EA043746B1 - NEW T-CELL RECEPTORS OBTAINED BY GENETIC ENGINEERING METHODS AND IMMUNE THERAPY USING THEIR - Google Patents

Description

Field of invention

The present invention relates to antigen recognition structures for COL6A3 antigens. In particular, the invention provides new genetically engineered T-cell receptor (TCR)-based molecules that are selective and specific for the tumor-expressed antigen COL6A3. The TCR of the invention and COL6A3 antigen-binding fragments derived from this TCR are useful for the diagnosis, treatment and prevention of cancers in which COL6A3 is expressed. Also provided are nucleic acids encoding the antigen recognition structures of the invention, vectors including these nucleic acids, recombinant cells expressing these antigen recognition structures, and pharmaceutical compositions including the compounds of the invention.

Description of the invention

Collagens are a superfamily of proteins that play a role in maintaining the integrity of various tissues. Collagens are extracellular matrix proteins and have a triple helix domain as their common structural element. Collagen VI is the main structural component of microfibrils. The main structural unit of collagen VI is a heterotrimer of alpha 1(VI), alpha 2(VI) and alpha 3(VI) collagen chains. Alpha 1(VI) and alpha 2(VI) chains encode the COL6A1 and COL6A2 genes, respectively. The protein encoded by the COL6A3 gene is the alpha-3 subunit of type VI collagen (collagen 3(VI) alpha chain) (Bertini et al., 2002, Eur. J. Paediatr. Neurol., 6:193-8). As previously demonstrated, COL6A3 gene expression is associated with breast cancer progression, and its levels were increased in colon cancer (Smith M.J., et al. Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification, British journal of cancer. 2009; 100:1452-1464; Tilman G. et al Human periostin gene expression in normal tissues, tumors and melanoma: evidence for periostin production by both stromal and melanoma cells, Mol. Cancer. 2007; 6:80) , and also acts as a prognostic marker of colorectal carcinoma (Qiao J. et al. Stroma derived COL6A3 is a potential prognosis marker of colorectal carcinoma revealed by quantitative proteomics, Oncotarget. 2015 Oct 6; 6(30):29929-29946). COL6A3 is located on chromosome 2q37 of the human genome and contains 44 exons. The COL6A3 protein consists of 3177 amino acids and contains 12 von Willebrand factor A (vWA) type domains, one fibronectin type 3 domain, and one serine protease inhibitor (KU) domain of the BPTI/Kunitz family.

The targets of T cell-based immunotherapy are represented by peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by major histocompatibility complex (MHC) molecules. These tumor-associated antigens (TAAs) can be peptides derived from any class of proteins, such as enzymes, receptors, transcription factors, etc., that are expressed and usually have increased levels in cells of the corresponding tumor compared to intact cells of the same origin.

Specific elements of cellular immune responses are capable of selective recognition and destruction of tumor cells. Isolation of T cells specific for tumor antigens from populations of tumor-infiltrating cells or from peripheral blood suggests that such cells play an important role in the natural immune defense against cancer. In particular, CD8-positive T cells, which recognize peptides bound to major histocompatibility complex (MHC) class I molecules, play an important role in this response. These peptides typically consist of 8–10 amino acid residues derived from proteins or defective ribosomal products (DRiPs) found in the cytosol. Human MHC molecules are also called human leukocyte antigens (HLA).

TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily that associates with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist as αβ- and γδ-heterodimers, which are similar in structure but have very different anatomical locations and likely functions. The extracellular portion of a naturally occurring heterodimeric aTCR consists of two polypeptide chains, each of which has a constant domain located closer to the membrane and a variable domain located further from the membrane. Each of the constant and variable domains includes a disulfide bond within the chain. Variable domains contain highly polymorphic loops similar to the complementarity determining regions (CDRs) of antibodies. The use of TCR-based gene therapy can overcome many of the existing barriers. It allows the patient's own T cells to be equipped with the desired specificity and a sufficient number of T cells to be produced in a short period of time without depleting them. TCRs are transduced into central memory T cells or T cells with stem cell characteristics, which may provide better stability and retention of functionality upon transfer. For cancer patients with lymphopenia caused by chemotherapy or radiation therapy, TCR-embedded T cells are infused to allow successful engraftment while inhibiting immunosuppression.

Despite advances in the development of molecularly targeted anticancer drugs

- 1 043746 therapy, in this area there remains a need for the development of new anticancer drugs, the targets of which would be molecules that are highly specific for cancer cells. The present disclosure addresses this need by providing novel modified COL6A3-specific TCRs, corresponding recombinant TCR structures, nucleic acids, vectors and host cells that specifically bind to the COL6A3 epitopes of the invention; and methods of using such molecules in the treatment of cancer.

In the first aspect, the object of the invention is achieved using an antigen recognition structure comprising a first domain comprising three complementary determinant regions (CDRs) in accordance with SEQ ID NO: 5 (CDRa1), 6 (CDRa2) and 7 (CDRa3), and a second domain comprising three complementary determinant regions (CDRs) in accordance with SEQ ID NO: 13 (CDRb1), 14 (CDRb2) and 15 (CDRb3), where at least one of these complementary determinant regions is replaced by at least one sequence selected from the group : a) SEQ ID NO: 26 (CDRa1-mut1) and SEQ ID NO: 37-49 (CDRb1-mut1 to CDRb1-mut13), preferably SEQ ID NO: 40; and b) sequences with mutations SEQ ID NO: 26 and SEQ ID NO: 37-49, preferably SEQ ID NO: 40, including conservative amino acid substitutions.

In another aspect, CDRa1 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% , is at least about 99% identical to the amino acid sequence according to SEQ ID NO: 5.

In another aspect, CDRa2 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % is at least about 99% identical to the amino acid sequence according to SEQ ID NO: 6.

In another aspect, CDRa3 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % is at least about 99% identical to the amino acid sequence according to SEQ ID NO: 7.

In another aspect, the CDRb1 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % is at least about 99% identical to the amino acid sequence according to SEQ ID NO: 13.

In another aspect, the CDRb2 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % is at least about 99% identical to the amino acid sequence according to SEQ ID NO: 14.

In another aspect, the CDRb3 of the antigen recognition structure may be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99% identified

- 2 043746 chen amino acid sequence in accordance with SEQ ID NO: 15.

In the context of the present invention, the inventors have identified improved genetically engineered R4P3F9 anti-COL6A3 TCR variants comprising CDR1 sequences with mutations in the alpha and beta chain(s) that improve the stability, recognition ability and selectivity of the parent R4P3F9. The selection of these mature TCR variants was carried out using a two-stage method, in which, at the first stage, variants are selected for stability, and in the second, for affinity (see examples). Since the TCRs of the invention include mutated CDR1 regions, it is likely that CDR2 and/or CDR3 may also be mutated to increase binding affinity, specificity and/or selectivity, and such mutated CDRs could ideally be incorporated into existing structures.

Due to affinity maturity, variants with significantly stronger binding activity towards HLA-A*02/COL6A3-peptide were identified while maintaining or even improving specificity. Compared to the parent C-1 TCR (R4P3F9 TCR incorporating the wild-type CDR, see Table 5), all variants of the invention improved IFN-gamma release, reaching higher levels even at lower loading peptide concentrations.

Within the variable domain, CDR1 and CDR2 are located in the variable domain (V) of the polypeptide chain, and CDR3 includes some of the V domains, all diversity segments (D), and junction segments (J). CDR3 is proposed to be the most variable and major CDR responsible for specific and selective antigen recognition. Surprisingly, in the case of some CDR1 receptors, the TCR also appears to contact the peptide and is thus also responsible for selective recognition. In this case, without being bound by any particular theory, the mutated CDR1b appears to interact with position 8 of the COL6A3-peptide complex.

Naturally occurring alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Each alpha chain includes variable, junction and constant segments, and the beta chain usually also includes a short diversity segment between the variable and junction segments, but this diversity segment is often considered part of the junction segment. Each variable region includes three CDR regions (Complementarity Determining Regions) embedded in the framework sequence, one of which is a hypervariable region called CDR3. There are several types of alpha chain variable regions (Va) and several types of beta chain variable regions (νβ), distinguished by their framework regions, CDR1 and CDR2 sequences, and the somewhat defined CDR3 sequence. In the IMGT (International Immunogenetic Information System) nomenclature, Va species have a unique TRAV number, νβ species have a unique TRBV number.

Preferably, the antigen recognition structure of the invention comprises the corresponding CDR1 to CDR3 sequences of one distinct modified TCR variable region of the invention disclosed herein. Preferred are the antigen recognition structures (eg, αβ and γδ TCRs) of the invention that include at least one, preferably two, affinity matured CDR1 sequences.

The CDR variants disclosed herein, particularly the CDR1 variants, may be modified by substituting one or more residues at various, possibly selective, regions of the peptide chain unless otherwise stated. Such substitutions are conservative in nature when one amino acid is replaced by an amino acid with similar structure and characteristics, as occurs when a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative is the substitution of amino acids of the same or similar size and chemical character, such as the substitution of isoleucine for leucine. In studies of sequence variation within families of naturally occurring homologous proteins, certain amino acid substitutions are tolerated more often than others, and they are often associated with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its substitution; and this is the basis for the definition of conservative substitutions. Preferred conservative substitutions are defined herein as exchanges within one of the following five groups:

group 1 - small, aliphatic, nonpolar or weakly polar residues (Ala, Ser, Thr, Pro, Gly);

group 2 - polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln);

group 3 - polar, positively charged residues (His, Arg, Lys);

group 4 - large, aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys);

group 5 - large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may involve replacing one amino acid with another that has similar characteristics but differs to some extent in size, as in the case of replacing an alanine with an isoleucine residue.

The terms specificity, or antigen-specific, or specific to a given antigen, as used in the present context, mean that the antigen recognition structure can specifically bind to said antigen, preferably the COL6A3 antigen, more preferably

- 3 043746 especially with high avidity, when the specified antigen is presented in complex with HLA, preferably with HLA A2. For example, a TCR presented as an antigen recognition structure can be considered to have antigen specificity for COL6A3 antigens if T cells expressing the TCR in response to an HLA presenting COL6A3 secrete at least 200 pg/ml or more (e.g., 250 pg/ml or more, 300 pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600 pg/ml or more, 700 pg/ml or more, 1000 pg/ml or more, 2000 pg /ml or more, 2500 pg/ml or more, 5000 pg/ml or more) interferon γ (IFN-γ) when co-cultured with HLA A2 target cells loaded with a low concentration of COL6A3 antigen, such as COL6A3 epitopes and antigens proposed in the present context (eg about 10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 mol/l). Alternatively or additionally, a TCR can be considered to have antigen specificity for COL6A3 if T cells expressing the TCR secrete at least twice the amount of IFN-γ compared to the background level of IFN-γ in non-transduced cells when co-cultured with the cells -targets loaded with a low concentration of the COL6A3 antigen. The analysis of such specificity described above can be carried out, for example, using the ELISA method.

In one alternative or additional embodiment of the invention, the antigen recognition structure is stable and capable of specifically and/or selectively binding to the COL6A3 antigen; preferably, the COL6A3 antigen is an epitope or peptide of a protein with the amino acid sequence represented by SEQ ID NO: 1, or a variant thereof, where the variant comprises a deletion, addition, insertion or substitution of an amino acid in no more than three, preferably two, and most preferably no more than at one amino acid position.

By selectivity or selective recognition/binding is meant the property of an antigen recognition structure, such as a TCR or antibody, to selectively recognize or bind preferentially to only one specific epitope and preferably not or substantially not to cross-react with another epitope. Preferably selectivity or selective recognition/binding means that the antigen recognition structure (e.g., TCR) selectively recognizes or binds preferably to only one specific epitope and preferably exhibits no or substantially no cross-reaction with another epitope, wherein said epitope is unique to one protein , such that the antigen recognition structure does not exhibit, or substantially does not exhibit, cross-reaction with another epitope and another protein.

The antigen recognition structure according to the invention is preferably selected from an antibody, or a derivative thereof, or a fragment thereof, a bispecific molecule, or a T cell receptor (TCR), or a derivative thereof, or a fragment thereof. The antibody or TCR derivative or fragment thereof of the invention preferably retains the antigen binding/recognition ability of the parent molecule, in particular its specificity and/or selectivity, as explained above.

In one embodiment, the genetically engineered TCRs of the invention are capable of recognizing COL6A3 antigens through major histocompatibility complex (MHC) class I. The term "major histocompatibility complex (MHC) class I" in the context of the present invention means that TCR elicits an immune response upon binding to COL6A3 peptide antigens in the context of a class I MHC molecule. The MHC class I molecule can be any MHC class I molecule known in the art, such as HLA-A molecules. In a preferred embodiment of the invention, the MHC class I molecule is an HLA-A2 molecule.

The invention proposes both single-chain antigen recognition structures and double-chain recognition structures, as well as other variants of molecules. The invention, in particular, provides an engineered TCR, or a fragment thereof, or a derivative thereof, as an antigen recognition structure. Preferably, it is an engineered TCR of human origin, which means that it is derived from the human TCR locus and thus includes human TCR sequences. Moreover, the TCR of the invention is characterized as an affinity mature TCR that is capable of specifically and selectively recognizing the COL6A3 peptide antigen.

In another embodiment, the invention additionally or alternatively provides an antigen recognition structure described above that induces an immune response, preferably where the immune response is characterized by an increase in interferon (IFN)-gamma levels.

The TCRs of the invention may be provided as single chain α or β or γ or δ molecules or alternatively as a double chain structure consisting of both chains, an α and β chain, or a γ and δ chain.

Most preferably, in certain further embodiments, the invention provides antigen recognition structures comprising any one, two, or all of the CDR1 to CDR3 regions of the engineered TCR chains disclosed herein (see Table 1). Preferred antigen recognition structures may be those that include a natural or engineered CDR sequence having three, two, and preferably only one modification

- 4 043746 amino acid residue. The modified amino acid residue may be selected from insertion, deletion or substitution of an amino acid. Most preferably, all three, two, preferably one, modified amino acid residue is the first or last amino acid residue of the corresponding CDR sequence. If the modification is a substitution, then in some embodiments it is preferred that the substitution be a conservative amino acid substitution.

The TCRs of the invention may further include a constant region derived from any suitable species, such as any mammal, such as human, rat, monkey, rabbit, donkey or mouse. In one embodiment, the TCRs of the invention further include a human constant region. In some preferred embodiments, the TCR constant region of the invention may be slightly modified, for example by introducing heterologous sequences, preferably mouse sequences, which may enhance TCR expression and stability.

Other stabilizing mutations known in the art (eg DE 102016123893.7) can also be introduced, for example the replacement of unfavorable amino acids in the V segments and/or the introduction of a disulfide bridge between the TCR C domains and the removal of unpaired cysteines.

As used herein, the terms murine or human when they refer to an antigen recognition structure, or a TCR, or any of the TCR components described herein (e.g., complementarity determining region (CDR), variable region, constant region, α- chain and/or β-chain) denotes a TCR (or component thereof) that is derived from the murine or human TCR locus without rearrangements, respectively.

In one embodiment of the invention, chimeric TCRs are provided, wherein the TCR chains include sequences from multiple biological species. Preferably, the TCR of the invention may comprise an α chain, including a human α chain variable region and, for example, a mouse TCR α chain constant region.

In one embodiment, the TCR of the invention is a human TCR, including human variable regions in accordance with the above embodiments and human constant regions.

The TCR of the invention may be provided as a single-chain TCR. A single-chain TCR may include a variable region polypeptide of the first TCR chain (eg, the alpha chain) and a full-length polypeptide of the entire second TCR chain (eg, the beta chain), or vice versa. Moreover, a single chain TCR may optionally include one or more linkers that connect two or more polypeptides to each other. The linker may be, for example, a peptide that joins together two single chains as described herein. Also provided is a single chain TCR of the invention that is fused to a human cytokine such as IL-2, IL-7 or IL-15.

The antigen recognition structure according to the invention may also be provided in the form of a multimeric complex comprising at least two single-chain TCR molecules, wherein each of said single-chain TCR molecules is fused to at least one biotin component and wherein said single-chain TCR molecules are linked to each other by interaction biotin-streptavidin, allowing the formation of this multimeric complex. Similar approaches for producing multimeric TCRs are also possible and are included in the present invention. Higher order multimeric complexes comprising more than two double-stranded TCRs of the invention are also provided.

For purposes of the present invention, a TCR is a component having at least one TCR alpha or gamma chain variable domain and/or a TCR beta or delta chain variable domain. They generally include both the TCR alpha variable domain and the TCR beta variable domain. They may be αβ heterodimers or may be in single chain form. For use in adoptive therapy, αβ-heterodimeric TCRs can, for example, be transfected as full-length chains having both cytoplasmic and transmembrane domains. Optionally, an introduced disulfide bond may be present between residues of the respective constant domains.

In a preferred embodiment, the antigen recognition structure is a human TCR, or a fragment or derivative thereof. A human TCR, or fragment thereof, or derivative thereof is a TCR that comprises greater than 50% of the corresponding human TCR sequence. Preferably, only a small part of the TCR sequence is of artificial origin or derived from other species. However, it is known that chimeric TCRs, for example, of human origin with murine sequences in the constant domains, have advantages. Therefore, particularly preferred are TCRs according to the present invention that contain murine sequences in the extracellular portion of their constant domains.

Thus, it is also preferred that the antigen recognition structure of the invention is capable of recognizing its peptide antigen by a human leukocyte antigen (HLA) dependent mechanism, preferably by the HLA-A02-3aeucuMOMy mechanism. Concept by

HLA-dependent mechanism in the context of the present description means that the antigen recognition structure binds to the antigen only if the antigenic peptide is presented by the specified HLA.

The antigen recognition structure according to the invention in one embodiment preferably induces an immune response, preferably where the immune response is characterized by an increase in interferon (IFN)-gamma levels.

The term polypeptide as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds. With respect to the polypeptides of the invention, the functional region can be any region comprising contiguous amino acids of the TCR (or functional variant thereof) of which it is a part, provided that the functional region specifically binds to the COL6A3 antigen, preferably as disclosed herein in Table. 1. The term functional region, when used in relation to a TCR (or a functional variant thereof), refers to any part or fragment of a TCR (or a functional variant thereof) of the invention, a part or fragment of which retains the biological activity of the TCR (or a functional variant thereof), of which it is a part (the original TKR or its original functional version). Functional regions cover, for example, those portions of the TCR (or a functional variant thereof) that retain the ability to specifically bind to the COL6A3 antigen (via an HLA-A2-dependent mechanism) or to detect, treat, or prevent cancer to a similar, equal, or greater extent degree than the original TKR (or its functional variant).

The functional region may include additional amino acids at the amino or carboxyl end of the region, or at both ends, at which additional amino acids are not found in the amino acid sequence of the original TCR or a functional variant thereof. It is desirable that the additional amino acids do not interfere with the biological function of the functional site, such as specific binding to COL6A3 antigens; and/or existing ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity relative to the biological activity of the original TCR or functional variant thereof.

As mentioned above, the TCR binding functionality of the invention may be provided by an antibody scaffold. The term antibody in its various grammatical forms is used in the present context to refer to immunoglobulin molecules and the immunologically active regions of immunoglobulin molecules, i.e. molecules that contain an antigen-binding site or paratope. Such molecules are also called antigen-binding fragments of immunoglobulin molecules. The invention further provides an antibody or antigen-binding portion thereof that specifically binds to the antigens described herein. The antibody may be any type of immunoglobulin known in the art. For example, the antibody can be of any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. The antibody may be monoclonal or polyclonal. The antibody may be a naturally occurring antibody, such as an antibody isolated and/or purified from cells of a mammal, such as mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody may be produced by genetic engineering techniques, such as a humanized antibody or a chimeric antibody. The antibody may be in the form of a monomer or a polymer.

The invention also provides antigen binding regions of any of the antibodies described in this context. The antigen binding site can be any region that has at least one antigen binding site, such as Fab, F(ab') ₂ , dsFv, scFv, diabodies and tribodies. A single chain antibody fragment variable region (scFv), which consists of a truncated Fab fragment comprising the antibody heavy chain variable domain (V) linked to the antibody light chain V domain by a synthetic peptide, can be produced using standard recombinant DNA techniques. Similarly, disulfide bond-stabilized variable region fragments (dsFv) can be produced by recombinant DNA technology; the antibody fragments of the invention are, however, not limited to these particular types of antibody fragments. Also, the antibody or antigen-binding portion thereof may be modified to include a detectable label, such as, for example, a radioisotope, a fluorophore (for example, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (for example, alkaline phosphatase, horseradish peroxidase) and elemental particles (eg gold particles).

Suitable methods for producing antibodies are known in the art. For example, standard hybridoma technologies are described, for example, in Kohler and Milstein, Eur. J. Immunol, 5, 51:1-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988) and C.A. Janeway et al. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, NY (2011)). Alternatively, other methods such as EBV hybridoma technologies (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984) and Roder et al., Methods Enzymol, 121, 140-67 (1986) ) and vector-based expression systems

- 6,043,746 bacteriophages (see, for example, Huse et al., Science, 246, 1275-81 (1989)), are known from the prior art.

In addition, methods for producing antibodies from non-human animals are described, for example, in US patent No. 5545806, 5569825 and 5714352 and US patent application No. 2002/0197266.

Some embodiments of the invention also relate to TCRs or functional fragments thereof and polypeptides thereof, which are soluble TCRs. As used herein, the term soluble T cell receptor refers to single chain or heterodimeric truncated variants of naturally occurring TCRs that include at least the TCR α chain and β chain variable domains linked by a polypeptide linker (SEQ ID NOs: 22, 24, 25 and 27). Soluble TCR variants typically lack at least the transmembrane and cytosolic domains of the native protein; sometimes such soluble structures preferably do not contain any constant domain sequences. The soluble T cell receptor structures of the invention in preferred embodiments include structures consisting of α- and β-chain variable domain sequences as provided herein linked by a suitable linker sequence. The variable domain (amino acid or nucleic acid) sequence of the soluble TCR α chain and β chain may be identical to the corresponding natural TCR sequences or may include variant sequences of the soluble TCR variable domain α chain and β chain as compared to the corresponding natural TCR sequences. The term soluble T cell receptor as used in the present context includes soluble TCRs with variant or non-variant α chain and β chain sequences of the soluble TCR variable domain. Variations may be in the framework and/or CDR regions of the soluble TCR α chain and β chain variable domain sequences and may include, but are not limited to, amino acid deletion, insertion, substitution mutations, and changes in the nucleic acid sequence that do not alter amino acid sequence. The soluble TCR of the invention in any case retains or advantageously improves the binding functionality of its parent TCR molecules.

The above problem is also solved by using a nucleic acid encoding the antigen recognition structure of the invention or any of the previously mentioned protein or polypeptide structures. The nucleic acid may preferably (a) have a strand encoding an antigen recognition structure in accordance with the invention; (b) has a strand complementary to the strand in (a); or (c) has a strand that, under stringent conditions, hybridizes with the molecule described in (a) or (b). Severe conditions are known to one skilled in the art, particularly from Sambrook et al., Molecular Cloning. In addition, the nucleic acid optionally has additional sequences that are necessary for expression of the nucleic acid sequence corresponding to the protein, particularly for expression in a mammalian/human cell. The nucleic acid used may be included in a vector suitable for expressing the nucleic acid sequence corresponding to the polypeptide in a cell. However, nucleic acids can also be used to transform antigen presenting cells, which are not limited to classical antigen presenting cells such as dendritic cells, so that they themselves produce the corresponding proteins on their cell surface.

The term nucleic acid as used herein includes a polynucleotide, oligonucleotide and nucleic acid molecule and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthesized or derived (e.g., isolated and/or purified) from natural sources, which may contain naturally occurring naturally occurring, non-naturally occurring, or modified nucleotides and may contain a naturally occurring, non-naturally occurring, or modified internucleotide linkage, such as a phosphoramidate linkage or phosphorothioate linkage in place of the phosphodiester linkage existing between the nucleotides of the unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. In the present context, the term recombinant means (i) molecules whose constructs are obtained outside living cells by combining fragments of naturally occurring or synthetic nucleic acids with nucleic acid molecules that can replicate in a living cell; or (ii) molecules resulting from the replication of those described in (i) above. For the purposes of the present invention, the replication may be in vitro replication or in vivo replication. A nucleic acid may include any nucleotide sequence that encodes any of the TCRs, polypeptides or proteins or functional fragments or functional variants thereof described herein.

The invention further provides a vector comprising a nucleic acid according to the invention as described above. Desirably, the vector is an expression vector or a recombinant expression vector. The term recombinant expression vector in the context of the present invention refers to a nucleic acid construct that allows the expression

- 7 043746 mRNA, protein or polypeptide in a suitable host cell. The recombinant expression vector of the invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those that are designed for propagation and propagation or expression, or both, such as plasmids and viruses. Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo. Preferably, the recombinant expression vector is a viral vector, for example a retroviral vector. A recombinant expression vector includes regulatory sequences, such as transcription and translation initiation and stop codons, that are specific to the host cell species (for example, bacterial, fungal, plant or animal) into which the vector is to be introduced and in which expression of the nucleic acid can be effected. acids according to the invention. In addition, the vector of the invention may include one or more marker genes that allow selection of transformed or transfected hosts. A recombinant expression vector may include a native or normal promoter operably linked to a nucleotide sequence encoding structures of the invention or to a nucleotide sequence that is complementary to or which hybridizes to a nucleotide sequence encoding structures of the invention. The choice of promoters includes, for example, strong, weak, inducible, tissue- and organ-specific promoters. The promoter may be of viral or non-viral origin. Recombinant expression vectors of the invention can be designed for transient or sustained expression, or both. Also, recombinant expression vectors can be designed for constitutive expression or for inducible expression.

The invention also relates to a host cell including an antigen recognition structure in accordance with the invention. In particular, the host cell of the invention contains a nucleic acid or vector, as described herein above. The host cell may be a eukaryotic cell, such as a plant, animal, fungal, or algal cell, or may be a prokaryotic cell, such as a bacterial or protozoan cell. The host cell may be a culture cell or a primary cell, i.e. isolated directly from an organism, for example, a person. The host cell may be an adherent cell or a suspended cell, i.e. cell grown in suspension. For the purpose of producing a recombinant TCR, polypeptide or protein, the host cell is preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell. Most preferably, the host cell is a human cell. Although the host cell can be any type of cell, can be derived from any type of tissue, and can be at any stage of development, it is preferable that the host cell is a peripheral blood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. The T cell may be any T cell, such as a T cell from a cell culture, such as a primary T cell, or a T cell from a T cell line culture, such as Jurkat, SupT1, etc., or a T cell derived from a mammal, preferably a T cell or T cell precursor from a human patient. If the T cell is derived from a mammal, it may be derived from numerous sources, including, but not limited to, blood, bone marrow, lymph node, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, the T cell is a T cell of human origin. More preferably, the T cell is a T cell isolated from a human body. The T cell may be any type of T cell and any stage of development, including CD4-positive and/or CD8-positive, CD4-positive helper T cells, such as Th1 and Th2 cells, CD8-positive T cells, such as cytotoxic T cells -cells), tumor infiltrating cells (TILs), memory T cells, naïve T cells, etc., but are not limited to the above. Preferably, the T cell is a CD8 positive T cell or a CD4 positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably a T lymphocyte, such as a CD4 positive or CD8 positive T cell. The host cell is further preferably a tumor-reactive T cell specific for tumor cells expressing COL6A3.

In yet another aspect, the present invention relates to antigen recognition structures, nucleic acids, vectors, pharmaceutical compositions and/or host cells as provided herein for use in medicine. Medical use, in one preferred embodiment, includes use in the diagnosis, prevention and/or treatment of a neoplastic disease, such as a malignant or benign neoplastic disease. A tumor disease is, for example, a tumor disease characterized by the expression of COL6A3 in a cancer or tumor cell of the tumor disease.

With respect to the aforementioned medical use of antigen recognition structures and other materials derived from them, the cancer to be treated and/or diagnosed may

- 8 043746 be any type of cancer, including any of the diseases: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectal area, eye cancer, cancer of the intrahepatic bile ducts duct cancer, joint cancer, neck, gall bladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer , gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer gland, peritoneum, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and urinary cancer bubble The preferred type of cancer is cancer of the cervix, oropharynx, anus, anal canal, anorectal area, vagina, vulva or penis. A particularly preferred cancer is COL6A3-positive cancer, including gastrointestinal and gastric cancer.

The structures, proteins, anti-TCR antibodies, polypeptides and nucleic acids of the invention are intended, in particular, for use in immune therapy, preferably in adoptive T-cell therapy. Administration of the compounds of the invention may, for example, involve infusion of T cells of the invention into said patient. Preferably, such T cells are autologous cells from the patient, transduced in vitro with a nucleic acid or antigen recognition structure according to the present invention.

Application WO 2016/011210 proposes cells engineered for adoptive therapy, including NK cells and T cells, and compositions containing these cells, as well as methods for administering them to subjects. These cells may contain genetically engineered antigen receptors that specifically bind to antigens, such as chimeric antigen receptors (CARs) and costimulatory receptors.

The object of the invention is also solved by proposing a method for obtaining a cell line expressing a COL6A3-specific antigen recognition structure, including:

a) providing a suitable host cell;

b) providing a genetic construct including a coding sequence encoding an antigen recognition structure in accordance with any of claims 1 to 4;

c) introducing said genetic construct into said suitable host cell and

d) expression of said genetic construct by said suitable host cell.

The method may further include the step of presenting said antigen recognition structure on the surface of said suitable host cell.

In other preferred embodiments, the genetic construct is an expression construct comprising a promoter sequence operably linked to said coding sequence.

Preferably, said antigen recognition structure is derived from a mammal, preferably from a human. A preferred suitable host cell for use in the method of the invention is a mammalian cell, such as a human cell, in particular a human T lymphocyte. T cells for use in the invention are described above in the present context.

The invention also includes embodiments wherein said antigen recognition structure is a modified TCR, wherein said modification is the addition of functional domains such as a label or a therapeutically active substance. In addition, TCRs with alternative domains have been proposed, such as an alternative membrane anchor domain instead of the endogenous transmembrane region.

Desirably, the transfection system for introducing the genetic construct into said suitable host cell is a retroviral vector system. Such systems are well known to those skilled in the art.

The present invention also includes, in one embodiment, the additional method step of isolating and purifying an antigen recognition structure from a cell and, optionally, recovering translated fragments of the antigen recognition structure in the T cell.

An alternative aspect of the invention provides a T cell obtained or that can be obtained by a method for obtaining a T cell receptor (TCR) that is specific for tumor cells and has high avidity, as previously described in the present context. Such a T cell depends on the host cell used in the method of the invention, for example a T cell of human or non-human origin, preferably a TCR of human origin.

TCRs, polypeptides, proteins (including functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof) and antibodies (including antigen binding fragments thereof) of the invention can be isolated and/or cleared. The term isolated, as used in the present context, means removal from the natural environment.

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The term purified as used in the present context means an increase in the degree of purity, purity being a relative concept and not necessarily construed as absolute purity. For example, the purity may be at least about 50, may be more than 60, 70,

80, 90, 95 or maybe 10o%.

Antigen recognition structures, TCRs, polypeptides, proteins (including functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof) and antibodies (including antigen binding fragments thereof) of the invention, all of which hereinafter referred to collectively as the TCR materials of the invention, may be formulated in a composition, such as a pharmaceutical composition. In this regard, the invention also provides a pharmaceutical composition comprising any of antigen recognition structures, TCRs, polypeptides, proteins, functional fragments, functional variants, nucleic acids, expression vectors, host cells (including populations thereof) and antibodies (including their antigen binding fragments) described in the present context, and a pharmaceutically acceptable carrier, excipient and/or stabilizer. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may include more than one TCR material of the invention, such as a polypeptide and a nucleic acid or two or more different TCRs (including functional fragments and functional variants thereof). Alternatively, pharmaceutical compositions may include the TCR material of the invention in combination with other pharmaceutically active substance(s) or drug(s), such as chemotherapeutic agents, for example asparaginase, busulfan, carboplatin, cisplatin , daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those commonly used for the particular TCR material of the invention in question. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are publicly available. Preferably, the pharmaceutically acceptable carrier is one that does not have adverse side effects or toxicity under the conditions of use.

Thus, also provided is a pharmaceutical composition comprising any of the products of the invention and the TCR materials of the invention described herein, in particular any proteins, nucleic acids or host cells. In a preferred embodiment, the pharmaceutical composition is for immune therapy, preferably adoptive cell therapy.

Preferably, the TCR material of the invention is administered by injection, for example intravenously. If the TCR material of the invention is a host cell expressing the TCR of the invention (or a functional variant thereof), the pharmaceutically acceptable carrier for the cells for injection may include any isotonic vehicle, such as, for example, normal saline (about 0.90% wt. ./vol NaCl in water, about 300 mOsm/L NaCl in water or about 9.0 g NaCl per 1 L of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL, USA), PLASMA-LYTE A (Baxter, Deerfield, Illinois, USA), about 5% dextrose in water or Ringer's lactate. In one embodiment, human serum albumin is added to the pharmaceutically acceptable carrier.

For purposes of the present invention, the amount or dose (e.g., the number of cells when the TCR material of the invention is one or more cells) of the TCR material of the invention administered may be sufficient to produce an effect, such as a therapeutic or prophylactic response, in a subject or animal within a reasonable period of time. period of time. For example, a dose of the TCR material of the invention should be sufficient to bind to a cancer antigen or to detect, treat, or prevent cancer over a period of about 2 hours or longer, such as 12 to 24 hours or more, from the time of administration. In certain embodiments, the time period may be even longer. The dose is determined by the effectiveness of the particular TCR material of the invention and the condition of the animal (eg, human), as well as the body weight of the animal (eg, human) being treated.

It is appreciated that the pharmaceutical compositions of the invention, TCRs (including functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells or cell populations may be used in methods of treating or preventing cancer or COL6A3- positive pre-tumor state. The TCRs of the invention (and functional variants thereof) are believed to specifically bind to the COL6A3 antigen such that the TCR (or a related polypeptide or protein of the invention and functional variants thereof), when expressed by a cell, is capable of mediating an immune response to the cell. a target expressing the COL6A3 antigens of the invention. In this regard, the invention provides a method of treating or preventing a condition, in particular cancer, in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, antigen recognition structures, in particular TCRs (and functional variants thereof), polypeptides or proteins described in the present context, any a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs (and functional variants thereof), polypeptides, proteins described herein, or any host cell or population of cells comprising the recombinant vector that encodes any of the structures of the invention (and functional variants thereof), polypeptides or proteins described herein, in an amount effective to treat or prevent a condition in a mammal where the condition is cancer, preferably COL6A3-positive cancer.

Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPHA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing products such such as bovine whey or skim milk, and buffers (eg phosphate buffer).

The terms treat and prevent, as well as words derived from them, in the context of this description do not necessarily imply 100% or complete cure or prevention. Rather, they are varying degrees of cure or prevention that the average person in the art determines to have potential benefit or therapeutic effect. In this regard, the methods of the invention can provide any degree of cure or prevention of a pathological condition in a mammal. In addition, the treatment or prevention provided by the method of the invention may include treatment or prevention of one or more pathological conditions or symptoms of these conditions, such as cancer, being treated or prevented. For example, treatment or prevention may include promoting tumor regression. Also for the purposes of the present invention, the warning may include delaying the development of a pathological condition or a symptom or condition thereof.

The present invention also relates to a method of treating cancer, comprising administering a TCR, nucleic acid or host cell as described herein in combination with at least one chemotherapy drug and/or radiation therapy.

Also provided is a method of treating cancer in a subject in need thereof, comprising:

a) isolating a cell from the body of the specified subject;

b) transforming the cell with at least one vector encoding an antigen recognition structure according to the present invention to obtain a transformed cell;

c) culturing the transformed cell to obtain a plurality of transformed cells;

d) administering a plurality of transformed cells to said subject.

Also provided is a method of treating cancer in a subject in need thereof, comprising:

a) isolating a cell from a healthy donor’s body;

b) transforming the cell with a vector encoding the antigen recognition structure according to the present invention to obtain a transformed cell;

c) culturing the transformed cell to obtain a plurality of transformed cells; And

d) administering a plurality of transformed cells to said subject.

A method for detecting cancer in a biological sample is also proposed, including:

a) contacting a biological sample with an antigen recognition structure according to the present description;

b) identifying the binding of the antigen recognition structure to the biological sample.

In some embodiments, the method of detecting cancer is performed in vitro, in vivo, or in situ.

A method for identifying a pathological condition in a mammal is also proposed. The method includes (i) contacting a sample comprising one or more mammalian cells with any of TCRs (and functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or antigen binding fragments thereof. the invention or pharmaceutical compositions described in the present context, which leads to the formation of a complex; and (ii) detecting the complex, wherein detection of the complex is an indicator of the presence of a pathological condition in the mammal, where the condition is a cancer, such as a COL6A3-positive cancer.

With respect to a method for detecting a pathological condition in a mammal, the cell sample may be a sample containing whole cells, lysates thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction.

For the purposes of the detection method of the invention, contact with a mammal can take place in vitro or in vivo. It is preferable if the contact takes place in vitro.

Detection of the complex can also be accomplished using any number of methods known in the art. For example, antigen recognition structures (and functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or antibodies or TCRs or antigen binding fragments thereof described herein may be labeled with a suitable detecting a label such as, for example, a radioisotope, a fluorophore (for example, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (for example, alkaline phosphatase, horseradish peroxidase) and elemental particles (for example, gold particles).

For purposes of the methods of the invention, when host cells or populations of cells are administered, the cells may be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

With respect to the previously mentioned medical uses of the TCR material according to the invention, the cancer requiring treatment and/or diagnosis may be any cancer, including acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer anus, anal canal or anorectal area, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder or pleural cancer, cancer of the nose, nasal cavity or middle ear, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia , chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer , non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic, peritoneal, omental and mesenteric cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, cancer stomach, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. The preferred type of cancer is cancer of the cervix, oropharynx, anus, anal canal, anorectal area, vagina, vulva or penis. A particularly preferred cancer is COL6A3-positive cancer, such as gastrointestinal cancer and gastric cancer.

In general, the invention provides a method of treating a subject suffering from a tumor or neoplastic disease, which includes the administration of antigen recognition structures, nucleic acids, vectors, pharmaceutical compositions and/or a host cell, as disclosed in the present invention. Preferably, the subject is a subject in need of such treatment. The subject in preferred embodiments is a mammalian subject, preferably a patient of human origin, suffering from a tumor or neoplastic disease that is COL6A3 positive.

The present invention will be further described by means of the following examples with reference to the accompanying figures and sequences, however, without limiting the invention thereto. In accordance with the purposes of the present invention, all references cited are incorporated herein in their entirety by reference. In figures and in sequences:

In fig. 1 shows the conversion of TCR to Va/Ve stabilized single chain TCR (scTv) by yeast based surface display. ScTv molecules presented on the surface of transformed Saccharomyces cerevisiae EBY100 cells were stained with FITC-labeled anti-Vbeta1 antibody and PE-labeled HLA-A*02/COL6A3-002 tetramer. The unmodified scTv R4P3F9 (left section, SEQ ID NO: 22) is compared to an scTv clone with single point mutations to stabilize the scTv backbone (right section), which was obtained by selection from a random scTv mutation library.

In fig. Figure 2 shows scTv affinity maturation using a yeast-based surface display. For stabilized scTv molecules, with or without the immature affinity matured CDR1 beta chain, staining was done with HLA-A*02 tetramers containing COL6A3-002 (SEQ ID NO: 1) and counterstaining with a mixture of HLA-A*02 tetramers containing 9 peptides (SEQ ID NO: 28-36) with high sequence similarity to COL6A3-002. Stabilized scTv (SEQ ID NO: 27) with non-affinity matured CDR1 beta chain sequence, RSGDLS (SEQ ID NO: 13) is compared with scTv clones having affinity matured CDR1 beta chain sequences, AMDHPY (SEQ ID NO: 40 ) and ARWHRN (SEQ ID NO: 39).

In fig. Figure 3 shows elution profiles obtained by size exclusion chromatography for anti-CD3 Fab-scTv hybrid variants R4P3F9S from 75-1 to 75-25.

In fig. Figure 4 shows the kinetics of binding of HLA-A*02/COL6A3-002 to anti-CD3 Fab-scTv R4P3F9S in hybrid variants 75-1 to 75-25, measured using biolayer interferometry. The analyzed concentrations of HLA-A*02/COL6A3-002 are indicated.

In fig. 5 shows binding analysis of anti-CD3 Fab-scTv R4P3F9S in hybrid variants 75-1 to 75-25 as measured by biolayer interferometry (BLI). 1 μM HLA-A*02 was analyzed in complex with the indicated similar peptides.

In fig. 6 shows a comparison of the binding kinetics of HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 (SEQ ID NO: 30) with anti-CD3 Fab-scTv R4P3F9S in various hybrid variants. The analyzed concentrations of Fab-scTv molecules are indicated.

In fig. Figure 7 shows PE-labeled HLA-A*02/COL6A3-002 tetramer staining of human T cells expressing the affinity matured TCR variant R4P3F9. Absence of TCR (mock) or expression of NYESO1-001-specific TCR 1G4 and staining with PE-labeled tetramers

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HLA-A*02/NYESO1-001 was used for control purposes.

In fig. 8 shows IFN-gamma release from the affinity matured TCR variant R4P3F9 expressed by human CD8+ T cells in response to COL6A3-002. For control purposes, the TCR was not expressed (mock) or the NYESO1-001-specific TCR 1G4 was expressed. IFN-γ release was determined by ELISA after coculture of electroporated CD8+ T cells with T2 cells loaded with serial dilutions of COL6A3-002.

In fig. 9 shows IFN-gamma release from the affinity matured TCR variant R4P3F9 expressed by human CD8+ T cells in response to COL6A3-002 and various related peptides. For control purposes, the TCR was not expressed (mock) or the NYESO1-001-specific TCR 1G4 was expressed. IFN-γ release was determined by ELISA after coculture of electroporated CD8+ T cells with T2 cells loaded with 10 μM COL6A3-002 or related peptides.

In fig. 10 shows PE-labeled peptide-HLA-A*02 tetramer staining of human T cells expressing the affinity matured TCR variant R4P3F9. For control purposes, TCR was not expressed (mock) or TCR 1G4, specific for NYESO1-001, was expressed and stained with PE-labeled HLA-A*02/NYESO1-001 tetramers.

In fig. 11 shows IFN-gamma release from the affinity matured TCR variant R4P3F9 expressed by human CD8+ T cells in response to COL6A3-002 or COL6A1-001. For control purposes, the TCR was not expressed (mock) or the NYESO1-001-specific TCR 1G4 was expressed. IFN-γ release was determined by ELISA after coculture of electroporated CD8+ T cells with T2 cells loaded with serial dilutions of COL6A3-002 or COL6A1-001.

In fig. 12 shows the release of IFN-γ by primary human CD8+ T cells expressing R4P3F9 TCR variants after co-culture with various tumor cell lines. T cells of the SF539, SW982 and Hs840 lines present the target peptide at different levels. MCF-7 cells do not present the target peptide. As controls, effector cells without exogenous TCR were analyzed together with cells with the studied TCR. IFN-γ release was determined by ELISA. * Marked point is out of scale.

Table 1 Sequences of the peptide according to the invention (positions according to IMGT numbering (Frangois Ehrenmann, Patrice Duroux, Chantal Ginestoux; Protein displays: human (Homo sapiens) TRAV; IMGT Repertoire. IMGT®, the international ImMunoGenetics information system® http://www.imgt .org.; Created: 03/16/2011. Version: 06/03/2016; Frangois Ehrenmann, Patrice Duroux, Chantal Ginestoux; Protein displays: human (Homo sapiens) TRBV; IMGT Repertoire. IMGT®, the international ImMunoGenetics information system ® http://www.imgt.org.; Created: 03/16/2011. Version: 06/03/2016.

SEQ ID NO Name Description Subsequence 1 COL6A3002 FLLDGSANV 2 R4P3F9 TKR R4P3F9, MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIAS alpha alpha chain - complete length LNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTF GKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA CANAFNNSIIPEDTFFPSPESSCDVKLVE KSFETDTNLNFQ NLSVIGFRILLLKVAGFNLLMTLRLWSS 3 R4P3F9, alpha, leader TCR R4P3F9, alpha chain - leader peptide MKSLRVLLVILWLQLSWVWSQ 4 R4P3F9, TKR R4P3F9, QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG alpha, alpha chain KSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS variable variable domain DSATYLCAAYSGAGSYQLTFGKGTKLSVIP 5 R4P3F9, CDRal TCR R4P3F9, alpha chain - CDR1 DRGSQS 6 R4P3F9,CDRa2 TCR R4P3F9, alpha chain - CDR2 IYSNGD 7 R4P3F9, CDRa3 TCR R4P3F9, alpha chain - CDR3 CAAYSGAGSYQLT 8 R4P3F9 - TKR R4P3F9, NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD alpha, alpha chain VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSII constant - PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL th constant domain LLKVAGFNLLMTLRLWSS 9 R4P3F9 alpha, constant, start TCR R4P3F9, alpha chain constant NIQN

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domain, beginning 10 R4P3F9, beta TCR R4P3F9, full length beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLR CSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILER FSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFG SGTRLTWEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVSTDPQPL KEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKA T LYAVLVSALVLMAMVKRKD F eleven R4P3F9, beta, leader TCR R4P3F9, beta chain leader peptide MGFRLLCCVAFCLLGAGPV 12 R4P3F9, beta, variable TCR R4P3F9, beta chain variable domain DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQ GLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTW 13 R4P3F9, CDRbl TCR R4P3F9, beta chain CDR1 RSGDLS 14 R4P3F9, CDRb2 TCR R4P3F9, beta chain CDR2 YYNGEE 15 R4P3F9, CDRb3 TCR R4P3F9, CDR3 beta chain CASSVESSYGYT 16 R4P3F9, beta, constants TCR R4P3F9, beta chain constant domain EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHV ELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVS ALVLMAMMVKRKDF 17 R4P3F9, beta, constants, start 1 TCR R4P3F9, beta chain constant domain, start 1 EDLNK 18 R4P3F9, beta, constant, start 2 TCR R4P3F9, beta chain constant domain, beginning 2 EDLKN 19 Ada2p R4P3F9 Ada2p fusion protein with scTv R4P3F9 and tags MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLS TTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPIN TQYVFGGGGSDYKDDDDKGGGASQKEVEQNSGPLSVPEGAI ASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDG RFTAQLNKASQYVSLLIRDSQPSDS ATYLCAAYSGAGSYQL TFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGVTQT PKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQ YYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALY FCASSVESSYGYTFGSGTRLTWEDLNKAAAGGSGGEQKLI SEEDL 20 Ada2r Leadership sequence and Ada2p MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLS TTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPIN TQYVF 21 FLAG tag FLAG tag plus linkers GGGGSDYKDDDDKGGGAS 22 scTv R4P3F9 Single chain variable QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS DSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGG

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nal domains of R4P3F9 with a linker; aF55S in the alpha chain variable domain GGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDL SVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPD LHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTW EDLNK 23 Mus Label Linker and mus tag AAAGGSGGEQKLISEEDL 24 scTv R4P3F9 bQ43K scTv R4P3F9 with a stabilizing mutation bQ43K in the beta chain variable domain QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS DSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGG GGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDL SVYWYKQSLDQGLQFLIQYYNG EERAKGNILERFSAQQFPD LHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTW EDLNK 25 scTv R4P3F9 bL72S scTv R4P3F9 with stabilizing mutation bL72S in the beta chain variable domain QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS DSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGG GGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDL SVYWYQQSLDQGLQFLIQYYNG EERAKGNISERFSAQQFPD LHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTW EDLNK 26 CDRal mutant! aG29R mutation DRRSQS 27 scTv R4P3F9S Stabilized version of scTv R4P3F9 QKEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYSG KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPS DSATYLCAAYSGAGSYQLTFGKGTKLSVIPGGGGSGGGGSG GGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGD LSVYWYKQSLDQGLQFLIQYYNGEE RAKGNISERFSAQQFP DLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTV V 28 AGRN-001 Similar peptides ALLDGRVQL 29 CLAS Pl001 Similar peptides RLLDGAFKL thirty COL6A1001 Similar peptides ILLDGSASV 31 COL6A2001 Similar peptides FLLDGSERL 32 COL6A3006 Similar peptides FLFDGSANLV 33 COL6A3008 Similar peptides FLFDGSAN 34 COL6A3014 Similar peptides FLLDGSEGV 35 VWA2-001 Similar peptides FLLDGSNSV 36 VWF-001 Similar peptides FLLDGSSRL 37 CDRbl mutant 1 Beta chain CDR1 variant 1 ARWHNN 38 CDRbl mutant 2 Beta chain CDR1 option 2 AKDHLN

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39 CDRbl mutant 3 Beta chain CDR1 variant 3 ARWHRN 40 CDRbl mutant 4 Beta chain CDR1 option 4 AMDHPY 41 CDRbl mutant 5 Beta chain CDR1 variant 5 ATDHYN 42 CDRbl mutant 6 Beta chain CDR1 variant 6 ARYHTN 43 CDRbl mutant 7 Beta chain CDR1 variant 7 APYHLN 44 CDRbl mutant 8 Beta chain CDR1 variant 8 AKDHTN 45 CDRbl mutant 9 Beta chain CDR1 variant 9 ARYHRN 46 CDRbl mutant 10 Beta chain CDR1 variant 10 ARWHSN 47 CDRbl mutant 11 Beta chain CDR1 variant 11 ATDHYN 48 CDRbl mutant 12 Beta chain CDR1 variant 12 RWGDLN 49 CDRbl mutant 13 Beta chain CDR1 variant 13 ARDHLN 50 75-1 Fab heavy chain with stabilized scTv R4P3F9S MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCA ASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASLNCTYSDR GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKAS QYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSV IPNIQNGGG GSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLI TATGQRVTLRCSPRSGDLSVYWYKQSLDQGLQFLIQYYNGE ERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASS VESSYGYTFGSGTRLTWEDLKN 51 75- Fab heavy chain Fab Heavy Chain MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCA ASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTSPPSPAPPVAG 52 75- Fab light chain Light chain Fab MKWVTFISLLFLFSSAYSDIQMTQSPSSLSASVGDRVTITC RASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSG SGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLS SPVTKS FNRGEC 53 1G4, alpha TCR 1G4, alpha chain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLN CSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTF

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- full length GRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKTVLDMRSMDFKSSNSAVAWSNKSDFA CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQ NLSVIGFRILLLKVAGFNLLMTLRLWSS 54 1G4, alpha, leader TCR 1G4, alpha chain - leader peptide METLLGLLILWLQLQWVSSK 55 1G4, alpha, variable TCR 1G4, alpha chain variable domain QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGK GLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPG DSATYLCAVRPTSGGSYIPTFGRGTSLIVHP 56 1G4, alpha, constants TCR 1G4, alpha chain constant domain YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSII PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS 57 1G4, beta TCR 1G4, full length beta chain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQ CAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNG YNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFF GEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKE QPALNDSR YCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA KPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGK ATLYAVLVSALVLMAMMVKRKDSRG 58 1G4, beta, leader TCR 1G4, beta chain leader peptide MSIGLLCCAALS LLWAGPVNA 59 1G4, beta, variable TCR 1G4, beta chain variable domain GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGL RLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPS QTSVYFCASSYVGNTGELFFGEGSRLTVL 60 1G4, beta, constant Beta chain constant domain EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHV ELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS ALVLMAMMVKRKD S RG 61 NYESO1001 Control peptide SLLMWITQV 62 S-14, beta; C5, beta Full length beta chain TCR C-14; S-5 with CDRbl, mutant 4 MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLR CSPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNILER FSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFG SGTRLTWEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDF 63 S-14, alpha S-14; Full length TCR beta chain with CDRal mutant 1 MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIAS LNCTYSDRRSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTF GKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKTVLD MRSMDFKSSNSAVAWSNKSDFA CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQ NLSVIGFRILLLKVAGFNLLMTLRLWSS 64 75-5 Fab heavy chain with stabilized scTv R4P3F9S and MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCA ASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

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CDRbl mutant 4 HTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASLNCTYSDR GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKAS QYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSV IPNIQNGGGGSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLI TATGQRVTLRCSPAMDHPYVYWYKQ SLDQGLQFLIQYYNGE ERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASS VESSYGYTFGSGTRLTWEDLKN 65 75-14 Fab heavy chain with stabilized scTv R4P3F9S, CDRal, mutant 1, and CDRbl, mutant 4 MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCA ASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASLNCTYSDR RSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKAS QYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSV IPNIQNGGG GSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLI TATGQRVTLRCSPAMDHPYVYWYKQSLDQGLQFLIQYYNGE ERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASS VESSYGYTFGSGTRLTWEDLKN 66 75-25 Fab heavy chain with stabilized scTv R4P3F9S in beta/alpha orientation, CDRal, mutant 1 MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCA ASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTSPPSPAPPVAGGVTQTPKHLITATGQRVTLRCSPRSGDL SVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFSAQQFPD LHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTW EDLKNGGGGSGGGG SGGGGSGGGGSGGGGSQKEVEQNSGPL SVPEGAIASLNCTYSDRRSQSFFWYRQYSGKSPELIMSIYS NGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYS GAGSYQLTFGKGTKLSVIPNIQN

Examples

Naturally occurring T cell receptors (TCRs) for cancer antigens often have lower affinity compared to TCRs targeting viral antigens, and this may be one possible explanation for tumor cell immune evasion (Aleksic et al., 2012). . Thus, it is desirable to have higher affinity TCR variants designed for use as antigen recognition structures in adoptive cell therapy, or as recognition modules in a soluble circuit approach, such as using bispecific molecules (Hickman et al., 2016 ). The present invention thus relates to the modification and optimization of the naturally occurring T cell receptor R4P3F9 (SEQ ID NO: 2 and 10), the target of which is the tumor associated peptide COL6A3-002 (SEQ ID NO: 1) with an affinity of about 60 μM ( DE102016115246).

Example 1. Preparation of stable scTv.

For the purposes of the present invention, the previously studied R4P3F9 TCR (SEQ ID NO: 2 and 10) was converted into a single chain TCR structure (scTv, SEQ ID NO: 22) for maturation by yeast-based surface display technology using a variable alpha combination (SEQ ID NO: 4) and beta (SEQ ID NO: 12) domain with the addition of corresponding constant domains (SEQ ID NO: 9 and 17) and a suitable glycine-serine linker sequence. DNA of the corresponding sequence was synthesized and transformed into Saccharomyces cerevisiae EBY100 (MATa AGA1::GAL1AGA1::URA3 ura352 trp1 leu2delta200 his3delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC® MYA 4941™) along with a yeast display vector containing leader sequence and the yeast mating type protein Aga2p (SEQ ID NO: 20), based on pCT302 (Boder et al., 2000). The resulting fusion protein after homologous recombination in yeast cells (SEQ ID NO: 19) contains a leader peptide at the N-terminus of the Aga2p protein, which is responsible for displaying the protein under study (Boder et al., 1997), short peptide tags including linker sequences ( SEQ ID NO: 21 and 23) for the purpose of expressing controls and the protein of interest, namely scTv R4P3F9 (SEQ ID NO: 22) or variants thereof. The transformation was carried out as described in application DE 102016121899, resulting in up to 109 yeast clones per library. The libraries were obtained by random mutagenesis during PCR, covering the entire scTv R4P3F9 gene sequence.

The selection process for yeast clones with the best expressing scTv, which selectively binds to COL6A3-002 with the participation of HLA-A*02, was carried out essentially as described in Smith et al., 2015. In order to confirm the high level of expression and the correct conformation of the R4P3F9 scTv variant , exposed on the surface of yeast, an antibody to Vbeta1 (Beckman Coulter, clone BL37.2) was used together with the HLA-A*02/COL6A3-002 tetramer (Fig. 1). Transformation of scTv by yeast-based surface display revealed two key stabilizing mutations in the framework region required along with the initial CDR sequences for proper presentation of scTv to the cell surface, namely bQ43K (SEQ ID NO: 24) and bL72S (SEQ ID NO: 25 ), both of which are located on the beta strand. Additionally, during stability maturation, a conversion was made at position 29 in CDR1 of the alpha chain (SEQ ID NO: 5) to glycine to arginine (CDRa1 mutant 1, SEQ ID NO: 26), resulting in improved binding with a tetramer.

Example 2: Affinity maturation of stabilized scTvs.

In order to obtain scTv molecules with higher binding affinity towards HLA-A*02/COL6A3-002, CDRb1 (SEQ ID NO: 13) was degenerated using the previously identified stabilized scTv scaffold R4P3F9S (SEQ ID NO: 27) expressing the stabilizing mutations aG29R, bQ43K and bL72S. CDRb1 residues were randomized using degenerate DNA oligonucleotide primers essentially as described previously ( Smith et al., 2015 ). The resulting DNA library was transformed as described in Example 1. To maintain scTv binding selectivity, negative selection was used against HLA-A*02 tetramers comprising peptides derived from normal tissues (SEQ ID NO: 28-36) that have high similarity sequences with the COL6A3-002 peptide.

To screen for scTv R4P3F9S variants with increased affinity and selectivity, the HLA-A*02/COL6A3-002 tetramer was used, with decreasing concentrations for each screening step. After three rounds of selection, individual scTv clones were isolated and sequenced, resulting in a set of affinity matured CDRb1 sequences (SEQ ID NO: 37 to 49). For scTvs with affinity matured CDRb1 sequences, a strong improvement in binding ability to COL6A3-002 could be demonstrated, whereas selectivity for binding to COL6A3-002 was maintained as no binding was observed for 9 similar peptides (Fig. 2).

Example 3: Production of bispecific antibody-scTv fusion proteins.

Stabilized HLA-A*02/COL6A3-002 affinity-matured scTv can be expressed in fusion form with an anti-CD3 antibody fragment, allowing tumor-specific targeting and activation of T cells regardless of their natural specificity. We have produced bispecific antibody-TCR fusion proteins comprising an antibody heavy chain with an anti-CD3 Fab fragment (UCHT1) (SEQ ID NO: 51) fused to scTv R4P3F9S variants (SEQ ID NOs: 50, 64, 65 and 66) and an anti-CD3 Fab fragment antibody light chain (UCHT1) (SEQ ID NO: 52). The resulting Fab-scTv fusion proteins have a molecular mass of approximately 75 kDa. Based on the different CDR1 sequences of scTv R4P3F9S alpha (SEQ ID NO: 5 and 26) and beta chain (SEQ ID NO: 13 and 37-49), various variants of the Fab-scTv fusion (75-1 to 75-25, table. 2) were expressed in transiently transfected ExpiCHO cells according to the manufacturer's recommendations. Proteins were purified by protein L chromatography and size exclusion chromatography. All fusions could be obtained with yields ranging from 80 μg to 1 mg (Table 2) and homogeneously shaped heterodimers of the expected size as determined by size exclusion chromatography analysis (Figure 3).

table 2

Nomenclature and yield of bispecific Fab-scTv fusion proteins. The molecule is based on the sequences SEQ ID NO 50 and 52 and the indicated variants CDRa1 and CDRb1

Option CDRal/SEQ CDRbl/SEQ Output [μ 75-1 DRGSQS (SEQ Ш NO. 5) RSGDLS (SEQ ID NO. 13) 267.9 75-2 DRGSQS (SEQ Ш NO. 5) ARWHNN (SEQ ID NO. 37) 78.4 75-3 DRGSQS (SEQ ID NO. 5) AKDHLN (SEQ ID NO. 38) 646.7 75-4 DRGSQS (SEQ ID NO. 5) ARWHRN (SEQ ID NO. 39) 704.3 75-5 DRGSQS (SEQ ID NO. 5) AMDHPY (SEQ ID NO. 40) 397.2 75-6 DRGSQS (SEQ ID NO. 5) ATDHYN (SEQ ID NO. 41) 268.1 75-7 DRGSQS (SEQ ID NO. 5) ARYHTN (SEQ ID NO. 42) 83.2 75-8 DRGSQS (SEQ ID NO. 5) APYHLN (SEQ ID NO. 43) 765.7 75-9 DRGSQS (SEQ ID NO. 5) AKDHTN (SEQ ID NO. 44) 1067.2 75-10 DRRSQS (SEQ ID NO. 26) RSGDLS (SEQ ID NO. 13) 389.6 75-11 DRRSQS (SEQ ID NO. 26) ARWHNN (SEQ ID NO. 37) 270.4 75-12 DRRSQS (SEQ ID NO. 26) AKDHLN (SEQ ID NO. 38) 943.6 75-13 DRRSQS (SEQ ID NO. 26) ARWHRN (SEQ ID NO. 39) 560.3 75-14 DRRSQS (SEQ ID NO. 26) AMDHPY (SEQ ID NO. 40) 360.7 75-15 DRRSQS (SEQ ID NO. 26) ATDHYN (SEQ ID NO. 41) 541.5 75-16 DRRSQS (SEQ ID NO. 26) ARYHTN (SEQ ID NO. 42) 403.6 75-17 DRRSQS (SEQ ID NO. 26) APYHLN (SEQ ID NO. 43) 195.5 75-18 DRRSQS (SEQ ID NO. 26) AKDHTN (SEQ ID NO. 44) 731.3 75-19 DRRSQS (SEQ ID NO. 26) ARYHRN (SEQ ID NO. 45) 794 75-20 DRRSQS (SEQ ID NO. 26) ARWHSN (SEQ ID NO. 46) 85.5 75-21 DRRSQS (SEQ ID NO. 26) ATDHYN (SEQ ID NO. 47) 276 75-22 DRRSQS (SEQ ID NO. 26) RWGDLN (SEQ ID NO. 48) 255 75-23 DRRSQS (SEQ ID NO. 26) ARDHLN (SEQ ID NO. 49) 217 75-24 ^a DRGSQS (SEQ ID NO: 5) RSGDLS (SEQ ID NO. 13) 166.6 75-25 ^a DRRSQS (SEQ ID NO. 26) RSGDLS (SEQ ID NO. 13) 267

^a Beta-alpha orientation of scTv.

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Example 4 Binding of Fab-scTv Fusion Proteins to COL6A3-002 and Related Peptides.

The binding affinity of anti-CD3-scTv R4P3F9S fusion proteins to HLA-A*02 monomers with the COL6A3-002 peptide or various related peptides was measured using biolayer interferometry. Measurements were carried out on an Octet RED384 system using settings recommended by the manufacturer. Briefly, purified Fab-scTv molecules were loaded onto biosensors (FAB2G) prior to serial dilution analysis of HLA-A*02/COL6A3-002. Compared with variants 75-1 and 75-24, which include wild-type CDRa1 and wild-type CDRb1, Fab-scTv variants with affinity matured CDRa1 and/or CDRb1 sequences showed up to a 40-fold increase in binding affinity (Table 3 Fig. 4). In order to assess the selectivity of binding to HLA-A*02/COL6A3-002, purified Fab-scTv molecules loaded onto FAB2G biosensors were screened against binding to 1 μM similar peptides (SEQ ID NO: 28-36), each of which was in complex with HLA-A*02. With the exception of HLA-A*02/COL6A1-001 (SEQ ID NO: 30), to which the majority of Fab-scTv variants containing mature CDRa1 with affinity matured bound (SEQ ID NO: 26), Fab-scTv variants did not demonstrate binding to similar peptides (Fig. 5), indicating high selectivity of binding. For some Fab-scTv variants, the therapeutic window between binding to HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 was examined by loading biotinylated peptide-HLA complexes onto biosensors (SA) and analyzing serial dilutions of Fab variants -scTv. Whereas the binding affinity of variant 75-10, comprising the affinity matured CDRa1 sequence (SEQ ID NO: 26) and the wild-type CDRb1 sequence (SEQ ID NO: 13), was 8-fold higher with HLA-A*02/COL6A3-002 than with HLA-A*02/COL6A1-001, the binding affinity detected for the 75-13 Fab-scTv variant involving CDRb1 (SEQ ID NO: 39) with maturity was up to 57-fold higher, supporting improved therapeutic windows (Table 4, Fig. 6).

Table 3

Binding affinity of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002

Option KD (M) kopeck (1/Ms) koff (1/s) 75-1 8.06E-06 1.01E+05 8D7E-01 75-2 3.69E-06 1.59E+05 5.86E-01 75-3 4.92E-06 9.71E+04 4.78E-01 75-4 5.76E-06 9.78E+04 5.63E-01 75-5 4.32E-04 2.21E+03 9.55E-01 75-6 1.1 ZE-06 2.06E+05 2.32E-01 75-7 1.79E-06 1.93E+05 3.44E-01 75-8 3.45E-06 1.36E+05 4.69E-01 75-9 1.41E-05 6.02E+04 8.51E-01 75-10 1.78E-06 1.69E+05 3.01E-01 75-11 2.82E-07 4D6E+05 1D8E-01 75-12 3.74E-07 2.67E+05 1.00E-01 75-13 4.05E-07 3.28E+05 1,ЗЗЭ-01 75-14 3.10E-06 8.41E+04 2.61E-01 75-15 7.78E-07 2,ЗЗЭ+05 1.81E-01 75-16 5.87E-07 3.37E+05 1.98E-01 75-17 2.27E-07 3.62E+05 8.20E-02 75-18 1.93E-06 1.51E+05 2.91E-01 75-19 6.00E-07 2.96E+05 1.78E-01 75-20 5.31E-07 6.08E+05 3.23E-01 75-21 5.52E-07 2.72E+05 1.50E-01 75-22 8.22E-07 2.48E+05 2.04E-01 75-23 3.24E-07 3.18E+05 1.03E-01 75-24 5.20E-06 1.08E+05 5.62E-01 75-25 8,ZZE-06 6.23E+04 5D9E-01

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Table 4

Comparative binding affinities of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001

Option pHLA-A*02 KD (M) KDcOL6A1-001/KDcOL6A3-002 75-10 COL6A3-002 1.37E-05 8 COL6A1-001 1.08E-04 75-11 COL6A3-002 8.50E-07 8 COL6A1-001 6.46E-06 75-12 COL6A3-002 7.24E-07 12 COL6A1-001 8.98E-06 75-13 COL6A3-002 7.39E-07 57 COL6A1-001 4.23E-05 75-17 COL6A3-002 8.25E-07 9 COL6A1-001 7.10E-06 75-23 COL6A3-002 1D5E-06 22 COL6A1-001 2.55E-05

Example 5: Use of Affinity Mature TCRs for Cellular Expression.

Modification of T cells to express TCRs that recognize the tumor-specific peptide-HLA complex is a promising alternative for redirecting T cells to cancer cells. Since the use of affinity matured CDR1 sequences could improve cell-associated TCRs to HLA-A*02/COL6A3-002, the identified CDRa1 and CDRb1 sequences with mutations were introduced into the parent R4P3F9 TCR (SEQ ID NO: 2 and 10). The resulting TCR variants with mutations (C-1 to C-18, Table 5) were expressed in human CD8+ T cells after electroporation of the corresponding mRNA obtained by in vitro transcription of PCR-amplified DNA constructs. For control purposes, TCR 1G4 (SEQ ID NO: 53 and 57) was expressed to the NYESO1-001 peptide (SEQ ID NO: 61). After overnight incubation of RNA-electroporated CD8+ T cells, expression of the introduced TCR variants was analyzed by staining with PE-labeled HLA-A*02/COL6A3-002 tetramers or HLA-A*02/NYESO1-001 tetramers. While the parental R4P3F9 TCR variant C-1 showed only minimal staining with HLA-A*02/COL6A3-002 tetramers, R4P3F9 TCR variants C-2 to C-18 with CDRa1 and/or affinity-matured CDRb1 showed increased tetramer staining ( Fig. 7). The functional activation of CD8+ T cells (20,000 cells/well) expressing different affinity-matured R4P3F9 TCR variants was examined by determining the levels of released IFN-γ after coculture with T2 cells (20,000 cells/well) loaded with either serial dilutions of COL6A3-002 (SEQ ID NO: 1), or 10 μM COL6A3-002 and related peptides (SEQ ID NO: 28-36). Compared with the parent R4P3F9 TCR variant C-1, the affinity matured TCR variants C-2 to C-18 exhibited increased IFN-gamma release, reaching maximal levels even at lower peptide concentrations (Fig. 8). As expected, no IFN-γ release was observed in T cells not expressing the TCR or expressing the control TCR 1G4 specific for NYESO1-001. To analyze the selectivity of COL6A3-002 recognition by affinity-matured R4P3F9 TCR variants, IFN-gamma release assays were performed in response to T2 cells loaded with various similar peptides (SEQ ID NOs: 28-36), revealing different selectivity profiles for R4P3F9 TCR variants matured affinity. Most interestingly, TCR variants C-5 (SEQ ID NO: 62 and 2) and C-14 (SEQ ID NO: 62 and 63), including the same affinity-matured CDRb1 (SEQ ID NO: 40), did not demonstrate no cross-reaction with COL6A1-001 or other similar peptides (Fig. 9), making the R4P3F9 TCR variants with affinity matured C-5 and C14 the most promising candidates for TCR-based intracellular tumor targeting.

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Table 5

Nomenclature of TCR cell variants. The molecule is based on the sequences SEQ ID NO: 2 and 10 and the indicated variants CDRa1 and CDRb1

Option CDRal CDRbl S-1 DRGSQS (SEQ Ш NO. 5) RSGDLS (SEQ Ш NO. 13) S-2 DRGSQS (SEQ Ш NO. 5) ARWHNN (SEQ ID NO. 37) S-3 DRGSQS (SEQ ID NO. 5) AKDHLN (SEQ ID NO. 38) S-4 DRGSQS (SEQ ID NO. 5) ARWHRN (SEQ ID NO. 39) S-5 DRGSQS (SEQ ID NO. 5) AMDHPY (SEQ ID NO. 40) S-6 DRGSQS (SEQ ID NO. 5) ATDHYN (SEQ ID NO. 41) S-7 DRGSQS (SEQ ID NO. 5) ARYHTN (SEQ ID NO. 42) S-8 DRGSQS (SEQ ID NO. 5) APYHLN (SEQ ID NO. 43) S-9 DRGSQS (SEQ ID NO. 5) AKDHTN (SEQ ID NO. 44) S-10 DRRSQS (SEQ ID NO. 26) RSGDLS (SEQ Ш NO. 13) S-11 DRRSQS (SEQ ID NO. 26) ARWHNN (SEQ ID NO. 37) S-12 DRRSQS (SEQ ID NO. 26) AKDHLN (SEQ ID NO. 38) S-13 DRRSQS (SEQ ID NO. 26) ARWHRN (SEQ ID NO. 39) S-14 DRRSQS (SEQ ID NO. 26) AMDHPY (SEQ ID NO. 40) S-15 DRRSQS (SEQ ID NO. 26) ATDHYN (SEQ ID NO. 41) S-16 DRRSQS (SEQ ID NO. 26) ARYHTN (SEQ ID NO. 42) S-17 DRRSQS (SEQ ID NO. 26) APYHLN (SEQ ID NO. 43) S-18 DRRSQS (SEQ ID NO. 26) AKDHTN (SEQ ID NO. 44)

Example 6. Window for recognition of COL6A3-002 and COL6A1-001 by cellular TCR variants.

Cellular expression and analysis of R4P3F9 variants were performed as described above. Consistent with previous experiments (Fig. 7), the level of staining of T cells expressing TCR R4P3F9 variants C-2 to C-18 with PE-labeled HLA-A*02/COL6A3-002 tetramers was higher compared to the parent TCR C -1. In addition, TCR variants C-12 and C-17 showed binding to HLA-A*02/COL6A1-001 (Fig. 10). Expression of all affinity-matured R4P3F9 variants improved functional activation of CD8+ T cells in response to T2 cells loaded with serial dilutions of COL6A3-002 (SEQ ID NO: 1), achieving EC50 values 5-90 times lower than the parent TKR C-1 (Fig. 11, Table 6). The lowest EC ₅₀ level was recorded for variant C-14. Again, TCR variants C-5 (SEQ ID NO: 62 and 2) and C-14 (SEQ ID NO: 62 and 63), including the same affinity-matured CDRb1 (SEQ ID NO: 40), did not demonstrate no cross-talk with COL6A1-001, whereas other variants showed strong recognition with EC ₅₀ windows (COL6A3-002 versus COL6A1-001) at levels as low as a factor of 5.

Table 6

EC ₅₀ [nM] values of IFN-γ release by T cells expressing R4P3F9 variants after co-culture with T2 cells loaded with COL6A3-002 or COL6A1-001

Option ECso for COL6A3-002 |nT ECso for COL6A1-001 [nT C-1 2.51 - C-2 0.16 - C-3 0.14 87 l ^a C-4 0.13 - C-5 0.15 - C-6 0.48 - C-7 0.29 - C-8 0.20 350 C-9 0.55 - C-10 0.32 1.5 C-ll 0.32 8.2 C-12 0.20 1.9 C-13 0.23 9.7 C-14 0.03 - C-15 0.31 69 C-16 0.34 78 C-17 0.33 4.1 C-18 0.14 ^280089a

^{a The} plateau level has not been reached.

Example 7: Efficacy of affinity matured R4P3F9 variants C-5 and C-14 on tumor cell lines.

Cellular expression and analysis of R4P3F9 variants were performed as described above. Expression of affinity matured variants R4P3F9C-5 (SEQ ID NOs: 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) improved functional activation of CD8+ T cells in response to tumor cell lines presenting COL6A3-002 (SEQ ID NO: 1), in comparison with the original TCR C-1 (Fig. 12). The tumor cell lines used during this study present varying amounts of the target peptide. SF539 cells have ~4000 copies of HLA-A*02/COL6A3-002 per cell and SW982 cells have ~460 copies per cell. While the parent TCR C-1 did not mediate strong T cell activation after coculture with target-positive cell lines, the TCR C-14 variant showed an even stronger improvement in functional activation than the TCR C-5 variant. These data are comparable to the improvement in EC50, from TKR C-1 to C-5 to C-14 (Table 6). The target-negative cell line MCF-7 was not recognized by any of the TCR data.

Literature:

Aleksic et al. 2012: Different affinity windows for virus and cancer-specific T-cell receptors implications for therapeutic strategies, Eur J Immunol. 2012 Dec;42(12):3174-9;

Hickman et al. 2016: Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer

Therapies, JBiomol Screen. 2016 Sep;21(8):769-85;

Boder and Wittrup 2000: Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol. 2000;328:430-44;

Boder and Wittrup 1997: Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol. 1997 Jun;15(6):553-7;

Smith et al. 2015: T Cell Receptor Engineering and Analysis Using the Yeast Display

Platform, Methods Mol Biol. 2015;1319:95-141;

DE102016121899.5

DE102016115246

List of sequences < 110> Immatics Biotechnologies GmbH < 120> NEW GENETIC ENGINEERING T-CELL RECEPTORS AND IMMUNE THERAPY USING THEIR < 130> I33054WO < 150> DE 102017125888.4 < 15 1> 2017-11-06 < 150> US 62/582,202 < 151> 2017-11-06 < 160>66 < 170> PatentIn version 3.5 < 210>1 < 211>9 < 212> PRT < 213> Homo sapiens < 400>1

Phe Leu Leu Asp Gly Ser Ala Asn Val 15 <210> 2 <211> 274 <212> PRT <213> Homo sapiens <400>2

- 23 043746

Met Lys Ser Leu Arg 1 5

Val Leu Leu Val Ile

Leu Trp Leu Gln Leu

Trp Val Trp Ser Gln

Gln Lys Glu Val Glu

Gln Asn Ser Gly Pro 30

Ser Val Pro Glu Gly 35

Ala Ile Ala Ser Leu 40

Asn Cys Thr Tyr Ser 45

Arg Gly Ser Gln Ser 50

Phe Phe Trp Tyr Arg 55

Gln Tyr Ser Gly Lys 60

Pro Glu Leu Ile Met 65

Phe Ile Tyr Ser Asn 70

Gly Asp Lys Glu Asp 75

Arg Phe Thr Ala Gln 85

Leu Asn Lys Ala Ser 90

Gln Tyr Val Ser Leu 95

Ile Arg Asp Ser Gln

100

Pro Ser Asp Ser Ala

105

Thr Tyr Leu Cys Ala

110

Tyr Ser Gly Ala Gly 115

Ser Tyr Gln Leu Thr 120

Phe Gly Lys Gly Thr 125

Leu Ser Val Ile Pro

130

Asn Ile Gln Asn Pro

135

Asp Pro Ala Val Tyr 140

Leu Arg Asp Ser Lys 145

Ser Ser Asp Lys Ser 150

Val Cys Leu Phe Thr 155

Phe Asp Ser Gln Thr

165

Asn Val Ser Gln Ser

170

Lys Asp Ser Asp Val

175

Ile Thr Asp Lys Thr

180

Val Leu Asp Met Arg

185

Ser Met Asp Phe Lys

190

Asn Ser Ala Val Ala

195

Trp Ser Asn Lys Ser 200

Asp Phe Ala Cys Ala 205

Ala Phe Asn Asn Ser

210

Ile Ile Pro Glu Asp

215

Thr Phe Phe Pro Ser

220

Glu Ser Ser Cys Asp 225

Val Lys Leu Val Glu 230

Lys Ser Phe Glu Thr 235

Thr Asn Leu Asn Phe

245

Gln Asn Leu Ser Val

250

Ile Gly Phe Arg Ile

255

Ser

Leu

Asp

Ser

Gly 80

Leu

Ala

Lys

Gln

Asp 160

Tyr

Ser

Asn

Pro

Asp 240

Leu

- 24 043746

Leu Leu Lys Val Ala Gly Phe Asn Leu

260 265

Ser Ser <210>3 <211>21 <212> PRT <213> Homo sapiens <400>3

Met Lys Ser Leu Arg Val Leu Leu Val 15

Trp Val Trp Ser Gln 20 <210>4 <211>112 <212> PRT <213> Homo sapiens <400>4

Gln Lys Glu Val Glu Gln Asn Ser Gly 15

Ala Ile Ala Ser Leu Asn Cys Thr Tyr 20 25

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly

40

Phe Ile Tyr Ser Asn Gly Asp Lys Glu

55

Leu Asn Lys Ala Ser Gln Tyr Val Ser 65 70

Pro Ser Asp Ser Ala Thr Tyr Leu Cys 85

Ser Tyr Gln Leu Thr Phe Gly Lys Gly

100 105

Met Thr Leu Arg Leu Trp

270

Leu Trp Leu Gln Leu Ser

Leu Ser Val Pro Glu Gly

Asp Arg Gly Ser Gln Ser 30

Ser Pro Glu Leu Ile Met

Gly Arg Phe Thr Ala Gln 60

Leu Ile Arg Asp Ser Gln 75 80

Ala Tyr Ser Gly Ala Gly 95

Lys Leu Ser Val Ile Pro

110 <210> 5 <211> 6 <212> PRT <213> Homo sapiens

- 25 043746 <400> 5

Asp Arg Gly Ser Gln Ser

5 <210>6 <211>5 <212> PRT < 213> Homo sapiens < 400>6

Tyr Ser Asn Gly Asp 15 <210>7 <211>13 <212> PRT <213> Homo sapiens <400>7

Cys Ala Ala Tyr Ser Gly Ala Gly Ser 15 <210>8 <211>141 <212> PRT <213> Homo sapiens <400>8

Asn Ile Gln Asn Pro Asp Pro Ala Val

Ser Ser Asp Lys Ser Val Cys Leu Phe 20 25

Asn Val Ser Gln Ser Lys Asp Ser Asp

40

Val Leu Asp Met Arg Ser Met Asp Phe

55

Trp Ser Asn Lys Ser Asp Phe Ala Cys 65 70

Ile Ile Pro Glu Asp Thr Phe Phe Pro 85

Val Lys Leu Val Glu Lys Ser Phe Glu

100 105

Gln Leu Thr

Gln Leu Arg Asp Ser Lys

Asp Phe Asp Ser Gln Thr 30

Tyr Ile Thr Asp Lys Thr 45

Ser Asn Ser Ala Val Ala

Asn Ala Phe Asn Asn Ser 75 80

Pro Glu Ser Ser Cys Asp 95

Asp Thr Asn Leu Asn Phe

110

- 26 043746

Gln Asn Leu

115

Gly Phe Asn

130

Ser Val Ile Gly Phe Arg Ile Leu 120

Leu Leu Met Thr Leu Arg Leu Trp 135

Leu Leu Lys Val

125

Ser Ser 140

Ala <210>9 <211>4 <212> PRT <213> Homo <400>9

Asn Ile Gln 1 sapiens

Asn <210>10 <211>308 <212> PRT <213> Homo <400>10

Met Gly Phe 1 sapiens

Gly Pro Val

Ala Thr Gly

Leu Ser Val

Leu Ile Gln 65

Glu Arg Phe

Leu Ser Ser

Ser Val Glu

115

Thr Val Val

130

Arg Leu 5

Asp Ser 20

Gln Arg

Tyr Trp

Tyr Tyr

Ser Ala 85

Leu Glu 100

Ser Ser

Glu Asp

Leu Cys

Gly Val

Val Thr

Tyr Gln 55

Asn Gly 70

Gln Gln

Leu Gly

Tyr Gly

Leu Asn

135

Cys Val

Thr Gln

Leu Arg 40

Gln Ser

Glu Glu

Phe Pro

Asp Ser

105

Tyr Thr 120

Lys Val

Ala Phe 10

Thr Pro

Cys Ser

Leu Asp

Arg Ala 75

Asp Leu 90

Ala Leu

Phe Gly

Phe Pro

Cys Leu

Leu Gly 15

Lys His

Leu Ile 30

Pro Arg 45

Gln Gly 60

Lys Gly

His Ser

Tyr Phe

Ser Gly

125

Pro Glu 140

Ser Gly

Leu Gln

Asn Ile

Glu Leu

Cys Ala 110

Thr Arg

Val Ala

Ala

Thr

Asp

Phe

Leu 80

Asn

Ser

Leu

Val

- 27 043746

Phe Glu Pro Ser Glu Ala Glu Ile Ser

145 150

Val Cys Leu Ala Thr Gly Phe Phe Pro

165

Trp Val Asn Gly Lys Glu Val His Ser

180 185

Pro Leu Lys Glu Gln Pro Ala Leu Asn

195 200

Ser Arg Leu Arg Val Ser Ala Thr Phe

210 215

Phe Arg Cys Gln Val Gln Phe Tyr Gly

225 230

Thr Gln Asp Arg Ala Lys Pro Val Thr

245

Trp Gly Arg Ala Asp Cys Gly Phe Thr

260 265

Val Leu Ser Ala Thr Ile Leu Tyr Glu

275 280

Leu Tyr Ala Val Leu Val Ser Ala Leu

290 295

Arg Lys Asp Phe 305 <210>11 <211>19 <212> PRT <213> Homo sapiens <400>11

Met Gly Phe Arg Leu Leu Cys Cys Val 15

Thr Gln Lys Ala Thr Leu

155 160

His Val Glu Leu Ser Trp 175

Val Ser Thr Asp Pro Gln

190

Ser Arg Tyr Cys Leu Ser

205

Gln Asn Pro Arg Asn His

220

Ser Glu Asn Asp Glu Trp

235 240

Ile Val Ser Ala Glu Ala

255

Val Ser Tyr Gln Gln Gly

270

Leu Leu Gly Lys Ala Thr

285

Leu Met Ala Met Val Lys 300

Phe Cys Leu Leu Gly Ala

Gly Pro Val <210> 12 <211> 112 <212> PRT <213> Homo sapiens

- 28 043746 <400> 12

Asp Ser Gly 1 Val Thr 5 Gln Thr Pro Lys His 10 Leu Ile Thr Ala Thr 15 Gln Arg Val Thr 20 Leu Arg Cys Ser Pro 25 Arg Ser Gly Asp Leu 30 Ser Tyr Trp Tyr 35 Gln Gln Ser Leu Asp 40 Gln Gly Leu Gln Phe 45 Leu Ile Tyr Tyr Asn 50 Gly Glu Glu Arg 55 Ala Lys Gly Asn Ile 60 Leu Glu Arg Ser Ala Gln 65 Gln Phe Pro 70 Asp Leu His Ser Glu 75 Leu Asn Leu Ser Leu Glu Leu Gly Asp 85 Ser Ala Leu Tyr Phe 90 Cys Ala Ser Ser Val 95 Ser Ser Tyr Gly 100 Tyr Thr Phe Gly Ser 105 Gly Thr Arg Leu Thr 110 Val <210> <211> <212> <213> 13 6 PRT Homo sapiens <400> 13 Arg Ser Gly 1 Asp Leu 5 Ser <210> <211> <212> <213> 14 5 PRT Homo sapiens <400> 14 Tyr Asn Gly 1 Glu Glu 5 <210> <211> <212> <213> 15 12 PRT Homo sapiens <400> 15 Cys Ala Ser 1 Ser Val 5 Glu Ser Ser Tyr Gly 10 Tyr Thr

Gly

Val

Gln

Phe

Ser 80

Glu

Val

- 29 043746

Val Ala Val Phe Glu <210>16 <211>177 <212> PRT <213> Homo sapiens <400>16

Glu Asp Leu Asn Lys 1 5

Val Phe Pro Pro Glu

Ser Glu Ala Glu Ile

Ser His Thr Gln Lys

Ala Thr Leu Val Cys 30

Ala Thr Gly Phe Phe 35

Pro Asp His Val Glu 40

Leu Ser Trp Trp Val 45

Gly Lys Glu Val His 50

Ser Gly Val Ser Thr 55

Asp Pro Gln Pro Leu 60

Glu Gln Pro Ala Leu 65

Asn Asp Ser Arg Tyr 70

Cys Leu Ser Ser Arg 75

Arg Val Ser Ala Thr 85

Phe Trp Gln Asn Pro 90

Arg Asn His Phe Arg 95

Gln Val Gln Phe Tyr

100

Gly Leu Ser Glu Asn

105

Asp Glu Trp Thr Gln

110

Arg Ala Lys Pro Val 115

Thr Gln Ile Val Ser

120

Ala Glu Ala Trp Gly 125

Ala Asp Cys Gly Phe 130

Thr Ser Val Ser Tyr

135

Gln Gln Gly Val Leu 140

Ala Thr Ile Leu Tyr 145

Glu Ile Leu Leu Gly 150

Lys Ala Thr Leu Tyr 155

Val Leu Val Ser Ala

165

Leu Val Leu Met Ala

170

Met Val Lys Arg Lys

175

Pro

Leu

Asn

Lys

Leu 80

Cys

Asp

Arg

Ser

Ala 160

Asp

Phe <210> 17 <211> 5 <212> PRT <213> Homo sapiens <400> 17

- 30 043746

Glu Asp Leu Asn Lys 1 5 <210>18 <211>5 <212> PRT <213> Homo sapiens <400>18

Glu Asp Leu Lys Asn 15 <210>19 <211>374 <212> PRT <213> Homo sapiens <400>19

Met Gln Leu Leu Arg 1 5

Cys Phe Ser Ile Phe

Ser Val Ile Ala Ser

Leu Ala Gln Glu Leu

Thr Thr Ile Cys Glu 25

Gln Ile Pro Ser Pro

Leu Glu Ser Thr Pro

Tyr Ser Leu Ser Thr 40

Thr Thr Ile Leu Ala

Gly Lys Ala Met Gln 50

Gly Val Phe Glu Tyr 55

Tyr Lys Ser Val Thr 60

Val Ser Asn Cys Gly 65

Ser His Pro Ser Thr 70

Thr Ser Lys Gly Ser 75

Ile Asn Thr Gln Tyr 85

Val Phe Gly Gly Gly 90

Gly Ser Asp Tyr Lys 95

Asp Asp Asp Lys Gly

100

Gly Gly Ala Ser Gln

105

Lys Glu Val Glu Gln

110

Ser Gly Pro Leu Ser 115

Val Pro Glu Gly Ala

120

Ile Ala Ser Leu Asn

125

Thr Tyr Ser Asp Arg

130

Gly Ser Gln Ser Phe

135

Phe Trp Tyr Arg Gln

140

Ser Gly Lys Ser Pro 145

Glu Leu Ile Met Ser 150

Ile Tyr Ser Asn Gly 155

Val

Thr

Asn

Phe

Pro 80

Asp

Asn

Cys

Tyr

Asp 160

Tyr

Lys Glu Asp Gly Arg

Phe Thr Ala Gln Leu

Asn Lys Ala Ser Gln

- 31 043746

165

175

Ala Thr

190

Val Ser Leu

Leu Ile 180

Arg Asp

Ser Gln

185

Leu Cys Ala

195

Ala Tyr

Ser Gly

Ala Gly 200

Lys Gly Thr

210

Lys Leu

Ser Val

215

Ile Pro

170

Pro Ser

Ser Tyr

Asn Ile

Asp Ser

Gln Leu

205

Gln Asn

220

Thr Phe

Gly Gly

Gly Ser Gly 225

Gly Gly

Gly Ser 230

Gly Gly

Gly Gly

235

Ser Gly

Gly Gly

Ser Gly Val

Thr Gln

245

Thr Pro

Lys His

Leu Ile

250

Thr Ala

Thr Gly

255

Arg Val Thr

Leu Arg 260

Cys Ser

Pro Arg

265

Ser Gly

Asp Leu

Ser Val

270

Trp Tyr Gln

275

Gln Ser

Leu Asp

Gln Gly 280

Leu Gln

Phe Leu

285

Ile Gln

Tyr Asn Gly

290

Glu Glu

Arg Ala

295

Lys Gly

Asn Ile

Leu Glu 300

Arg Phe

Ala Gln Gln 305

Phe Pro

Asp Leu 310

His Ser

Glu Leu

315

Asn Leu

Ser Ser

Glu Leu Gly

Asp Ser

325

Ala Leu

Tyr Phe

Cys Ala 330

Ser Ser

Val Glu

335

Ser Tyr Gly

Tyr Thr 340

Phe Gly

Ser Gly

345

Thr Arg

Leu Thr

Val Val 350

Asp Leu Asn

355

Lys Ala

Ala Ala

Gly Gly 360

Ser Gly

Gly Glu

365

Gln Lys

Tyr

Gly

Gly

Gly 240

Gln

Tyr

Tyr

Ser

Leu 320

Ser

Glu

Leu

Ile Ser Glu

370

Glu Asp

Leu <210>20 <211>87 <212> PRT <213> Homo <400>20

Met Gln Leu sapiens

Leu Arg

CysPhe

Ser Ile

Phe Ser

Val Ile

Ala Ser

Val

- 32 043746

Leu Ala Gln

Leu Glu Ser

Gly Lys Ala 50

Val Ser Asn 65

Ile Asn Thr <210>21 <211>18 <212> PRT <213> Homo <400>21

Gly Gly Gly 1

Glu Leu Thr 20

Thr Pro Tyr

Met Gln Gly

Cys Gly Ser 70

Gln Tyr Val 85 sapiens

Gly Ser Asp 5

Thr Ile Cys 25

Ser Leu Ser

Val Phe Glu 55

His Pro Ser

Phe

Tyr Lys Asp

Ala Ser <210>22 <211>251 <212> PRT <213> Homo <400>22

Gln Lys Glu 1 sapiens

Val Glu Gln

Asn Ser Gly

Ala Ile Ala

Ser Leu Asn 20

Cys Thr Tyr

Phe Phe Trp

Tyr Arg Gln

Tyr Ser Gly 40

Ser Ile Tyr 50

Ser Asn Gly

Asp Lys Glu 55

Leu Asn Lys 65

Ala Ser Gln

Tyr Val Ser

Glu Gln Ile

Thr Thr Thr

Tyr Tyr Lys 60

Thr Thr Ser 75

Asp Asp Asp 10

Pro Leu Ser 10

Ser Asp Arg

Lys Ser Pro

Asp Gly Arg

Leu Leu Ile

Pro Ser Pro 30

Ile Leu Ala 45

Ser Val Thr

Lys Gly Ser

Lys Gly Gly 15

Val Pro Glu

Gly Ser Gln 30

Glu Leu Ile 45

Phe Thr Ala

Arg Asp Ser

Thr

Asn

Phe

Pro 80

Gly

Gly

Ser

Met

Gln

Gln 80

- 33 043746

Pro Ser Asp Ser Ala Thr Tyr Leu Cys 85

Ser Tyr Gln Leu Thr Phe Gly Lys Gly

100 105

Asn Ile Gln Asn Gly Gly Gly Gly Ser

115 120

Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135

Leu Ile Thr Ala Thr Gly Gln Arg Val 145 150

Ser Gly Asp Leu Ser Val Tyr Trp Tyr

165

Leu Gln Phe Leu Ile Gln Tyr Tyr Asn

180 185

Asn Ile Leu Glu Arg Phe Ser Ala Gln

195 200

Glu Leu Asn Leu Ser Ser Leu Glu Leu

210 215

Cys Ala Ser Ser Val Glu Ser Ser Tyr

225 230

Thr Arg Leu Thr Val Val Glu Asp Leu

245 < 210>23 < 211>18 < 212> PRT < 213> Homo sapiens < 400>23

Ala Ala Ala Gly Gly Ser Gly Gly Glu

Asp Leu <210> 24 <211> 251

Ala Tyr Ser Gly Ala Gly 95

Lys Leu Ser Val Ile Pro 110

Gly Gly Gly Ser Gly Gly

125

Thr Gln Thr Pro Lys His

140

Leu Arg Cys Ser Pro Arg

155 160

Gln Ser Leu Asp Gln Gly

175

Glu Glu Arg Ala Lys Gly

190

Phe Pro Asp Leu His Ser

205

Asp Ser Ala Leu Tyr Phe

220

Tyr Thr Phe Gly Ser Gly

235 240

Lys

Lys Leu Ile Ser Glu Glu

Ser Val

Pro Glu <212> PRT <213> Homo <400> 24

Gln Lys Glu 1 sapiens

Ala Ile Ala

Phe Phe Trp

Ser Ile Tyr 50

Leu Asn Lys 65

Val Glu 5

Ser Leu 20

Tyr Arg

Ser Asn

Ala Ser

Gln Asn

Ser Gly

Asn Cys

Thr Tyr

Gln Tyr

Gly Asp 55

Gln Tyr 70

Pro Ser Asp

Ser Ala

Thr Tyr

Ser Tyr Gln

Leu Thr 100

Phe Gly

Ser Gly 40

Lys Glu

Val Ser

Leu Cys

Lys Gly

105

Asn Ile Gln

115

Asn Gly

Gly Gly

Gly Ser 120

Gly Gly Ser

130

Gly Gly

Gly Gly

135

Ser Gly

Leu Ile Thr 145

Ala Thr

Gly Gln 150

Arg Val

Ser Gly Asp

Leu Ser

165

Val Tyr

Trp Tyr

Leu Gln Phe

Leu Ile 180

Gln Tyr

Tyr Asn

185

Asn Ile Leu

195

Glu Arg

Phe Ser

Ala Gln 200

Glu Leu Asn

210

Leu Ser

Ser Leu

215

Glu Leu

Cys Ala Ser 225

Ser Val

Glu Ser

230

Ser Tyr

Pro Leu 10

Ser Asp

Lys Ser

Asp Gly

Arg Gly

Pro Glu

Arg Phe 60

Ser Gln 30

Leu Ile

Thr Ala

Leu Leu

Ile Arg

Asp Ser

Ala Ala 90

Tyr Ser

Gly Ala 95

Thr Lys

Leu Ser

Val Ile

110

Gly Gly

Gly Gly

125

Ser Gly

Val Thr

Thr Leu

155

Lys Gln 170

Gly Glu

Gln Phe

Gly Asp

Gly Tyr

235

Gln Thr 140

Pro Lys

Arg Cys

Ser Pro

Ser Leu

Glu Arg

Pro Asp

205

Ser Ala

220

Thr Phe

Asp Gln

175

Ala Lys 190

Leu His

Leu Tyr

Gly Ser

Gly

Ser

Met

Gln

Gln 80

Gly

Pro

Gly

His

Arg 160

Gly

Gly

Ser

Phe

Gly 240

- 35 043746

Thr Arg Leu

Thr Val Val Glu Asp Leu Asn Lys

245 250 <210>25 <211>251 <212> PRT <213> Homo <400>25

Gln Lys Glu 1 sapiens

Val Glu Gln

Asn Ser Gly

Pro Leu Ser 10

Val Pro Glu

Ala Ile Ala

Ser Leu Asn 20

Cys Thr Tyr 25

Ser Asp Arg

Gly Ser Gln 30

Phe Phe Trp

Tyr Arg Gln

Tyr Ser Gly 40

Lys Ser Pro

Glu Leu Ile 45

Ser Ile Tyr 50

Ser Asn Gly

Asp Lys Glu 55

Asp Gly Arg

Phe Thr Ala

Leu Asn Lys 65

Ala Ser Gln

Tyr Val Ser

Leu Leu Ile

Arg Asp Ser

Pro Ser Asp

Ser Ala Thr

Tyr Leu Cys

Ala Ala Tyr 90

Ser Gly Ala

Ser Tyr Gln

Leu Thr Phe 100

Gly Lys Gly

105

Thr Lys Leu

Ser Val Ile

110

Asn Ile Gln

115

Asn Gly Gly

Gly Gly Ser

120

Gly Gly Gly

Gly Ser Gly 125

Gly Gly Ser

130

Gly Gly Gly

Gly Ser Gly 135

Val Thr Gln

140

Thr Pro Lys

Leu Ile Thr 145

Ala Thr Gly

150

Gln Arg Val

Thr Leu Arg

155

Cys Ser Pro

Ser Gly Asp

Leu Ser Val

165

Tyr Trp Tyr

Gln Gln Ser 170

Leu Asp Gln

175

Leu Gln Phe

Leu Ile Gln 180

Tyr Tyr Asn

185

Gly Glu Glu

Arg Ala Lys

190

Asn Ile Ser

195

Glu Arg Phe

Ser Ala Gln

200

Gln Phe Pro

Asp Leu His 205

Gly

Ser

Met

Gln

Gln 80

Gly

Pro

Gly

His

Arg 160

Gly

Gly

Ser

- 36 043746

Glu Leu Asn

210

Leu

Ser

Ser

Leu

215

Glu

Leu

Gly

Asp

Ser 220

Ala

Leu

Tyr

Phe

Cys Ala Ser 225

Ser

Val

Glu

230

Ser

Ser

Tyr

Gly

Tyr 235

Thr

Phe

Gly

Ser

Gly 240

Thr Arg Leu

Thr

Val

245

Val

Glu

Asp

Leu

Asn

250

Lys <210>26 <211>6 <212> PRT <213> Homo <400>26

Asp Arg Arg 1 sapiens

Ser Gln

Ser <210>27 <211>247 <212> PRT <213> Homo <400>27

Gln Lys Glu 1 sapiens

Val

Glu 5

Gln

Asn

Ser

Gly

Pro 10

Leu

Ser

Val

Pro

Glu 15

Gly

Ala Ile Ala

Ser 20

Leu

Asn

Cys

Thr

Tyr 25

Ser

Asp

Arg

Arg

Ser 30

Gln

Ser

Phe Phe Trp

Tyr

Arg

Gln

Tyr

Ser 40

Gly

Lys

Ser

Pro

Glu 45

Leu

Ile

Met

Ser Ile Tyr 50

Ser

Asn

Gly

Asp 55

Lys

Glu

Asp

Gly

Arg 60

Phe

Thr

Ala

Gln

Leu Asn Lys 65

Ala

Ser

Gln 70

Tyr

Val

Ser

Leu

Leu 75

Ile

Arg

Asp

Ser

Gln 80

Pro Ser Asp

Ser

Ala 85

Thr

Tyr

Leu

Cys

Ala 90

Ala

Tyr

Ser

Gly

Ala 95

Gly

Ser Tyr Gln

Leu

100

Thr

Phe

Gly

Lys

Gly 105

Thr

Lys

Leu

Ser

Val

110

Ile

Pro

Gly Gly Gly

115

Gly

Ser

Gly

Gly

Gly 120

Gly 125

Gly

Ser

Gly

Gly

Gly

Ser

Gly

- 37 043746

Gly Gly Gly Ser Gly 130

Gly Gly Gly Ser Gly

135

Val Thr Gln Thr Pro

140

His Leu Ile Thr Ala 145

Thr Gly Gln Arg Val 150

Thr Leu Arg Cys Ser 155

Arg Ser Gly Asp Leu

165

Ser Val Tyr Trp Tyr

170

Lys Gln Ser Leu Asp

175

Gly Leu Gln Phe Leu

180

Ile Gln Tyr Tyr Asn

185

Gly Glu Glu Arg Ala

190

Gly Asn Ile Ser Glu 195

Arg Phe Ser Ala Gln 200

Gln Phe Pro Asp Leu

205

Ser Glu Leu Asn Leu

210

Ser Ser Leu Glu Leu

215

Gly Asp Ser Ala Leu

220

Phe Cys Ala Ser Ser 225

Val Glu Ser Ser Tyr 230

Gly Tyr Thr Phe Gly 235

Lys

Pro 160

Gln

Lys

His

Tyr

Ser 240

Gly Thr Arg Leu Thr

245

Val Val <210>28 <211>9 <212> PRT <213> Homo sapiens <400>28

Ala Leu Leu Asp Gly Arg Val Gln Leu <210>29 <211>9 <212> PRT <213> Homo sapiens <400>29

Arg Leu Leu Asp Gly Ala Phe Lys Leu <210>30 <211>9 <212> PRT <213> Homo sapiens <400>30

Ile Leu Leu Asp Gly Ser Ala Ser Val

- 38 043746 <210>31 <211>9 <212> PRT <213> Homo sapiens <400>31

Phe Leu Leu Asp Gly Ser Glu Arg Leu 15 < 210>32 < 211>10 < 212> PRT < 213> Homo sapiens < 400>32

Phe Leu Phe Asp Gly Ser Ala Asn Leu 15

Val 10 < 210>33 < 211>9 < 212> PRT < 213> Homo sapiens < 400>33

Phe Leu Phe Asp Gly Ser Ala Asn Leu 15 < 210>34 < 211>9 < 212> PRT < 213> Homo sapiens < 400>34

Phe Leu Leu Asp Gly Ser Glu Gly Val 15 < 210>35 < 211>9 < 212> PRT < 213> Homo sapiens < 400>35

Phe Leu Leu Asp Gly Ser Asn Ser Val <210>36 <211>9 <212> PRT <213> Homo sapiens

- 39 043746 <400>36

Phe Leu Leu Asp Gly Ser Ser Arg Leu 15 <210>37 <211>6 <212> PRT <213> Homo sapiens <400>37

Ala Arg Trp His Asn Asn <210>38 <211>6 <212> PRT <213> Homo sapiens <400>38

Ala Lys Asp His Leu Asn <210>39 <211>6 <212> PRT <213> Homo sapiens <400>39

Ala Arg Trp His Arg Asn <210>40 <211>6 <212> PRT <213> Homo sapiens <400>40

Ala Met Asp His Pro Tyr <210>41 <211>6 <212> PRT <213> Homo sapiens <400>41

Ala Thr Asp His Tyr Asn <210>42 <211>6

- 40 043746 <212> PRT <213> Homo sapiens <400>42

Ala Arg Tyr His Thr Asn <210>43 <211>6 <212> PRT <213> Homo sapiens <400>43

Ala Pro Tyr His Leu Asn <210>44 <211>6 <212> PRT <213> Homo sapiens <400>44

Ala Lys Asp His Thr Asn <210>45 <211>6 <212> PRT <213> Homo sapiens <400>45

Ala Arg Tyr His Arg Asn <210>46 <211>6 <212> PRT <213> Homo sapiens <400>46

Ala Arg Trp His Ser Asn <210>47 <211>6 <212> PRT <213> Homo sapiens <400>47

Ala Thr Asp His Tyr Asn

- 41 043746 <210>48 < 211>6 < 212> PRT < 213> Homo sapiens < 400>48

Arg Trp Gly Asp Leu Asn 15 < 210>49 < 211>6 < 212> PRT < 213> Homo sapiens < 400>49

Ala Arg Asp His Leu Asn <210>50 <211>514 <212> PRT <213> Homo sapiens <400>50

Met Lys Trp Val Thr Phe Ile Ser 1 5

Leu Leu Phe Leu Phe Ser Ser Ala

15

Tyr Glu Val Gln Leu Val Glu Ser 20

Gly Gly Gly Leu Val Gln Pro Gly 25 30

Gly Ser Leu Arg Leu Ser Cys Ala

40

Ala Ser Gly Tyr Ser Phe Thr Gly 45

Tyr Thr Met Asn Trp Val Arg Gln

55

Ala Pro Gly Lys Gly Leu Glu Trp 60

Val Ala Leu Ile Asn Pro Tyr Lys 65 70

Gly Val Ser Thr Tyr Asn Gln Lys

80

Phe Lys Asp Arg Phe Thr Ile Ser 85

Val Asp Lys Ser Lys Asn Thr Ala

95

Tyr Leu Gln Met Asn Ser Leu Arg

100

Ala Glu Asp Thr Ala Val Tyr Tyr

105 110

Cys Ala Arg Ser Gly Tyr Tyr Gly

115 120

Asp Ser Asp Trp Tyr Phe Asp Val

125

Trp Gly Gln Gly Thr Leu Val Thr

Val Ser Ser Ala Ser Thr Lys Gly

- 42 043746

Lys Ser Thr

155

Ser Gly Gly

160

130

135

140

Pro 145

Thr

Thr

Pro

Thr

Asn 225

Ser

Ala

Glu

Gln

Ile 305

Ala

Ser

Ala

Ile

Ser Val Phe

Pro Leu Ala

150

Pro Ser Ser

Ala Ala Leu

Val Ser Trp

180

Ala Val Leu

195

Val Pro Ser 210

His Lys Pro

Cys Asp Lys

Gly Gln Lys

260

Gly Ala Ile

275

Ser Phe Phe 290

Met Ser Ile

Gln Leu Asn

Gln Pro Ser

340

Gly Ser Tyr

355

Pro Asn Ile 370

Gly Cys Leu 165

Val Lys Asp

170

Tyr Phe Pro

Glu Pro Val

175

Asn Ser Gly

Ala Leu Thr

185

Ser Gly Val

His Thr Phe 190

Gln Ser Ser

Gly Leu Tyr 200

Ser Leu Ser

205

Ser Val Val

Ser Ser Leu

215

Ser Asn Thr

230

Thr His Thr 245

Glu Val Glu

Ala Ser Leu

Trp Tyr Arg

295

Tyr Ser Asn

310

Lys Ala Ser 325

Asp Ser Ala

Gln Leu Thr

Gln Asn Gly

375

Gly Thr Gln

Thr Tyr Ile

220

Cys Asn Val

Lys Val Asp

Lys Lys Val 235

Glu Pro Lys

240

Ser Pro Pro

250

Ser Pro Ala

Pro Pro Val

255

Gln Asn Ser

265

Gly Pro Leu

Ser Val Pro 270

Asn Cys Thr 280

Tyr Ser Asp

285

Arg Gly Ser

Gln Tyr Ser

Gly Lys Ser

300

Pro Glu Leu

Gly Asp Lys

Gln Tyr Val

330

Thr Tyr Leu

345

Phe Gly Lys 360

Gly Gly Gly

Glu Asp Gly 315

Ser Leu Leu

Cys Ala Ala

Gly Thr Lys

365

Ser Gly Gly

380

Arg Phe Thr

320

Ile Arg Asp

335

Tyr Ser Gly 350

Leu Ser Val

Gly Gly Ser

- 43 043746

Gly 385

Gly

Gly

Gly

Ser

Gly 390

Gly

Gly

Gly

Ser

Gly 395

Gly

Gly

Gly

Ser

Val

Thr

Gln

Thr

Pro

405

Lys

His

Leu

Ile

Thr

410

Ala

Thr

Gly

Gln

Arg 415

Thr

Leu

Arg

Cys 420

Ser

Pro

Arg

Ser

Gly 425

Asp

Leu

Ser

Val

Tyr 430

Trp

Lys

Gln

Ser

435

Leu

Asp

Gln

Gly

Leu

440

Gln

Phe

Leu

Ile

Gln

445

Tyr

Tyr

Gly Glu

450

Glu Arg Ala Lys Gly Asn Ile Ser

455

Glu Arg Phe Ser Ala 460

Gln Phe 465

Pro Asp Leu His Ser Glu Leu Asn 470

Leu Ser Ser Leu Glu 475

Gly Asp

Ser Ala Leu Tyr Phe Cys Ala Ser

485 490

Ser Val Glu Ser Ser

495

Gly Tyr

Thr Phe Gly Ser Gly Thr Arg Leu

500 505

Thr Val Val Glu Asp

510

Gly 400

Val

Tyr

Asn

Gln

Leu 480

Tyr

Leu

Lys Asn <210>51 <211>258 <212> PRT <213> Homo sapiens <400>51

Met Lys Trp Val Thr 15

Phe Ile Ser Leu Leu

Phe Leu Phe Ser Ser

Tyr Glu Val Gln Leu

Val Glu Ser Gly Gly

Gly Leu Val Gln Pro 30

Gly Ser Leu Arg Leu 35

Ser Cys Ala Ala Ser 40

Gly Tyr Ser Phe Thr 45

Tyr Thr Met Asn Trp 50

Val Arg Gln Ala Pro 55

Gly Lys Gly Leu Glu 60

Val Ala Leu Ile Asn 65

Pro Tyr Lys Gly Val 70

Ser Thr Tyr Asn Gln 75

Ala

Gly

Gly

Trp

Lys 80

- 44 043746

Phe Lys Asp Arg Phe Thr Ile Ser Val 85

Tyr Leu Gln Met Asn Ser Leu Arg Ala

100 105

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp

115 120

Trp Gly Gln Gly Thr Leu Val Thr Val

130 135

Pro Ser Val Phe Pro Leu Ala Pro Ser

145 150

Thr Ala Ala Leu Gly Cys Leu Val Lys

165

Thr Val Ser Trp Asn Ser Gly Ala Leu

180 185

Pro Ala Val Leu Gln Ser Ser Gly Leu

195 200

Thr Val Pro Ser Ser Ser Leu Gly Thr

210 215

Asn His Lys Pro Ser Asn Thr Lys Val 225 230

Ser Cys Asp Lys Thr His Thr Ser Pro

245

Ala Gly <210>52 <211>232 <212> PRT <213> Homo sapiens <400>52

Met Lys Trp Val Thr Phe Ile Ser Leu 15

Tyr Ser Asp Ile Gln Met Thr Gln Ser 20 25

Lys Ser Lys Asn Thr Ala 95

Asp Thr Ala Val Tyr Tyr

110

Asp Trp Tyr Phe Asp Val

125

Ser Ala Ser Thr Lys Gly

140

Lys Ser Thr Ser Gly Gly

155 160

Tyr Phe Pro Glu Pro Val

175

Ser Gly Val His Thr Phe

190

Ser Leu Ser Ser Val Val

205

Thr Tyr Ile Cys Asn Val

220

Lys Lys Val Glu Pro Lys

235 240

Ser Pro Ala Pro Pro Val

255

Phe Leu Phe Ser Ser Ala

Ser Ser Leu Ser Ala Ser

- 45 043746

Val Gly Asp Arg Val 35

Thr Ile Thr Cys Arg 40

Ala Ser Gln Asp Ile 45

Asn Tyr Leu Asn Trp 50

Tyr Gln Gln Lys Pro 55

Gly Lys Ala Pro Lys 60

Leu Ile Tyr Tyr Thr 65

Ser Arg Leu Glu Ser 70

Gly Val Pro Ser Arg 75

Ser Gly Ser Gly Ser 85

Gly Thr Asp Tyr Thr 90

Leu Thr Ile Ser Ser

Gln Pro Glu Asp Phe

100

Ala Thr Tyr Tyr Cys

105

Gln Gln Gly Asn Thr

110

Pro Trp Thr Phe Gly 115

Gln Gly Thr Lys Val 120

Glu Ile Lys Arg Thr

125

Ala Ala Pro Ser Val

130

Phe Ile Phe Pro Pro

135

Ser Asp Glu Gln Leu

140

Ser Gly Thr Ala Ser 145

Val Val Cys Leu Leu 150

Asn Asn Phe Tyr Pro 155

Glu Ala Lys Val Gln

165

Trp Lys Val Asp Asn

170

Ala Leu Gln Ser Gly

175

Ser Gln Glu Ser Val

180

Thr Glu Gln Asp Ser

185

Lys Asp Ser Thr Tyr

190

Leu Ser Ser Thr Leu

195

Thr Leu Ser Lys Ala 200

Asp Tyr Glu Lys His 205

Val Tyr Ala Cys Glu

210

Val Thr His Gln Gly

215

Leu Ser Ser Pro Val

220

Arg

Leu

Phe 80

Leu

Leu

Val

Lys

Arg 160

Asn

Ser

Lys

Thr

Lys Ser Phe Asn Arg 225

Gly Glu Cys 230 <210>53 <211>274 <212> PRT <213> Homo sapiens <400>53

Met Glu Thr Leu Leu Gly Leu Leu 1 5

Ile Leu Trp Leu Gln Leu Gln

15

Trp

- 46 043746

Val Ser Ser Lys Gln

Glu Val Thr Gln Ile

Pro Ala Ala Leu Ser

Pro Glu Gly Glu Asn 35

Leu Val Leu Asn Cys 40

Ser Phe Thr Asp Ser 45

Ile Tyr Asn Leu Gln 50

Trp Phe Arg Gln Asp 55

Pro Gly Lys Gly Leu 60

Ser Leu Leu Leu Ile 65

Gln Ser Ser Gln Arg 70

Glu Gln Thr Ser Gly 75

Leu Asn Ala Ser Leu

Asp Lys Ser Ser Gly 90

Arg Ser Thr Leu Tyr 95

Ala Ala Ser Gln Pro

100

Gly Asp Ser Ala Thr

105

Tyr Leu Cys Ala Val

110

Pro Thr Ser Gly Gly 115

Ser Tyr Ile Pro Thr 120

Phe Gly Arg Gly Thr 125

Leu Ile Val His Pro

130

Tyr Ile Gln Asn Pro

135

Asp Pro Ala Val Tyr 140

Leu Arg Asp Ser Lys 145

Ser Ser Asp Lys Ser 150

Val Cys Leu Phe Thr 155

Phe Asp Ser Gln Thr

165

Asn Val Ser Gln Ser

170

Lys Asp Ser Asp Val

175

Ile Thr Asp Lys Thr

180

Val Leu Asp Met Arg

185

Ser Met Asp Phe Lys

190

Asn Ser Ala Val Ala

195

Trp Ser Asn Lys Ser 200

Asp Phe Ala Cys Ala 205

Ala Phe Asn Asn Ser

210

Ile Ile Pro Glu Asp

215

Thr Phe Phe Pro Ser

220

Glu Ser Ser Cys Asp 225

Val Lys Leu Val Glu 230

Lys Ser Phe Glu Thr 235

Thr Asn Leu Asn Phe

245

Gln Asn Leu Ser Val

250

Ile Gly Phe Arg Ile

255

Leu Leu Lys Val Ala

260

Gly Phe Asn Leu Leu

265

Met Thr Leu Arg Leu

270

Val

Ala

Thr

Arg 80

Ile

Arg

Ser

Gln

Asp 160

Tyr

Ser

Asn

Pro

Asp 240

Leu

Trp

- 47 043746

Ser Ser <210>54 <211>20 <212> PRT <213> Homo sapiens <400>54

Met Glu Thr Leu Leu Gly Leu Leu Ile 15

Val Ser Ser Lys 20 <210>55 <211>113 <212> PRT <213> Homo sapiens <400>55

Gln Glu Val Thr Gln Ile Pro Ala Ala

Asn Leu Val Leu Asn Cys Ser Phe Thr 20 25

Gln Trp Phe Arg Gln Asp Pro Gly Lys

40

Ile Gln Ser Ser Gln Arg Glu Gln Thr

55

Leu Asp Lys Ser Ser Gly Arg Ser Thr 65 70

Pro Gly Asp Ser Ala Thr Tyr Leu Cys 85

Gly Ser Tyr Ile Pro Thr Phe Gly Arg

100 105

Trp Leu Gln Leu Gln Trp

Ser Val Pro Glu Gly Glu

Ser Ala Ile Tyr Asn Leu 30

Leu Thr Ser Leu Leu Leu

Gly Arg Leu Asn Ala Ser 60

Tyr Ile Ala Ala Ser Gln 75 80

Val Arg Pro Thr Ser Gly 95

Thr Ser Leu Ile Val His

110

Pro <210> 56 <211> 141 <212> PRT <213> Homo sapiens

- 48 043746 <400> 56

Tyr Ile Gln Asn Pro 1 5

Asp Pro Ala Val Tyr

Gln Leu Arg Asp Ser

Ser Ser Asp Lys Ser 20

Val Cys Leu Phe Thr

Asp Phe Asp Ser Gln 30

Asn Val Ser Gln Ser 35

Lys Asp Ser Asp Val 40

Tyr Ile Thr Asp Lys 45

Val Leu Asp Met Arg 50

Ser Met Asp Phe Lys 55

Ser Asn Ser Ala Val

Trp Ser Asn Lys Ser 65

Asp Phe Ala Cys Ala 70

Asn Ala Phe Asn Asn 75

Ile Ile Pro Glu Asp 85

Thr Phe Phe Pro Ser

Pro Glu Ser Ser Cys 95

Val Lys Leu Val Glu

100

Lys Ser Phe Glu Thr

105

Asp Thr Asn Leu Asn

110

Gln Asn Leu Ser Val

115

Ile Gly Phe Arg Ile 120

Leu Leu Leu Lys Val 125

Lys

Thr

Thr

Ala

Ser 80

Asp

Phe

Ala

Gly Phe Asn Leu Leu 130

Met Thr Leu Arg Leu

135

Trp Ser Ser

140 <210>57 <211>311 <212> PRT <213> Homo <400>57

Met Ser Ile 1

Gly Pro Val

Lys Thr Gly

Glu Tyr Met 50

Ile His Tyr sapiens

Gly Leu Leu 5

Asn Ala Gly 20

Gln Ser Met

Ser Trp Tyr

Ser Val Gly

Cys Cys Ala

Val Thr Gln

Thr Leu Gln

Arg Gln Asp 55

Ala Gly Ile

Ala Leu Ser 10

Thr Pro Lys

Cys Ala Gln

Pro Gly Met 60

Thr Asp Gln

Leu Leu Trp 15

Phe Gln Val

Asp Met Asn 45

Gly Leu Arg

Gly Glu Val

Ala

Leu

His

Leu

Pro

- 49 043746

Glu Asp Phe Pro Leu

Asn Gly Tyr Asn Val 85

Ser Arg Ser Thr Thr 90

Leu Leu Ser Ala Ala

100

Pro Ser Gln Thr Ser

105

Val Tyr Phe Cys Ala

110

Ser Tyr Val Gly Asn

115

Thr Gly Glu Leu Phe

120

Phe Gly Glu Gly Ser 125

Leu Thr Val Leu Glu

130

Asp Leu Lys Asn Val

135

Phe Pro Pro Glu Val

140

Val Phe Glu Pro Ser 145

Glu Ala Glu Ile Ser 150

His Thr Gln Lys Ala 155

Leu Val Cys Leu Ala

165

Thr Gly Phe Tyr Pro

170

Asp His Val Glu Leu

175

Trp Trp Val Asn Gly

180

Lys Glu Val His Ser

185

Gly Val Ser Thr Asp

190

Gln Pro Leu Lys Glu 195

Gln Pro Ala Leu Asn

200

Asp Ser Arg Tyr Cys 205

Ser Ser Arg Leu Arg

210

Val Ser Ala Thr Phe

215

Trp Gln Asn Pro Arg

220

His Phe Arg Cys Gln 225

Val Gln Phe Tyr Gly 230

Leu Ser Glu Asn Asp 235

Trp Thr Gln Asp Arg

245

Ala Lys Pro Val Thr

250

Gln Ile Val Ser Ala

255

Ala Trp Gly Arg Ala

260

Asp Cys Gly Phe Thr

265

Ser Glu Ser Tyr Gln

270

Gly Val Leu Ser Ala

275

Thr Ile Leu Tyr Glu 280

Ile Leu Leu Gly Lys

285

Thr Leu Tyr Ala Val

290

Leu Val Ser Ala Leu

295

Val Leu Met Ala Met

300

Arg

Ser

Arg

Ala

Thr 160

Ser

Pro

Leu

Asn

Glu 240

Glu

Gln

Ala

Val

Lys Arg Lys Asp Ser 305

Arg Gly 310

- 50 043746

Leu Ser Leu Leu Trp Ala <210>58 <211>21 <212> PRT <213> Homo sapiens <400>58

Met Ser Ile Gly Leu Leu Cys Cys Ala 15

Gly Pro Val Asn Ala 20 <210>59 <211>111 <212> PRT <213> Homo sapiens <400>59

Gly Val Thr Gln Thr Pro Lys Phe Gln

Met Thr Leu Gln Cys Ala Gln Asp Met 20 25

Tyr Arg Gln Asp Pro Gly Met Gly Leu

40

Gly Ala Gly Ile Thr Asp Gln Gly Glu

55

Ser Arg Ser Thr Thr Glu Asp Phe Pro 65 70

Pro Ser Gln Thr Ser Val Tyr Phe Cys 85

Thr Gly Glu Leu Phe Phe Gly Glu Gly

100 105 <210>60 <211>179 <212> PRT <213> Homo sapiens <400>60

Glu Asp Leu Lys Asn Val Phe Pro Pro

Ser Glu Ala Glu Ile Ser His Thr Gln

25

Leu Lys Thr Gly Gln Ser

His Glu Tyr Met Ser Trp 30

Leu Ile His Tyr Ser Val 45

Pro Asn Gly Tyr Asn Val 60

Arg Leu Leu Ser Ala Ala 75 80

Ser Ser Tyr Val Gly Asn 95

Arg Leu Thr Val Leu

110

Val Ala Val Phe Glu Pro

Ala Thr Leu Val Cys Leu 30

- 51 043746

Leu Ser Trp Trp Val Asn

Ala Thr Gly 35 Phe Tyr Pro Asp His 40 Val Gly Lys 50 Glu Val His Ser Gly 55 Val Ser Glu 65 Gln Pro Ala Leu Asn 70 Asp Ser Arg Arg Val Ser Ala Thr 85 Phe Trp Gln Asn Gln Val Gln Phe 100 Tyr Gly Leu Ser Glu 105 Arg Ala Lys 115 Pro Val Thr Gln Ile 120 Val Ala Asp 130 Cys Gly Phe Thr Ser 135 Glu Ser Ala 145 Thr Ile Leu Tyr Glu 150 Ile Leu Leu Val Leu Val Ser Ala 165 Leu Val Leu Met Ser Arg Gly <210> <211> <212> <213> 61 9 PRT Homo sapiens <400> 61 Ser 1 Leu Leu Met Trp 5 Ile Thr Gln Val <210> <211> <212> <213> 62 308 PRT Homo sapiens <400> 62 Met 1 Gly Phe Arg Leu 5 Leu Cys Cys Val

Asp Pro Gln Pro Leu Lys 60

Cys Leu Ser Ser Arg Leu 75 80

Arg Asn His Phe Arg Cys 95

Asp Glu Trp Thr Gln Asp

110

Ala Glu Ala Trp Gly Arg

125

Gln Gln Gly Val Leu Ser

140

Lys Ala Thr Leu Tyr Ala

155 160

Met Val Lys Arg Lys Asp

175

Phe Cys Leu Leu Gly Ala

- 52 043746

Gly

Ala

Pro

Leu 65

Glu

Leu

Ser

Thr

Phe 145

Val

Trp

Pro

Ser

Phe 225

Thr

Trp

Pro Val Asp Ser Gly 20

Thr Gly Gln Arg Val 35

Tyr Val Tyr Trp Tyr 50

Ile Gln Tyr Tyr Asn 70

Arg Phe Ser Ala Gln 85

Ser Ser Leu Glu Leu

100

Val Glu Ser Ser Tyr 115

Val Val Glu Asp Leu 130

Glu Pro Ser Glu Ala

150

Cys Leu Ala Thr Gly

165

Val Asn Gly Lys Glu

180

Leu Lys Glu Gln Pro

195

Arg Leu Arg Val Ser 210

Arg Cys Gln Val Gln

230

Gln Asp Arg Ala Lys

245

Gly Arg Ala Asp Cys 260

Val Thr Gln Thr Pro

Thr Leu Arg Cys Ser 40

Gln Gln Ser Leu Asp 55

Gly Glu Glu Arg Ala

Gln Phe Pro Asp Leu

Gly Asp Ser Ala Leu 105

Gly Tyr Thr Phe Gly 120

Asn Lys Val Phe Pro 135

Glu Ile Ser His Thr

155

Phe Phe Pro Asp His

170

Val His Ser Gly Val

185

Ala Leu Asn Asp Ser 200

Ala Thr Phe Trp Gln 215

Phe Tyr Gly Leu Ser

235

Pro Val Thr Gln Ile

250

Gly Phe Thr Ser Val

265

Lys His Leu Ile Thr 30

Pro Ala Met Asp His 45

Gln Gly Leu Gln Phe 60

Lys Gly Asn Ile Leu 80

His Ser Glu Leu Asn

Tyr Phe Cys Ala Ser

110

Ser Gly Thr Arg Leu

125

Pro Glu Val Ala Val 140

Gln Lys Ala Thr Leu

160

Val Glu Leu Ser Trp

175

Ser Thr Asp Pro Gln 190

Arg Tyr Cys Leu Ser 205

Asn Pro Arg Asn His 220

Glu Asn Asp Glu Trp

240

Val Ser Ala Glu Ala

255

Ser Tyr Gln Gln Gly

270

- 53 043746

Val Leu Ser Ala Thr Ile Leu Tyr Glu

275 280

Leu Tyr Ala Val Leu Val Ser Ala Leu

290 295

Arg Lys Asp Phe 305 <210>63 <211>274 <212> PRT <213> Homo sapiens <400>63

Met Lys Ser Leu Arg Val Leu Leu Val 15

Trp Val Trp Ser Gln Gln Lys Glu Val 20 25

Ser Val Pro Glu Gly Ala Ile Ala Ser

40

Arg Arg Ser Gln Ser Phe Phe Trp Tyr

55

Pro Glu Leu Ile Met Phe Ile Tyr Ser 65 70

Arg Phe Thr Ala Gln Leu Asn Lys Ala 85

Ile Arg Asp Ser Gln Pro Ser Asp Ser

100 105

Tyr Ser Gly Ala Gly Ser Tyr Gln Leu

115 120

Leu Ser Val Ile Pro Asn Ile Gln Asn

130 135

Leu Arg Asp Ser Lys Ser Ser Asp Lys 145 150

Phe Asp Ser Gln Thr Asn Val Ser Gln

165

Leu Leu Gly Lys Ala Thr

285

Leu Met Ala Met Val Lys 300

Leu Trp Leu Gln Leu Ser

Gln Asn Ser Gly Pro Leu 30

Asn Cys Thr Tyr Ser Asp 45

Gln Tyr Ser Gly Lys Ser 60

Gly Asp Lys Glu Asp Gly 75 80

Gln Tyr Val Ser Leu Leu 95

Thr Tyr Leu Cys Ala Ala

110

Phe Gly Lys Gly Thr Lys

125

Asp Pro Ala Val Tyr Gln

140

Val Cys Leu Phe Thr Asp

155 160

Lys Asp Ser Asp Val Tyr

175

- 54 043746

Ile Thr Asp

Asn Ser Ala

195

Ala Phe Asn

210

Glu Ser Ser 225

Thr Asn Leu

Leu Leu Lys

Lys 180

Val

Asn

Cys

Asn

Val 260

Thr

Ala

Ser

Asp

Phe 245

Ala

Val

Trp

Ile

Val 230

Gln

Gly

Leu

Ser

Ile 215

Lys

Asn

Phe

Asp

Asn 200

Pro

Leu

Leu

Asn

Met 185

Lys

Glu

Val

Ser

Leu 265

Arg

Ser

Asp

Glu

Val 250

Leu

Ser

Asp

Thr

Lys 235

Ile

Met

Met Asp Phe Lys

190

Phe Ala Cys Ala

205

Phe Phe Pro Ser 220

Ser Phe Glu Thr

Gly Phe Arg Ile

255

Thr Leu

Arg Leu 270

Ser

Asn

Pro

Asp 240

Leu

Trp

Ser Ser <210>64 <211>515 <212> PRT <213> Homo <400>64

Met Lys Trp 1 sapiens

Val Thr Phe

Ile Ser Leu

Leu Phe Leu 10

Phe Ser Ser

Tyr Ser Glu

Val Gln Leu 20

Val Glu Ser

Gly Gly Gly

Leu Val Gln

Gly Gly Ser

Leu Arg Leu

Ser Cys Ala 40

Ala Ser Gly

Tyr Ser Phe 45

Gly Tyr Thr 50

Met Asn Trp

Val Arg Gln 55

Ala Pro Gly 60

Lys Gly Leu

Trp Val Ala 65

Leu Ile Asn

Pro Tyr Lys

Gly Val Ser

Thr Tyr Asn

Lys Phe Lys

Asp Arg Phe 85

Thr Ile Ser

Val Asp Lys 90

Ser Lys Asn

Ala Tyr Leu

Gln Met Asn 100

Ser Leu Arg

105

Ala Glu Asp

Thr Ala Val

110

Ala

Pro

Thr

Glu

Gln 80

Thr

Tyr

- 55 043746

Tyr Cys Ala Arg Ser 115

Gly Tyr Tyr Gly Asp

120

Ser Asp Trp Tyr Phe

125

Val Trp Gly Gln Gly

130

Thr Leu Val Thr Val

135

Ser Ser Ala Ser Thr

140

Gly Pro Ser Val Phe 145

Pro Leu Ala Pro Ser 150

Ser Lys Ser Thr Ser 155

Gly Thr Ala Ala Leu

165

Gly Cys Leu Val Lys

170

Asp Tyr Phe Pro Glu

175

Val Thr Val Ser Trp

180

Asn Ser Gly Ala Leu

185

Thr Ser Gly Val His

190

Phe Pro Ala Val Leu

195

Gln Ser Ser Gly Leu 200

Tyr Ser Leu Ser Ser 205

Val Thr Val Pro Ser

210

Ser Ser Leu Gly Thr

215

Gln Thr Tyr Ile Cys 220

Val Asn His Lys Pro 225

Ser Asn Thr Lys Val 230

Asp Lys Lys Val Glu 235

Lys Ser Cys Asp Lys

245

Thr His Thr Ser Pro

250

Pro Ser Pro Ala Pro

255

Val Ala Gly Gln Lys

260

Glu Val Glu Gln Asn

265

Ser Gly Pro Leu Ser

270

Pro Glu Gly Ala Ile

275

Ala Ser Leu Asn Cys 280

Thr Tyr Ser Asp Arg

285

Ser Gln Ser Phe Phe

290

Trp Tyr Arg Gln Tyr

295

Ser Gly Lys Ser Pro

300

Leu Ile Met Ser Ile 305

Tyr Ser Asn Gly Asp 310

Lys Glu Asp Gly Arg 315

Thr Ala Gln Leu Asn

325

Lys Ala Ser Gln Tyr

330

Val Ser Leu Leu Ile

335

Asp Ser Gln Pro Ser

340

Asp Ser Ala Thr Tyr

345

Leu Cys Ala Ala Tyr

350

Gly Ala Gly Ser Tyr

Gln Leu Thr Phe Gly

Lys Gly Thr Lys Leu

Asp

Lys

Gly 160

Pro

Thr

Val

Asn

Pro 240

Pro

Val

Gly

Glu

Phe 320

Arg

Ser

Ser

- 56 043746

Ser Gly

380

Gly Gly

355

Val Ile Pro

370

Asn Ile

Gln Asn

375

Ser Gly Gly 385

Gly Gly

Ser Gly 390

Gly Val Thr

Gln Thr

405

Pro Lys

Val Thr Leu

Arg Cys 420

Ser Pro

Tyr Lys Gln

435

Ser Leu

Asp Gln

Asn Gly Glu

450

Glu Arg

Ala Lys

455

Gln Gln Phe 465

Pro Asp

Leu His 470

Leu Gly Asp

Ser Ala

485

Leu Tyr

Tyr Gly Tyr

Thr Phe 500

Gly Ser

360

Gly Gly

Gly Gly

His Leu

Ala Met 425

Gly Leu 440

Gly Asn

Ser Glu

Phe Cys

Gly Thr 505

Gly Gly

Gly Ser

395

Ile Thr 410

Asp His

Gln Phe

Ile Ser

Leu Asn

475

Ala Ser 490

Arg Leu

365

Gly Gly

Ala Thr

Pro Tyr

Leu Ile

445

Glu Arg 460

Leu Ser

Ser Val

Thr Val

Gly Gly

Gly Gln

415

Val Tyr 430

Gln Tyr

Phe Ser

Ser Leu

Glu Ser

495

Val Glu 510

Gly

Ser 400

Arg

Trp

Tyr

Ala

Glu 480

Ser

Asp

Leu Lys Asn

515 <210>65 <211>515 <212> PRT <213> Homo <400>65

Met Lys Trp 1

Tyr Ser Glu

Gly Gly Ser

Gly Tyr Thr sapiens

Val Thr Phe 5

Val Gln Leu 20

Leu Arg Leu

Met Asn Trp

Ile Ser Leu

Val Glu Ser 25

Ser Cys Ala 40

Val Arg Gln

Leu Phe Leu 10

Gly Gly Gly

Ala Ser Gly

Ala Pro Gly

Phe Ser Ser

Leu Val Gln

Tyr Ser Phe 45

Lys Gly Leu

Ala

Pro

Thr

Glu

- 57 043746

Val Ser Thr Tyr Asn

Trp Val Ala Leu Ile 65

Asn Pro Tyr Lys Gly 70

Lys Phe Lys Asp Arg 85

Phe Thr Ile Ser Val

Asp Lys Ser Lys Asn 95

Ala Tyr Leu Gln Met

100

Asn Ser Leu Arg Ala

105

Glu Asp Thr Ala Val

110

Tyr Cys Ala Arg Ser 115

Gly Tyr Tyr Gly Asp

120

Ser Asp Trp Tyr Phe

125

Val Trp Gly Gln Gly

130

Thr Leu Val Thr Val

135

Ser Ser Ala Ser Thr

140

Gly Pro Ser Val Phe 145

Pro Leu Ala Pro Ser 150

Ser Lys Ser Thr Ser 155

Gly Thr Ala Ala Leu

165

Gly Cys Leu Val Lys

170

Asp Tyr Phe Pro Glu

175

Val Thr Val Ser Trp

180

Asn Ser Gly Ala Leu

185

Thr Ser Gly Val His

190

Phe Pro Ala Val Leu

195

Gln Ser Ser Gly Leu 200

Tyr Ser Leu Ser Ser 205

Val Thr Val Pro Ser

210

Ser Ser Leu Gly Thr

215

Gln Thr Tyr Ile Cys

220

Val Asn His Lys Pro 225

Ser Asn Thr Lys Val 230

Asp Lys Lys Val Glu 235

Lys Ser Cys Asp Lys

245

Thr His Thr Ser Pro

250

Pro Ser Pro Ala Pro

255

Val Ala Gly Gln Lys

260

Glu Val Glu Gln Asn

265

Ser Gly Pro Leu Ser

270

Pro Glu Gly Ala Ile

275

Ala Ser Leu Asn Cys 280

Thr Tyr Ser Asp Arg

285

Ser Gln Ser Phe Phe

290

Trp Tyr Arg Gln Tyr

295

Ser Gly Lys Ser Pro

300

Gln 80

Thr

Tyr

Asp

Lys

Gly 160

Pro

Thr

Val

Asn

Pro 240

Pro

Val

Arg

Glu

- 58 043746

Leu

305

Ile

Met

Ser

Ile

Tyr 310

Ser

Asn

Gly

Asp

Lys 315

Glu

Asp

Gly

Arg

Phe

320

Thr

Ala

Gln

Leu

Asn

325

Lys

Ala

Ser

Gln

Tyr 330

Val

Ser

Leu

Leu

Ile

335

Arg

Asp

Ser

Gln

Pro

340

Ser

Asp

Ser

Ala

Thr

345

Tyr

Leu

Cys

Ala

Ala

350

Tyr

Ser

Gly Ala Gly Ser

355

Tyr Gln Leu

Thr Phe Gly 360

Lys Gly Thr

365

Lys Leu Ser

Val Ile Pro Asn

370

Ile Gln Asn

375

Gly Gly Gly

Gly Ser Gly

380

Gly Gly Gly

Ser Gly Gly Gly 385

Gly Ser Gly

390

Gly Gly Gly

Ser Gly Gly 395

Gly Gly Ser

400

Gly Val Thr Gln

Thr Pro Lys 405

His Leu Ile

410

Thr Ala Thr

Gly Gln Arg

415

Val Thr Leu Arg

420

Cys Ser Pro

Ala Met Asp

425

His Pro Tyr

Val Tyr Trp 430

Tyr Lys

Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu 435 440

Ile Gln Tyr Tyr 445

Asn

Gln 465

Leu

Tyr

Gly Glu Glu Arg Ala Lys Gly Asn Ile Ser 450 455

Gln Phe Pro Asp Leu His Ser Glu Leu Asn

470 475

Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser

485 490

Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu

500 505

Leu Lys Asn

515 <210> 66 <211> 515 <212> PRT <213> Homo sapiens

Glu Arg Phe Ser Ala 460

Leu Ser Ser Leu Glu

480

Ser Val Glu Ser Ser

495

Thr Val Val Glu Asp 510 <400> 66

- 59 043746

Met

Tyr

Gly

Gly

Trp 65

Lys

Ala

Tyr

Val

Gly 145

Gly

Val

Phe

Val

Val 225

Lys

Lys Trp Val Thr Phe 5

Ser Glu Val Gln Leu

Gly Ser Leu Arg Leu 35

Tyr Thr Met Asn Trp 50

Val Ala Leu Ile Asn

Phe Lys Asp Arg Phe 85

Tyr Leu Gln Met Asn

100

Cys Ala Arg Ser Gly 115

Trp Gly Gln Gly Thr 130

Pro Ser Val Phe Pro

150

Thr Ala Ala Leu Gly

165

Thr Val Ser Trp Asn

180

Pro Ala Val Leu Gln

195

Thr Val Pro Ser Ser 210

Asn His Lys Pro Ser

230

Ser Cys Asp Lys Thr

245

Ile Ser Leu Leu Phe

Leu Phe Ser Ser Ala

Val Glu Ser Gly Gly 25

Gly Leu Val Gln Pro 30

Ser Cys Ala Ala Ser 40

Gly Tyr Ser Phe Thr 45

Val Arg Gln Ala Pro 55

Pro Tyr Lys Gly Val

Thr Ile Ser Val Asp 90

Ser Leu Arg Ala Glu

105

Tyr Tyr Gly Asp Ser 120

Leu Val Thr Val Ser 135

Leu Ala Pro Ser Ser

155

Cys Leu Val Lys Asp

170

Ser Gly Ala Leu Thr

185

Ser Ser Gly Leu Tyr

200

Ser Leu Gly Thr Gln 215

Asn Thr Lys Val Asp

235

His Thr Ser Pro Pro

250

Gly Lys Gly Leu Glu 60

Ser Thr Tyr Asn Gln 80

Lys Ser Lys Asn Thr 95

Asp Thr Ala Val Tyr 110

Asp Trp Tyr Phe Asp 125

Ser Ala Ser Thr Lys 140

Lys Ser Thr Ser Gly

160

Tyr Phe Pro Glu Pro

175

Ser Gly Val His Thr 190

Ser Leu Ser Ser Val

205

Thr Tyr Ile Cys Asn 220

Lys Lys Val Glu Pro

240

Ser Pro Ala Pro Pro

255

- 60 043746

Val

Gly

Val

Gln 305

Phe

Ser

Glu

Val

Gly 385

Lys

Ile

Phe

Ile

Asn 465

Ser

Tyr

Ala Gly

Gln Arg

275

Tyr Trp 290

Tyr Tyr

Ser Ala

Leu Glu

Ser Ser

355

Glu Asp 370

Gly Gly

Glu Val

Ala Ser

Trp Tyr

435

Tyr Ser 450

Lys Ala

Asp Ser

Gln Leu

Gly Val 260

Val Thr

Tyr Lys

Asn Gly

Gln Gln

325

Leu Gly 340

Tyr Gly

Leu Lys

Gly Ser

Glu Gln

405

Leu Asn 420

Arg Gln

Asn Gly

Ser Gln

Ala Thr

485

Thr Phe 500

Thr Gln

Leu Arg

Gln Ser

295

Glu Glu 310

Phe Pro

Asp Ser

Tyr Thr

Asn Gly

375

Gly Gly 390

Asn Ser

Cys Thr

Tyr Ser

Asp Lys

455

Tyr Val 470

Tyr Leu

Gly Lys

Thr Pro

265

Cys Ser 280

Leu Asp

Arg Ala

Asp Leu

Ala Leu

345

Phe Gly 360

Gly Gly

Gly Gly

Gly Pro

Tyr Ser

425

Gly Lys 440

Glu Asp

Ser Leu

Cys Ala

Gly Thr

505

Lys His

Leu Ile

Pro Arg

Gln Gly

Lys Gly

315

His Ser 330

Tyr Phe

Ser Gly

Gly Ser

Ser Gly

395

Leu Ser 410

Asp Arg

Ser Pro

Gly Arg

Leu Ile

475

Ala Tyr 490

Lys Leu

Ser Gly

285

Leu Gln 300

Asn Ile

Glu Leu

Cys Ala

Thr Arg

365

Gly Gly 380

Gly Gly

Val Pro

Arg Ser

Glu Leu

445

Ph Thr 460

Arg Asp

Ser Gly

Ser Val

Thr Ala 270

Asp Leu

Phe Leu

Ser Glu

Asn Leu

335

Ser Ser

350

Leu Thr

Gly Gly

Gly Ser

Glu Gly

415

Gln Ser 430

Ile Met

Ala Gln

Ser Gln

Ala Gly

495

Ile Pro

510

Thr

Ser

Ile

Arg 320

Ser

Val

Val

Ser

Gln 400

Ala

Phe

Ser

Leu

Pro 480

Ser

Asn

-

Claims (33)

Ile Gln Asn 515 CLAIM 1. An antigen recognition structure that is stable and capable of specifically and/or selectively binding to a COL6A3 antigenic peptide, comprising a first domain and a second domain, each comprising three complementary determinant regions (CDRs), wherein the first domain contains amino acid sequences in accordance with SEQ ID NO : 6 (CDRa2) and 7 (CDRa3) and the second domain contains amino acid sequences in accordance with SEQ ID NO: 14 (CDRb2) and 15 (CDRb3), and: a) CDRa1 of the first domain is set forth in SEQ ID NO: 5 and CDRb1 of the second domain is selected from one of the amino acid sequences in accordance with SEQ ID NO: 37-44 or b) CDRa1 of the first domain is set forth in SEQ ID NO: 26 and CDRb1 of the second domain is selected from one of the amino acid sequences in accordance with SEQ ID NO: 13 and 37-49; wherein the antigen recognition structure is a T cell receptor (TCR), or a derivative or fragment thereof, wherein the derivative or fragment retains the antigen binding and/or recognition ability of the molecule to specifically and/or selectively bind to a COL6A3 antigenic polypeptide. 2. Antigen recognition structure according to claim 1, in which: (i) the first domain contains SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 and the second domain contains SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 40 or (ii) the first domain contains SEQ ID NO: 26, SEQ ID NO: 6 and SEQ ID NO: 7 and the second domain contains SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 40. 3. The antigen recognition structure according to claim 2, wherein the structure according to (i) contains SEQ ID NO: 2 and SEQ ID NO: 62 or wherein the structure according to (ii) contains SEQ ID NO: 62 and SEQ ID NO : 63. 4. Antigen recognition structure according to any one of claims 1 to 3, wherein said antigen recognition structure is stable and capable of specifically and/or selectively binding to the COL6A3 antigenic peptide comprising the amino acid sequence in accordance with SEQ ID NO: 1, in particular with the participation of MHC . 5. An antigen recognition structure according to any one of claims 1, 2 or 4, wherein said first domain is part of the TCR α- or γ-chain and/or wherein said second domain is part of the TCR β- or δ-chain. 6. An antigen recognition structure according to any one of claims 1 to 5, wherein the antigen recognition structure is a single chain antigen recognition structure. 7. The antigen recognition structure according to claim 6, wherein the single chain antigen recognition structure is a single chain TCR. 8. An antigen recognition structure according to any one of claims 1 to 7, wherein the antigen recognition structure is a soluble TCR. 9. A polypeptide chain that is stable and capable of specifically and/or selectively binding to a COL6A3 antigenic peptide, comprising a first domain and a second domain, each comprising three complementary determinant regions (CDRs), wherein the first domain contains amino acid sequences in accordance with SEQ ID NO : 6 (CDRa2) and 7 (CDRa3) and the second domain contains amino acid sequences in accordance with SEQ ID NO: 14 (CDRb2) and 15 (CDRb3), and: a) CDRa1 of the first domain is set forth in SEQ ID NO: 5 and CDRb1 of the second domain is selected from one of the amino acid sequences in accordance with SEQ ID NO: 37-44 or b) CDRa1 of the first domain is set forth in SEQ ID NO: 26 and CDRb1 of the second domain is selected from one of the amino acid sequences in accordance with SEQ ID NO: 13 and 37-49. 10. A nucleic acid encoding an antigen recognition structure according to any one of claims 1 to 8 or a polypeptide chain according to claim 9. 11. A nucleic acid vector containing the nucleic acid according to claim 10. 12. A recombinant host cell, comprising an antigen recognition structure according to any one of claims 1 to 8, a polypeptide chain according to claim 9, a nucleic acid according to claim 10, or a vector according to claim 11. 13. The host cell of claim 12, wherein said host cell is a mammalian cell. 14. The host cell according to claim 13, wherein said host cell is a human cell. - 62 043746 15. The host cell according to any one of claims 12-14, where the specified host cell is: a) a lymphocyte, preferably a T lymphocyte or a T lymphocyte precursor cell, more preferably a CD4 or CD8 positive T cell, or b) not a lymphocyte. 16. An antigen recognition structure according to any one of claims 1 to 8, a polypeptide chain according to claim 9, a nucleic acid according to claim 10, a vector according to claim 11 or a host cell according to any one of claims 12 to 15, where the antigen recognition structure is polypeptide the strand, nucleic acid, vector, or host cell is labeled with a detectable label. 17. The antigen recognition structure, polypeptide chain, nucleic acid, vector or host cell of claim 16, wherein the detectable label is selected from the group consisting of a radioisotope, a fluorophore, a phycoerythrin, an enzyme and an element particle. 18. A pharmaceutical composition comprising an antigen recognition structure according to any one of claims 1 to 8, a polypeptide chain according to claim 9, a nucleic acid according to claim 10, a vector according to claim 11 or a host cell according to claim 15a) and a pharmaceutically acceptable carrier, diluent, stabilizer and/or excipient. 19. The pharmaceutical composition according to claim 18, additionally containing another pharmaceutically active substance or drug. 20. The pharmaceutical composition according to claim 19, wherein the additional pharmaceutically active substance or drug is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and vincristine . 21. A method for obtaining a COL6A3-specific antigen recognition structure in accordance with any of claims 1 to 8, including: a) providing a suitable host cell; b) providing a genetic construct including a coding sequence encoding an antigen recognition structure in accordance with any of claims 1 to 8; c) introducing said genetic construct into said suitable host cell; d) expression of said genetic construct by said suitable host cell. 22. The method of claim 21, further comprising isolating and purifying the antigen recognition structure from a suitable host cell and, optionally, restoring the antigen recognition structure to the T cell. 23. Use of an antigen recognition structure according to any one of claims 1 to 8 in the prevention and/or treatment of a proliferative disease. 24. Use of the polypeptide chain according to claim 9 in the prevention and/or treatment of a proliferative disease. 25. The use of a nucleic acid according to claim 10 in the prevention and/or treatment of a proliferative disease. 26. Use of the vector according to claim 11 in the prevention and/or treatment of a proliferative disease. 27. Use of a host cell according to claim 15a) in the prevention and/or treatment of a proliferative disease. 28. Use of a pharmaceutical composition according to any one of claims 18-20 in the prevention and/or treatment of a proliferative disease. 29. Use according to claims 23 to 28, wherein the proliferative disease is a COL6A3 positive preneoplastic condition or cancer selected from the group consisting of breast cancer, colon cancer and colorectal carcinoma. 30. Use according to claim 29, wherein said cancer is selected from a cancer that has overexpression or mutations of COL6A3 and/or presents a COL6A3-derived tumor-associated antigen. 31. Use of a host cell according to claim 15a) for the manufacture of a medicament for the treatment of cancer or a COL6A3-positive pre-tumor condition in a subject in need thereof, wherein the host cell is an autologous cell from the patient. 32. Use of a host cell according to claim 15a) for the manufacture of a medicament for the treatment of cancer or a COL6A3-positive pre-tumor condition in a subject in need thereof, wherein the host cell is an allogeneic cell. 33. A method of treating cancer or a COL6A3-positive pre-neoplastic condition in a subject in need thereof, comprising: a) obtaining a cell from a specified subject; b) transforming the cell with at least one vector according to claim 11 to obtain a transformed cell; c) culturing the transformed cell to obtain a plurality of transformed cells; and d) administering a plurality of transformed cells to said subject. -