatcaaa 300agcaaaccgc tcggtgaaaa actcctcgaa atgattatca acggtatccc gtacctcggt 360gaccgtcgtg tcccgcttga agagttcaac accaacatcg caagcgtcac cgtcaacaaa 420ctcatcagca acccaggtga agtcgaacgt aaaaaaggta tcttcgcaaa cctcatcatc 480ttcggtccgg gtccggtcct caacgaaaac gaaaccatcg acatcggtat ccagaaccac 540ttcgcaagcc gtgaaggttt cggtggtatc atgcagatga aattctgccc ggaatacgtc 600agtgtcttca acaacgtcca ggaaaacaaa ggtgcaagca tcttcaaccg tcgtggttac 660ttcagcgacc cggcactcat cctcatgcat gaactcatcc acgtcctcca cggtctctac 720ggtatcaaag ttgacgacct cccgatcgtc ccgaacgaga agaaattctt catgcagagc 780accgacgcaa tccaggctga ggaactctac accttcggtg gccaagaccc aagtatcata 840accccgtcca ccgacaaaag catctacgac aaagtcctcc agaacttcag gggtatcgtg 900gacagactca acaaagtcct cgtctgcatc agcgacccga acatcaatat caacatatac 960aagaacaagt tcaaagacaa gtacaaattc gtcgaggaca gcgaaggcaa atacagcatc 1020gacgtagaaa gtttcgacaa gctctacaaa agcctcatgt tcggtttcac cgaaaccaac 1080atcgccgaga actacaagat caagacaagg gcaagttact tcagcgacag cctcccgcct 1140gtcaaaatca agaacctctt agacaacgag atttacacaa ttgaagaggg cttcaacatc 1200agtgacaaag acatggagaa ggaatacaga ggtcagaaca aggctatcaa caaacaggca 1260tacgaggaga tcagcaaaga acacctcgca gtctacaaga tccagatgtg cgtcgacggc 1320atcattacct ccaaaactaa atctgacgat gacgataaaa acaaagcgct gaacctgcag 1380tgcatcgacg ttgacaacga agacctgttc ttcatcgctg acaaaaacag cttcagtgac 1440gacctgagca aaaacgaacg tatcgaatac aacacccaga gcaactacat cgaaaacgac 1500ttcccgatca acgaactgat cctggacacc gacctgataa gtaaaatcga actgccgagc 1560gaaaacaccg aaagtctgac cgacttcaac gttgacgttc cggtttacga aaaacagccg 1620gctatcaaga aaatcttcac cgacgaaaac accatcttcc agtacctgta cagccagacc 1680ttcccgctgg acatccgtga catcagtctg accagcagtt tcgacgacgc tctgctgttc 1740agcaacaaag tttacagttt cttcagcatg gactacatca aaaccgctaa caaagttgtt 1800gaagcagggc tgttcgctgg ttgggttaaa cagatcgtta acgacttcgt tatcgaagct 1860aacaaaagca acactatgga caaaatcgct gacatcagtc tgatcgttcc gtacatcggt 1920ctggctctga acgttggtaa cgaaaccgct aaaggtaact ttgaaaacgc tttcgagatc 1980gctggtgcaa gcatcctgct ggagttcatc ccggaactgc tgatcccggt tgttggtgct 2040ttcctgctgg aaagttacat cgacaacaaa aacaagatca tcaaaaccat cgacaacgct 2100ctgaccaaac gtaacgaaaa atggagtgat atgtacggtc tgatcgttgc tcagtggctg 2160agcaccgtca acacccagtt ctacaccatc aaagaaggta tgtacaaagc tctgaactac 2220caggctcagg ctctggaaga gatcatcaaa taccgttaca acatctacag tgagaaggaa 2280aagagtaaca tcaacatcga cttcaacgac atcaacagca aactgaacga aggtatcaac 2340caggctatcg acaacatcaa caacttcatc aacggttgca gtgttagcta cctgatgaag 2400aagatgatcc cgctggctgt tgaaaaactg ctggacttcg acaacaccct gaaaaagaac 2460ctgctgaact acatcgacga aaacaagctg tacctgatcg gtagtgctga atacgaaaaa 2520agtaaagtga acaaatacct gaagaccatc atgccgttcg acctgagtat ctacaccaac 2580gacaccatcc tgatcgaaat gttcaacaaa tacaactct 261942616DNAArtificial SequenceDNA sequence of LHC 4atgacgtggc cagttaagga tttcaactac tcagatcctg taaatgacaa cgatattctg 60taccttcgca ttccacaaaa taaactgatc accacaccag tcaaagcatt catgattact 120caaaacattt gggtcattcc agaacgcttt tctagtgaca caaatccgag tttatctaaa 180cctccgcgtc cgacgtccaa atatcagagc tattacgatc cctcatatct cagtacggac 240gaacaaaaag atactttcct taaaggtatc attaaactgt ttaagcgtat taatgagcgc 300gatatcggga aaaagttgat taattatctt gttgtgggtt ccccgttcat gggcgatagc 360tctacccccg aagacacttt tgattttacc cgtcatacga caaacatcgc ggtagagaag 420tttgagaacg gatcgtggaa agtcacaaac atcattacac ctagcgtctt aatttttggt 480ccgctgccaa acatcttaga ttatacagcc agcctgactt tgcaggggca acagtcgaat 540ccgagtttcg aaggttttgg taccctgagc attctgaaag ttgccccgga atttctgctc 600actttttcag atgtcaccag caaccagagc tcagcagtat taggaaagtc aattttttgc 660atggacccgg ttattgcact gatgcacgaa ctgacgcact ctctgcatca actgtatggg 720atcaacatcc ccagtgacaa acgtattcgt ccccaggtgt ctgaaggatt tttctcacag 780gatgggccga acgtccagtt cgaagagttg tatactttcg gaggcctgga cgtagagatc 840attccccaga ttgagcgcag tcagctgcgt gagaaggcat tgggccatta taaggatatt 900gcaaaacgcc tgaataacat taacaaaacg attccatctt cgtggatctc gaatattgat 960aaatataaga aaatttttag cgagaaatat aattttgata aagataatac aggtaacttt 1020gtggttaaca ttgacaaatt caactccctt tacagtgatt tgacgaatgt aatgagcgaa 1080gttgtgtata gttcccaata caacgttaag aatcgtaccc attacttctc tcgtcactac 1140ctgccggttt tcgcgaacat ccttgacgat aatatttaca ctattcgtga cggctttaac 1200ttgaccaaca agggcttcaa tattgaaaat tcaggccaga acattgaacg caacccggcc 1260ttgcagaaac tgtcgagtga atccgtggtt gacctgttta ccaaagtctg cgtcgacaaa 1320agcgaagaga agctgtacga tgacgatgac aaagatcgtt ggggatcgtc cctgcagtgt 1380attaaagtga aaaacaatcg gctgccttat gtagcagata aagatagcat tagtcaggag 1440attttcgaaa ataaaattat cactgacgaa accaatgttc agaattattc agataaattt 1500tcactggacg aaagcatctt agatggccaa gttccgatta acccggaaat tgttgatccg 1560ttactgccga acgtgaatat ggaaccgtta aacctccctg gcgaagagat cgtattttat 1620gatgacatta cgaaatatgt ggactacctt aattcttatt actatttgga aagccagaaa 1680ctgtccaata acgtggaaaa cattactctg accacaagcg tggaagaggc tttaggctac 1740tcaaataaga tttatacctt cctcccgtcg ctggcggaaa aagtaaataa aggtgtgcag 1800gctggtctgt tcctcaactg ggcgaatgaa gttgtcgaag actttaccac gaatattatg 1860aaaaaggata ccctggataa aatctccgac gtctcggtta ttatcccata tattggccct 1920gcgttaaata tcggtaatag tgcgctgcgg gggaatttta accaggcctt tgctaccgcg 1980ggcgtcgcgt tcctcctgga gggctttcct gaatttacta tcccggcgct cggtgttttt 2040acattttact cttccatcca ggagcgtgag aaaattatca aaaccatcga aaactgcctg 2100gagcagcggg tgaaacgctg gaaagattct tatcaatgga tggtgtcaaa ctggttatct 2160cgcatcacga cccaattcaa ccatattaat taccagatgt atgatagtct gtcgtaccaa 2220gctgacgcca ttaaagccaa aattgatctg gaatataaaa agtactctgg tagcgataag 2280gagaacatca aaagccaggt ggagaacctt aagaatagtc tggatgtgaa aatctctgaa 2340gctatgaata acattaacaa attcattcgt gaatgttcgg tgacgtacct gttcaagaat 2400atgctgccaa aagttattga tgaactgaat aaatttgatc tgcgtaccaa aaccgaactt 2460atcaacctca tcgactccca caacattatc cttgtgggcg aagtggatcg tctgaaggcc 2520aaagtaaacg agagctttga aaatacgatg ccgtttaata ttttttcata taccaataac 2580tccttgctga aagatatcat caatgaatat ttcaat 261652616DNAArtificial SequenceDNA sequence of LHD 5atgacgtggc cagttaagga tttcaactac tcagatcctg taaatgacaa cgatattctg 60taccttcgca ttccacaaaa taaactgatc accacaccag tcaaagcatt catgattact 120caaaacattt gggtcattcc agaacgcttt tctagtgaca caaatccgag tttatctaaa 180cctccgcgtc cgacgtccaa atatcagagc tattacgatc cctcatatct cagtacggac 240gaacaaaaag atactttcct taaaggtatc attaaactgt ttaagcgtat taatgagcgc 300gatatcggga aaaagttgat taattatctt gttgtgggtt ccccgttcat gggcgatagc 360tctacccccg aagacacttt tgattttacc cgtcatacga caaacatcgc ggtagagaag 420tttgagaacg gatcgtggaa agtcacaaac atcattacac ctagcgtctt aatttttggt 480ccgctgccaa acatcttaga ttatacagcc agcctgactt tgcaggggca acagtcgaat 540ccgagtttcg aaggttttgg taccctgagc attctgaaag ttgccccgga atttctgctc 600actttttcag atgtcaccag caaccagagc tcagcagtat taggaaagtc aattttttgc 660atggacccgg ttattgcact gatgcacgaa ctgacgcact ctctgcatca actgtatggg 720atcaacatcc ccagtgacaa acgtattcgt ccccaggtgt ctgaaggatt tttctcacag 780gatgggccga acgtccagtt cgaagagttg tatactttcg gaggcctgga cgtagagatc 840attccccaga ttgagcgcag tcagctgcgt gagaaggcat tgggccatta taaggatatt 900gcaaaacgcc tgaataacat taacaaaacg attccatctt cgtggatctc gaatattgat 960aaatataaga aaatttttag cgagaaatat aattttgata aagataatac aggtaacttt 1020gtggttaaca ttgacaaatt caactccctt tacagtgatt tgacgaatgt aatgagcgaa 1080gttgtgtata gttcccaata caacgttaag aatcgtaccc attacttctc tcgtcactac 1140ctgccggttt tcgcgaacat ccttgacgat aatatttaca ctattcgtga cggctttaac 1200ttgaccaaca agggcttcaa tattgaaaat tcaggccaga acattgaacg caacccggcc 1260ttgcagaaac tgtcgagtga atccgtggtt gacctgttta ccaaagtctg cgtcgacaaa 1320agcgaagaga agctgtacga tgacgatgac aaagatcgtt ggggatcgtc cctgcagtgt 1380attaaagtga aaaacaatcg gctgccttat gtagcagata aagatagcat tagtcaggag 1440attttcgaaa ataaaattat cactgacgaa accaatgttc agaattattc agataaattt 1500tcactggacg aaagcatctt agatggccaa gttccgatta acccggaaat tgttgatccg 1560ttactgccga acgtgaatat ggaaccgtta aacctccctg gcgaagagat cgtattttat 1620gatgacatta cgaaatatgt ggactacctt aattcttatt actatttgga aagccagaaa 1680ctgtccaata acgtggaaaa cattactctg accacaagcg tggaagaggc tttaggctac 1740tcaaataaga tttatacctt cctcccgtcg ctggcggaaa aagtaaataa aggtgtgcag 1800gctggtctgt tcctcaactg ggcgaatgaa gttgtcgaag actttaccac gaatattatg 1860aaaaaggata ccctggataa aatctccgac gtctcggtta ttatcccata tattggccct 1920gcgttaaata tcggtaatag tgcgctgcgg gggaatttta accaggcctt tgctaccgcg 1980ggcgtcgcgt tcctcctgga gggctttcct gaatttacta tcccggcgct cggtgttttt 2040acattttact cttccatcca ggagcgtgag aaaattatca aaaccatcga aaactgcctg 2100gagcagcggg tgaaacgctg gaaagattct tatcaatgga tggtgtcaaa ctggttatct 2160cgcatcacga cccaattcaa ccatattaat taccagatgt atgatagtct gtcgtaccaa 2220gctgacgcca ttaaagccaa aattgatctg gaatataaaa agtactctgg tagcgataag 2280gagaacatca aaagccaggt ggagaacctt aagaatagtc tggatgtgaa aatctctgaa 2340gctatgaata acattaacaa attcattcgt gaatgttcgg tgacgtacct gttcaagaat 2400atgctgccaa aagttattga tgaactgaat aaatttgatc tgcgtaccaa aaccgaactt 2460atcaacctca tcgactccca caacattatc cttgtgggcg aagtggatcg tctgaaggcc 2520aaagtaaacg agagctttga aaatacgatg ccgtttaata ttttttcata taccaataac 2580tccttgctga aagatatcat caatgaatat ttcaat 2616653PRTArtificial SequenceEGFH16N 6Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 50753PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v1 7Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Gly Gly Val Cys Met Tyr Ile Lys Ala Val Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Thr 35 40 45Trp Trp Gly Pro Arg 50853PRTArtificial SequenceEGFH16Q 8Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 50953PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v2 9Ser Arg Gly Ser Lys Cys Pro Pro Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Gly Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ala Gly Glu Arg Cys Gln Tyr Arg Asp Leu Thr 35 40 45Trp Trp Gly Arg Arg 501053PRTArtificial SequenceEGFW49L 10Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Trp Glu Leu Arg 501153PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v3 11Asn Ser Asp Pro Lys Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 501253PRTArtificial SequenceEGFW49I 12Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ile Trp Glu Leu Arg 501353PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v4 13Asn Ser Tyr Ser Glu Cys Pro Pro Ser Tyr Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Arg Tyr Ile Glu Ala Leu Asp Ser Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ala Gly Glu Arg Cys Gln Tyr Arg Asp Leu Arg 35 40 45Trp Trp Gly Arg Arg 501453PRTArtificial SequenceEGFW49V 14Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Val Trp Glu Leu Arg 501553PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v5 15Asn Ser Asp Ser Gly Cys Pro Ser Phe His Asp Gly Tyr Cys Leu Asn1 5 10 15Gly Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Ile Gly Tyr Asn Gly Asp Arg Cys Gln Thr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 501653PRTArtificial SequenceEGFW49A 16Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ala Trp Glu Leu Arg 501753PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v6 (G12Q)

17Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gln Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 501853PRTArtificial SequenceEGFW49G 18Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Gly Trp Glu Leu Arg 501953PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v7 (H16D) 19Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asp1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 502053PRTArtificial SequenceEGFW49S 20Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ser Trp Glu Leu Arg 502153PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v8 (Y13W) 21Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Trp Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 502253PRTArtificial SequenceEGFW49T 22Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Thr Trp Glu Leu Arg 502353PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v9 (Q43A) 23Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Ala Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 502453PRTArtificial SequenceEGFW49N 24Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Asn Trp Glu Leu Arg 502553PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v10 (H16A) 25Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Ala1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 502653PRTArtificial SequenceEGF_W49Q 26Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Gln Trp Glu Leu Arg 502753PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v11 (L15A) 27Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Ala His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 502853PRTArtificial SequenceEGF_H16N_W49L 28Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Trp Glu Leu Arg 502953PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v12 (V19E) 29Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Glu Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 503053PRTArtificial SequenceEGF_H16Q_W49L 30Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Trp Glu Leu Arg 503153PRTArtificial SequenceProtein sequence of a EGF variant targeting moiety v13 (V34D) 31Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Asp Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 503253PRTArtificial SequenceEGF_H16N_W49I 32Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ile Trp Glu Leu Arg 5033937PRTArtificial SequenceProtein sequence of LHA-EGFv1 (Xa activation) 33Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Ile Asp Gly Arg Asn Lys Ala 435 440 445Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 450 455 460Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile465 470 475 480Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 485 490 495Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 500 505 510Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu 515 520 525Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 530 535 540Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His545 550 555 560Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 565 570 575Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 580 585 590Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln 595 600 605Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp 610 615 620Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu625 630 635 640Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 645 650 655Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 660 665 670Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val 675 680 685Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 690 695 700Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val705 710 715 720Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 725 730 735Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln 740 745 750Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu 755 760 765Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 770 775 780Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile785 790 795 800Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 805 810 815Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln 820 825 830Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 835 840 845Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr 850 855 860Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly865 870 875 880Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gln 885 890 895Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys 900 905 910Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr 915 920 925Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9353453PRTArtificial SequenceEGF_H16Q_W49I 34Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ile Trp Glu Leu Arg 5035937PRTArtificial SequenceProtein sequence of LHA-EGFv2 (Xa activation) 35Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Ile Asp Gly Arg Asn Lys Ala 435 440 445Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 450 455 460Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile465 470 475 480Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 485 490 495Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 500 505

510Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu 515 520 525Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 530 535 540Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His545 550 555 560Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 565 570 575Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 580 585 590Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln 595 600 605Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp 610 615 620Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu625 630 635 640Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 645 650 655Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 660 665 670Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val 675 680 685Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 690 695 700Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val705 710 715 720Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 725 730 735Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln 740 745 750Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu 755 760 765Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 770 775 780Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile785 790 795 800Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 805 810 815Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln 820 825 830Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 835 840 845Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr 850 855 860Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly865 870 875 880Ser Ala Leu Asp Ser Arg Gly Ser Lys Cys Pro Pro Ser His Asp Gly 885 890 895Tyr Cys Leu Gln Gly Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Arg 900 905 910Tyr Ala Cys Asn Cys Val Val Gly Tyr Ala Gly Glu Arg Cys Gln Tyr 915 920 925Arg Asp Leu Thr Trp Trp Gly Arg Arg 930 9353653PRTArtificial SequenceEGF_H16N_W50A 36Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Ala Glu Leu Arg 5037938PRTArtificial SequenceProtein sequence of LHA-EGFv3 (enhanced mutation) 37Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Leu Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys Asn Lys 435 440 445Ala Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Ala Leu Asp Asn Ser Asp Pro Lys Cys Pro Leu Ser His Glu 885 890 895Gly Tyr Cys Leu Asn Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp 900 905 910Arg Tyr Ala Cys Asn Cys Val Val Gly Tyr Val Gly Glu Arg Cys Gln 915 920 925Tyr Arg Asp Leu Lys Leu Ala Glu Leu Arg 930 9353853PRTArtificial SequenceEGF_H16Q_W50A 38Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Ala Glu Leu Arg 5039938PRTArtificial SequenceProtein sequence of LHA-EGFv4 39Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys Asn Lys 435 440 445Ala Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Ala Leu Asp Asn Ser Tyr Ser Glu Cys Pro Pro Ser Tyr Asp 885 890 895Gly Tyr Cys Leu His Asp Gly Val Cys Arg Tyr Ile Glu Ala Leu Asp 900 905 910Ser Tyr Ala Cys Asn Cys Val Val Gly Tyr Ala Gly Glu Arg Cys Gln 915 920 925Tyr Arg Asp Leu Arg Trp Trp Gly Arg Arg 930 9354053PRTArtificial SequenceEGF_H16N_W49L_W50A 40Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys

Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5041938PRTArtificial SequenceProtein sequence of LHA-EGFv5 41Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys Asn Lys 435 440 445Ala Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Ala Leu Asp Asn Ser Asp Ser Gly Cys Pro Ser Phe His Asp 885 890 895Gly Tyr Cys Leu Asn Gly Gly Val Cys Met Tyr Ile Glu Ala Leu Asp 900 905 910Lys Tyr Ala Cys Asn Cys Val Ile Gly Tyr Asn Gly Asp Arg Cys Gln 915 920 925Thr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9354253PRTArtificial SequenceEGF_H16Q_W49L_W50A 42Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5043938PRTArtificial SequenceProtein sequence of LHA-EGFv6 43Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys Asn Lys 435 440 445Ala Leu Asn Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp 885 890 895Gln Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp 900 905 910Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln 915 920 925Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9354453PRTArtificial SequenceEGF_H16N_W49I_W50A 44Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ile Ala Glu Leu Arg 5045939PRTArtificial SequenceProtein sequence of LHC-EGFv7 45Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp

Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu Asp Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9354653PRTArtificial SequenceEGF_H16Q_W49I_W50A 46Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Gln1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Ile Ala Glu Leu Arg 5047939PRTArtificial SequenceProtein sequence of LHC-EGFv8 47Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu Asp Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9354853PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G 48Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5049939PRTArtificial SequenceProtein sequence of LHC-EGFv9 49Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu

His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Ala Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9355053PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G_A25T 50Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5051939PRTArtificial SequenceProtein sequence of LHC-EGFv10 51Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu Ala Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9355253PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G_A25S 52Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Ser Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5053939PRTArtificial SequenceProtein sequence of LHC-EGFv11 53Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Ala His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9355453PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G_A25T_K28R 54Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5055939PRTArtificial SequenceProtein sequence of LHC-EGFv12 55Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn

Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu His Asp Gly Glu Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9355653PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G_A25T_K28R_S4P 56Asn Ser Asp Pro Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5057939PRTArtificial SequenceProtein sequence of LHC-EGFv13 57Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Leu Tyr Asn 435 440 445Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly865 870 875 880Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys Pro Leu Ser His 885 890 895Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu 900 905 910Asp Lys Tyr Ala Cys Asn Cys Asp Val Gly Tyr Ile Gly Glu Arg Cys 915 920 925Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 9355853PRTArtificial SequenceEGF_H16N_W49L_W50A_E24G_A25T_K28R_S4P_E5K 58Asn Ser Asp Pro Lys Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5059943PRTArtificial SequenceProtein sequence of LHB-EGFv1 59Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile385 390 395 400Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430Lys Ile Gln Met Cys Val Asp Glu Glu Lys Leu Tyr Asp Asp Asp Asp 435 440 445Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Ile Asp Val Asp Asn Glu 450 455 460Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser Asp Asp Leu Ser465 470 475 480Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn Tyr Ile Glu Asn 485 490 495Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp Leu Ile Ser Lys 500 505 510Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr Asp Phe Asn Val 515 520 525Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys Lys Ile Phe Thr 530 535 540Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln Thr Phe Pro Leu545 550 555 560Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp Asp Ala Leu Leu 565 570 575Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp Tyr Ile Lys Thr 580 585 590Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly Trp Val Lys Gln 595 600 605Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser Asn Thr Met Asp 610 615 620Ala Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile Gly Leu Ala Leu625 630 635 640Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu Asn Ala Phe Glu 645 650 655Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro Glu Leu Leu Ile 660 665 670Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile Asp Asn Lys Asn 675 680 685Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys Arg Asn Glu Lys 690 695 700Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp Leu Ser Thr Val705 710 715 720Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr Lys Ala Leu Asn 725 730 735Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr Arg Tyr Asn Ile 740 745 750Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp Phe Asn Asp Ile 755 760 765Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile Asp Asn Ile Asn 770 775 780Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met Lys Lys Met Ile785 790 795 800Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn Thr Leu Lys Lys 805 810 815Asn Leu Leu Asn

Tyr Ile Asp Glu Asn Lys Leu Tyr Leu Ile Gly Ser 820 825 830Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu Lys Thr Ile Met 835 840 845Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile Leu Ile Glu Met 850 855 860Phe Asn Lys Tyr Asn Ser Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Ser Arg Gly Ser Lys Cys 885 890 895Pro Pro Ser His Asp Gly Tyr Cys Leu Gln Gly Gly Val Cys Met Tyr 900 905 910Ile Glu Ala Leu Asp Arg Tyr Ala Cys Asn Cys Val Val Gly Tyr Ala 915 920 925Gly Glu Arg Cys Gln Tyr Arg Asp Leu Thr Trp Trp Gly Arg Arg 930 935 9406053PRTArtificial SequenceEGFv3 60Asn Ser Asp Pro Lys Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn1 5 10 15Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Leu Ala Glu Leu Arg 5061943PRTArtificial SequenceProtein sequence of LHB-EGFv5 61Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile385 390 395 400Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430Lys Ile Gln Met Cys Val Asp Glu Glu Lys Leu Tyr Asp Asp Asp Asp 435 440 445Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Ile Asp Val Asp Asn Glu 450 455 460Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser Asp Asp Leu Ser465 470 475 480Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn Tyr Ile Glu Asn 485 490 495Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp Leu Ile Ser Lys 500 505 510Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr Asp Phe Asn Val 515 520 525Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys Lys Ile Phe Thr 530 535 540Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln Thr Phe Pro Leu545 550 555 560Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp Asp Ala Leu Leu 565 570 575Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp Tyr Ile Lys Thr 580 585 590Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly Trp Val Lys Gln 595 600 605Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser Asn Thr Met Asp 610 615 620Ala Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile Gly Leu Ala Leu625 630 635 640Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu Asn Ala Phe Glu 645 650 655Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro Glu Leu Leu Ile 660 665 670Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile Asp Asn Lys Asn 675 680 685Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys Arg Asn Glu Lys 690 695 700Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp Leu Ser Thr Val705 710 715 720Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr Lys Ala Leu Asn 725 730 735Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr Arg Tyr Asn Ile 740 745 750Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp Phe Asn Asp Ile 755 760 765Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile Asp Asn Ile Asn 770 775 780Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met Lys Lys Met Ile785 790 795 800Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn Thr Leu Lys Lys 805 810 815Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr Leu Ile Gly Ser 820 825 830Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu Lys Thr Ile Met 835 840 845Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile Leu Ile Glu Met 850 855 860Phe Asn Lys Tyr Asn Ser Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly865 870 875 880Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Gly Cys 885 890 895Pro Ser Phe His Asp Gly Tyr Cys Leu Asn Gly Gly Val Cys Met Tyr 900 905 910Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Ile Gly Tyr Asn 915 920 925Gly Asp Arg Cys Gln Thr Arg Asp Leu Lys Trp Trp Glu Leu Arg 930 935 94062949PRTArtificial SequenceProtein sequence of Tetanus LHN-EGFv1 62Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn1 5 10 15Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr65 70 75 80Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe145 150 155 160Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His225 230 235 240Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp305 310 315 320Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe385 390 395 400Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430Ser Lys Leu Ile Gly Leu Cys Val Asp Gly Ile Ile Thr Ser Lys Thr 435 440 445Lys Ser Asp Asp Asp Asp Lys Asn Lys Ala Leu Asn Leu Gln Cys Ile 450 455 460Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe465 470 475 480Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn 485 490 495Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr 500 505 510Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val 515 520 525Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser 530 535 540Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu545 550 555 560Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn 565 570 575Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe 580 585 590Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe 595 600 605Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser 610 615 620Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro625 630 635 640Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn 645 650 655Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr 660 665 670Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu 675 680 685Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu 690 695 700Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala705 710 715 720Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln 725 730 735Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile 740 745 750Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala 755 760 765Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys 770 775 780Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe785 790 795 800Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe 805 810 815Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser 820 825 830Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn 835 840 845Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp 850 855 860Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Gly Leu Glu Gly Gly865 870 875 880Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp 885 890 895Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gln Tyr Cys Leu His 900 905 910Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 915 920 925Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 930 935 940Trp Trp Glu Leu Arg94563945PRTArtificial SequenceProtein sequence of LHD-EGFv6 (protease sensitivity site) 63Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65 70 75 80Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255Phe Phe Ser Leu Asp Gly Arg Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305 310 315 320Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370

375 380Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395 400Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430Phe Thr Lys Val Cys Val Asp Lys Ser Glu Glu Lys Leu Tyr Asp Asp 435 440 445Asp Asp Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Ile Lys Val Lys 450 455 460Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser Ile Ser Gln Glu465 470 475 480Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu Thr Asn Val Gln Asn Tyr 485 490 495Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile Leu Asp Gly Gln Val Pro 500 505 510Ile Asn Pro Glu Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met Glu 515 520 525Pro Leu Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr Asp Asp Ile Thr 530 535 540Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys545 550 555 560Leu Ser Asn Asn Val Glu Asn Ile Thr Leu Thr Thr Ser Val Glu Glu 565 570 575Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu Pro Ser Leu Ala 580 585 590Glu Lys Val Asn Lys Gly Val Gln Ala Gly Leu Phe Leu Asn Trp Ala 595 600 605Asn Glu Val Val Glu Asp Phe Thr Thr Asn Ile Met Lys Lys Asp Thr 610 615 620Leu Asp Lys Ile Ser Asp Val Ser Val Ile Ile Pro Tyr Ile Gly Pro625 630 635 640Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg Gly Asn Phe Asn Gln Ala 645 650 655Phe Ala Thr Ala Gly Val Ala Phe Leu Leu Glu Gly Phe Pro Glu Phe 660 665 670Thr Ile Pro Ala Leu Gly Val Phe Thr Phe Tyr Ser Ser Ile Gln Glu 675 680 685Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu Glu Gln Arg Val 690 695 700Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met Val Ser Asn Trp Leu Ser705 710 715 720Arg Ile Thr Thr Gln Phe Asn His Ile Asn Tyr Gln Met Tyr Asp Ser 725 730 735Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr 740 745 750Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu 755 760 765Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn 770 775 780Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn785 790 795 800Met Leu Pro Lys Val Ile Asp Glu Leu Asn Lys Phe Asp Leu Arg Thr 805 810 815Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val 820 825 830Gly Glu Val Asp Arg Leu Lys Ala Lys Val Asn Glu Ser Phe Glu Asn 835 840 845Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys 850 855 860Asp Ile Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly865 870 875 880Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser 885 890 895Glu Cys Pro Leu Ser His Asp Gln Tyr Cys Leu His Asp Gly Val Cys 900 905 910Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly 915 920 925Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu 930 935 940Arg94564945PRTArtificial SequenceProtein sequence of LHD-EGFv3 64Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65 70 75 80Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255Phe Phe Ser Leu Asp Gly Arg Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305 310 315 320Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395 400Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430Phe Thr Lys Val Cys Val Asp Lys Ser Glu Glu Lys Leu Tyr Asp Asp 435 440 445Asp Asp Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Ile Lys Val Lys 450 455 460Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser Ile Ser Gln Glu465 470 475 480Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu Thr Asn Val Gln Asn Tyr 485 490 495Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile Leu Asp Gly Gln Val Pro 500 505 510Ile Asn Pro Glu Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met Glu 515 520 525Pro Leu Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr Asp Asp Ile Thr 530 535 540Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys545 550 555 560Leu Ser Asn Asn Val Glu Asn Ile Thr Leu Thr Thr Ser Val Glu Glu 565 570 575Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu Pro Ser Leu Ala 580 585 590Glu Lys Val Asn Lys Gly Val Gln Ala Gly Leu Phe Leu Asn Trp Ala 595 600 605Asn Glu Val Val Glu Asp Phe Thr Thr Asn Ile Met Lys Lys Asp Thr 610 615 620Leu Asp Lys Ile Ser Asp Val Ser Val Ile Ile Pro Tyr Ile Gly Pro625 630 635 640Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg Gly Asn Phe Asn Gln Ala 645 650 655Phe Ala Thr Ala Gly Val Ala Phe Leu Leu Glu Gly Phe Pro Glu Phe 660 665 670Thr Ile Pro Ala Leu Gly Val Phe Thr Phe Tyr Ser Ser Ile Gln Glu 675 680 685Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu Glu Gln Arg Val 690 695 700Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met Val Ser Asn Trp Leu Ser705 710 715 720Arg Ile Thr Thr Gln Phe Asn His Ile Asn Tyr Gln Met Tyr Asp Ser 725 730 735Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr 740 745 750Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu 755 760 765Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn 770 775 780Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn785 790 795 800Met Leu Pro Lys Val Ile Asp Glu Leu Asn Lys Phe Asp Leu Arg Thr 805 810 815Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val 820 825 830Gly Glu Val Asp Arg Leu Lys Ala Lys Val Asn Glu Ser Phe Glu Asn 835 840 845Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys 850 855 860Asp Ile Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly865 870 875 880Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Pro 885 890 895Lys Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn Asp Gly Val Cys 900 905 910Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn Cys Val Val Gly 915 920 925Tyr Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Leu Ala Glu Leu 930 935 940Arg94565945PRTArtificial SequenceProtein sequence of LHD-EGFv11 65Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65 70 75 80Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305 310 315 320Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395 400Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430Phe Thr Lys Val Cys Val Asp Lys Ser Glu Glu Lys Leu Tyr Asp Asp 435 440 445Asp Asp Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Ile Lys Val Lys 450 455 460Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser Ile Ser Gln Glu465 470 475 480Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu Thr Asn Val Gln Asn Tyr 485 490 495Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile Leu Asp Gly Gln Val Pro 500 505 510Ile Asn Pro Glu Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met Glu 515 520 525Pro Leu Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr Asp Asp Ile Thr 530 535 540Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys545 550 555 560Leu Ser Asn Asn Val Glu Asn Ile Thr Leu Thr Thr Ser Val Glu Glu 565 570 575Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu Pro Ser Leu Ala 580 585 590Glu Lys Val Asn Lys Gly Val Gln Ala Gly Leu Phe Leu Asn Trp Ala 595 600 605Asn Glu Val Val Glu Asp Phe Thr Thr Asn Ile Met Lys Lys Asp Thr 610 615 620Leu Asp Lys Ile Ser Asp Val Ser Val Ile Ile Pro Tyr Ile Gly Pro625 630 635 640Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg Gly Asn Phe Asn Gln Ala 645 650 655Phe Ala Thr Ala Gly Val Ala Phe Leu Leu Glu Gly Phe Pro Glu Phe 660 665 670Thr Ile Pro Ala Leu Gly Val Phe Thr Phe Tyr Ser Ser Ile Gln Glu 675 680 685Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu Glu Gln Arg Val 690 695 700Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met Val Ser Asn Trp Leu Ser705 710 715 720Arg Ile Thr Thr Gln Phe Asn His Ile Asn Tyr Gln Met Tyr Asp Ser 725 730 735Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr 740 745 750Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu 755 760 765Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn 770 775 780Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn785 790 795 800Met Leu Pro Lys Val Ile Asp Glu Leu Asn Lys Phe Asp Leu Arg Thr 805 810 815Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val 820 825 830Gly Glu Val Asp Arg Leu Lys Ala Lys Val Asn Glu Ser Phe Glu Asn 835 840 845Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys 850 855 860Asp Ile Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly865 870 875 880Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser 885 890 895Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu Asp Asp Gly Val Cys 900 905 910Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly 915 920 925Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu 930 935 940Arg94566752PRTArtificial

SequenceProtein sequence of M26-IgA1-HC-EGFv3 66Met Glu Ser Asn Gln Pro Glu Lys Asn Gly Thr Ala Thr Lys Pro Glu1 5 10 15Asn Ser Gly Asn Thr Thr Ser Glu Asn Gly Gln Thr Glu Pro Glu Lys 20 25 30Lys Leu Glu Leu Arg Asn Val Ser Asp Ile Glu Leu Tyr Ser Gln Thr 35 40 45Asn Gly Thr Tyr Arg Gln His Val Ser Leu Asp Gly Ile Pro Glu Asn 50 55 60Thr Asp Thr Tyr Phe Val Lys Val Lys Ser Ser Ala Phe Lys Asp Val65 70 75 80Tyr Ile Pro Val Ala Ser Ile Thr Glu Glu Lys Arg Asn Gly Gln Ser 85 90 95Val Tyr Lys Ile Thr Ala Lys Ala Glu Lys Leu Gln Gln Glu Leu Glu 100 105 110Asn Lys Tyr Val Asp Asn Phe Thr Phe Tyr Leu Asp Lys Lys Ala Lys 115 120 125Glu Glu Asn Thr Asn Phe Thr Ser Phe Ser Asn Leu Val Lys Ala Ile 130 135 140Asn Gln Asn Pro Ser Gly Thr Tyr His Leu Ala Ala Ser Leu Asn Ala145 150 155 160Asn Glu Val Glu Leu Gly Pro Asp Glu Arg Ser Tyr Ile Lys Asp Thr 165 170 175Phe Thr Gly Arg Leu Ile Gly Glu Lys Asp Gly Lys Asn Tyr Ala Ile 180 185 190Tyr Asn Leu Lys Lys Pro Leu Phe Glu Asn Leu Ser Gly Ala Thr Val 195 200 205Glu Lys Leu Ser Leu Lys Asn Val Ala Ile Ser Gly Lys Asn Asp Ile 210 215 220Gly Ser Leu Ala Asn Glu Ala Thr Asn Gly Thr Lys Ile Lys Gln Val225 230 235 240His Val Asp Gly Cys Val Asp Glu Glu Lys Leu Tyr Asp Asp Asp Asp 245 250 255Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Arg Glu Leu Leu Val Lys 260 265 270Asn Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp 275 280 285Ile Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr 290 295 300Pro Asp Asn Val Ser Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser305 310 315 320Glu His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser 325 330 335Glu Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln 340 345 350Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu 355 360 365Ser Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala 370 375 380Leu Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn385 390 395 400Lys Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn 405 410 415Asp Val Val Glu Asp Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu 420 425 430Asp Lys Ile Ser Asp Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala 435 440 445Leu Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe 450 455 460Ala Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr465 470 475 480Ile Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg 485 490 495Asn Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys 500 505 510Arg Trp Lys Asp Ser Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg 515 520 525Ile Ile Thr Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu 530 535 540Asn Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys545 550 555 560Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn 565 570 575Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile 580 585 590Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met 595 600 605Leu Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys 610 615 620Ala Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly625 630 635 640Glu Val Asp Lys Leu Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr 645 650 655Ile Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp 660 665 670Ile Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly 675 680 685Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Pro Lys 690 695 700Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn Asp Gly Val Cys Met705 710 715 720Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn Cys Val Val Gly Tyr 725 730 735Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Leu Ala Glu Leu Arg 740 745 75067752PRTArtificial SequenceProtein sequence of M26-IgA1-HC-EGFv11 67Met Glu Ser Asn Gln Pro Glu Lys Asn Gly Thr Ala Thr Lys Pro Glu1 5 10 15Asn Ser Gly Asn Thr Thr Ser Glu Asn Gly Gln Thr Glu Pro Glu Lys 20 25 30Lys Leu Glu Leu Arg Asn Val Ser Asp Ile Glu Leu Tyr Ser Gln Thr 35 40 45Asn Gly Thr Tyr Arg Gln His Val Ser Leu Asp Gly Ile Pro Glu Asn 50 55 60Thr Asp Thr Tyr Phe Val Lys Val Lys Ser Ser Ala Phe Lys Asp Val65 70 75 80Tyr Ile Pro Val Ala Ser Ile Thr Glu Glu Lys Arg Asn Gly Gln Ser 85 90 95Val Tyr Lys Ile Thr Ala Lys Ala Glu Lys Leu Gln Gln Glu Leu Glu 100 105 110Asn Lys Tyr Val Asp Asn Phe Thr Phe Tyr Leu Asp Lys Lys Ala Lys 115 120 125Glu Glu Asn Thr Asn Phe Thr Ser Phe Ser Asn Leu Val Lys Ala Ile 130 135 140Asn Gln Asn Pro Ser Gly Thr Tyr His Leu Ala Ala Ser Leu Asn Ala145 150 155 160Asn Glu Val Glu Leu Gly Pro Asp Glu Arg Ser Tyr Ile Lys Asp Thr 165 170 175Phe Thr Gly Arg Leu Ile Gly Glu Lys Asp Gly Lys Asn Tyr Ala Ile 180 185 190Tyr Asn Leu Lys Lys Pro Leu Phe Glu Asn Leu Ser Gly Ala Thr Val 195 200 205Glu Lys Leu Ser Leu Lys Asn Val Ala Ile Ser Gly Lys Asn Asp Ile 210 215 220Gly Ser Leu Ala Asn Glu Ala Thr Asn Gly Thr Lys Ile Lys Gln Val225 230 235 240His Val Asp Gly Cys Val Asp Glu Glu Lys Leu Tyr Asp Asp Asp Asp 245 250 255Lys Asp Arg Trp Gly Ser Ser Leu Gln Cys Arg Glu Leu Leu Val Lys 260 265 270Asn Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp 275 280 285Ile Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr 290 295 300Pro Asp Asn Val Ser Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser305 310 315 320Glu His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser 325 330 335Glu Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln 340 345 350Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu 355 360 365Ser Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala 370 375 380Leu Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn385 390 395 400Lys Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn 405 410 415Asp Val Val Glu Asp Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu 420 425 430Asp Lys Ile Ser Asp Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala 435 440 445Leu Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe 450 455 460Ala Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr465 470 475 480Ile Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg 485 490 495Asn Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys 500 505 510Arg Trp Lys Asp Ser Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg 515 520 525Ile Ile Thr Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu 530 535 540Asn Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys545 550 555 560Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn 565 570 575Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile 580 585 590Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met 595 600 605Leu Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys 610 615 620Ala Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly625 630 635 640Glu Val Asp Lys Leu Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr 645 650 655Ile Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp 660 665 670Ile Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly 675 680 685Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu 690 695 700Cys Pro Leu Ser His Asp Gly Tyr Cys Ala His Asp Gly Val Cys Met705 710 715 720Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr 725 730 735Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 740 745 75068949PRTArtificial SequenceProtein sequence of Tetanus LHN-EGFv3 68Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn1 5 10 15Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr65 70 75 80Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe145 150 155 160Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His225 230 235 240Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp305 310 315 320Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe385 390 395 400Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430Ser Lys Leu Ile Gly Leu Cys Val Asp Gly Ile Ile Thr Ser Lys Thr 435 440 445Lys Ser Asp Asp Asp Asp Lys Asn Lys Ala Leu Asn Leu Gln Cys Ile 450 455 460Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe465 470 475 480Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn 485 490 495Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr 500 505 510Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val 515 520 525Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser 530 535 540Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu545 550 555 560Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn 565 570 575Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe 580 585 590Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe 595 600 605Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser 610 615 620Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro625 630 635 640Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn 645 650 655Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr 660 665 670Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu 675 680 685Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu 690 695 700Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala705 710 715 720Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln 725 730 735Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile 740 745 750Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala 755 760 765Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys 770 775 780Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe785 790 795 800Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe 805 810 815Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser 820 825 830Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn 835 840 845Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp 850 855 860Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Gly Leu Glu Gly Gly865 870 875 880Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp 885 890 895Asn Ser Asp Pro Lys Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn 900 905 910Asp Gly Val Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn 915 920 925Cys Val Val Gly Tyr Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 930 935 940Leu Ala Glu

Leu Arg94569948PRTArtificial SequenceProtein sequence of LHA-CP-EGFv2 69Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 435 440 445Asp Asp Asp Asp Lys Ser Arg Gly Ser Lys Cys Pro Pro Ser His Asp 450 455 460Gly Tyr Cys Leu Gln Gly Gly Val Cys Met Tyr Ile Glu Ala Leu Asp465 470 475 480Arg Tyr Ala Cys Asn Cys Val Val Gly Tyr Ala Gly Glu Arg Cys Gln 485 490 495Tyr Arg Asp Leu Thr Trp Trp Gly Arg Arg Pro Leu Ala Gly Gly Gly 500 505 510Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu 515 520 525Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu 530 535 540Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp545 550 555 560Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln 565 570 575Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser 580 585 590Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro 595 600 605Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr 610 615 620Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser625 630 635 640Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser 645 650 655Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys 660 665 670Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr 675 680 685Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala 690 695 700Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly705 710 715 720Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly 725 730 735Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu 740 745 750Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val Leu Thr Val 755 760 765Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu 770 775 780Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln785 790 795 800Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu Asn Gln Ala 805 810 815Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu 820 825 830Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys 835 840 845Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn Lys Phe Leu 850 855 860Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly865 870 875 880Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu 885 890 895Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln Val Asp Arg 900 905 910Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile Pro Phe Gln 915 920 925Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Leu Glu Ala 930 935 940Leu Ala Ser Gly94570930PRTArtificial SequenceProtein sequence of LHD-EGFv2 70Met Glu Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp 420 425 430Gly Gly Gly Gly Ser Ala Asp Asp Asp Asp Lys Ser Arg Gly Ser Lys 435 440 445Cys Pro Pro Ser His Asp Gly Tyr Cys Leu Gln Gly Gly Val Cys Met 450 455 460Tyr Ile Glu Ala Leu Asp Arg Tyr Ala Cys Asn Cys Val Val Gly Tyr465 470 475 480Ala Gly Glu Arg Cys Gln Tyr Arg Asp Leu Thr Trp Trp Gly Arg Arg 485 490 495Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 500 505 510Gly Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu 515 520 525Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly 530 535 540Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile545 550 555 560Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn 565 570 575Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly 580 585 590Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys 595 600 605Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu 610 615 620Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu625 630 635 640Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr 645 650 655Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp 660 665 670Val Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser 675 680 685Thr Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly 690 695 700Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly705 710 715 720Ala Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu 725 730 735Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala 740 745 750Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg 755 760 765Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu 770 775 780Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu785 790 795 800Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln 805 810 815Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile 820 825 830Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile 835 840 845Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn 850 855 860Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser865 870 875 880Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu 885 890 895Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser 900 905 910Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu 915 920 925Leu Ser 93071925PRTArtificial SequenceProtein sequence of LHC-CP-EGFv2 71Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Ser Arg Gly 435 440 445Ser Lys Cys Pro Pro Ser His Asp Gly Tyr Cys Leu Gln Gly Gly Val 450 455 460Cys Met Tyr Ile Glu Ala Leu Asp Arg Tyr Ala Cys Asn Cys Val Val465 470 475 480Gly Tyr Ala Gly Glu Arg Cys Gln Tyr Arg Asp Leu Thr Trp Trp Gly 485 490 495Arg Arg Ala Ala Leu Ala Gly Gly Gly Gly Ser Ala Leu Ala Leu Gln 500 505 510Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly Asp 515 520 525Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys Asp Ile Asn Glu 530 535 540Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln Val545 550 555 560Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu Asp Leu Leu Tyr 565

570 575Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn Gln Val 580 585 590Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr Tyr 595 600 605Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu Asp Phe Thr Phe 610 615 620Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr Thr625 630 635 640Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly Val Gln Gly Gly 645 650 655Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp Phe Thr Thr Asn 660 665 670Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala Ile 675 680 685Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn Ser Val Arg Arg 690 695 700Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val Thr Ile Leu Leu705 710 715 720Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val Ile 725 730 735Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys Thr Ile Asp Asn 740 745 750Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp Met 755 760 765Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe Asn Asn Ile Ser 770 775 780Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly Ala Ile Lys Ala785 790 795 800Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 805 810 815Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 820 825 830Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 835 840 845Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 850 855 860Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile Asp Ser865 870 875 880His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys Val 885 890 895Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr Thr 900 905 910Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe 915 920 92572925PRTArtificial SequenceProtein sequence of LHC-EGFv3 72Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys Val Asp Ala Asp Asp Asp Asp Lys Asn Ser Asp 435 440 445Pro Lys Cys Pro Leu Ser His Glu Gly Tyr Cys Leu Asn Asp Gly Val 450 455 460Cys Met Tyr Ile Gly Thr Leu Asp Arg Tyr Ala Cys Asn Cys Val Val465 470 475 480Gly Tyr Val Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Leu Ala Glu 485 490 495Leu Arg Ala Ala Leu Ala Gly Gly Gly Gly Ser Ala Leu Ala Leu Gln 500 505 510Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly Asp 515 520 525Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys Asp Ile Asn Glu 530 535 540Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln Val545 550 555 560Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu Asp Leu Leu Tyr 565 570 575Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn Gln Val 580 585 590Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr Tyr 595 600 605Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu Asp Phe Thr Phe 610 615 620Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr Thr625 630 635 640Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly Val Gln Gly Gly 645 650 655Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp Phe Thr Thr Asn 660 665 670Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala Ile 675 680 685Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn Ser Val Arg Arg 690 695 700Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val Thr Ile Leu Leu705 710 715 720Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val Ile 725 730 735Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys Thr Ile Asp Asn 740 745 750Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp Met 755 760 765Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe Asn Asn Ile Ser 770 775 780Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly Ala Ile Lys Ala785 790 795 800Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 805 810 815Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 820 825 830Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 835 840 845Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 850 855 860Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile Asp Ser865 870 875 880His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys Val 885 890 895Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr Thr 900 905 910Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe 915 920 92573159PRTArtificial SequenceDNA sequence of a EGF variant targeting moiety v3 73Ala Ala Thr Ala Gly Thr Gly Ala Cys Cys Cys Ala Ala Ala Gly Thr1 5 10 15Gly Thr Cys Cys Ala Thr Thr Ala Ala Gly Cys Cys Ala Thr Gly Ala 20 25 30Ala Gly Gly Ala Thr Ala Thr Thr Gly Thr Cys Thr Ala Ala Ala Cys 35 40 45Gly Ala Thr Gly Gly Thr Gly Thr Thr Thr Gly Thr Ala Thr Gly Thr 50 55 60Ala Cys Ala Thr Ala Gly Gly Gly Ala Cys Ala Thr Thr Gly Gly Ala65 70 75 80Thr Ala Gly Gly Thr Ala Thr Gly Cys Thr Thr Gly Cys Ala Ala Thr 85 90 95Thr Gly Cys Gly Thr Ala Gly Thr Gly Gly Gly Ala Thr Ala Cys Gly 100 105 110Thr Ala Gly Gly Thr Gly Ala Ala Cys Gly Ala Thr Gly Cys Cys Ala 115 120 125Ala Thr Ala Thr Ala Gly Ala Gly Ala Cys Thr Thr Ala Ala Ala Ala 130 135 140Cys Thr Gly Gly Cys Ala Gly Ala Gly Cys Thr Thr Ala Gly Ala145 150 155