阅读说明：本技术 用于癌症免疫疗法的mr1限制性t细胞受体 (MR 1-restricted T cell receptor for cancer immunotherapy ) 是由 加纳罗·得利博罗 马尔科·莱波雷 露西娅·莫利 于 2019-09-11 设计创作，主要内容包括：本发明涉及分离可表达T细胞受体的T细胞的方法,该T细胞受体能够特异性结合通过与MR分子相关联的癌细胞呈递的抗原。该方法包括以下步骤：(a)提供T细胞制剂；(b)使制剂与表达MR1蛋白的癌细胞接触；(c)分离与所述癌细胞产生特异反应的T细胞。本发明进一步涉及一种制备T细胞制剂的方法,该T细胞制剂在转基因表达载体上表达选择性识别MR1的T细胞受体,该T细胞制剂在癌症治疗中的应用,以及编码响应MR1的T细胞受体的核酸和细胞的集合。(The present invention relates to methods of isolating T cells that express a T cell receptor capable of specifically binding to an antigen presented by a cancer cell associated with an MR molecule. The method comprises the following steps: (a) providing a preparation of T cells; (b) contacting the preparation with a cancer cell expressing MR1 protein; (c) isolating T cells that specifically react with the cancer cells. The invention further relates to a method for the preparation of a T cell preparation expressing a T cell receptor that selectively recognizes MR1 on a transgenic expression vector, the use of the T cell preparation in the treatment of cancer, and a collection of nucleic acids and cells encoding a T cell receptor that responds to MR 1.)

1. A method of preparing a preparation of MR1T cells expressing a T cell receptor capable of binding to an antigen presented by a cancer cell associated with an MR1 molecule, the method comprising the steps of:

a. providing a tumor sample obtained from a patient;

b. contacting the tumor sample with:

i. a plurality of T cell clones, wherein each T cell clone is characterized by having an MR1T cell receptor capable of specifically binding to an antigen presented by a cancer cell associated with an MR1 molecule; or

A plurality of isolated, labeled and multimerized soluble T-cell receptors isolated from the MR1T cell receptor molecule;

wherein each said T cell clone or each said isolated, labeled and multimerized soluble T cell receptor is characterized by

-a CDR3 sequence fragment selected from any one of SEQ ID NO 065 to SEQ ID NO 096, or a CDR3 sequence fragment, characterized by a sequence identical to a sequence selected from any one of SEQ ID NO 065 to SEQ ID NO 096 but having one or two amino acid substitutions, in particular wherein said substitutions are selected according to the substitution rules given below; more particularly, the T cell clone or soluble TCR is characterized by a CDR3 sequence selected from any one of SEQ ID NO 065 to SEQ ID NO 096, wherein the sequence comprises a maximum total of 0, 1 or 2 substitutions at a position between the fourth N-terminal amino acid and the fifth C-terminal amino acid of the CDR3 α, γ or δ sequence, or the fourth N-terminal amino acid and the sixth C-terminal amino acid of the CDR3 β sequence, according to the following substitution rules:

-glycine (G) and alanine (a) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable, a and V are interchangeable;

tryptophan (W) and phenylalanine (F) are interchangeable, tyrosine (Y) and F are interchangeable;

-serine (S) and threonine (T) are interchangeable;

aspartic acid (D) and glutamic acid (E) are interchangeable

-asparagine (N) and glutamine (Q) are interchangeable; n and S are interchangeable; n and D are interchangeable; e and Q are interchangeable;

methionine (M) and Q are interchangeable;

cysteine (C), a and S are interchangeable;

-proline (P), G and a are interchangeable;

-arginine (R) and lysine (K) are interchangeable;

or

-wherein each of said T cell clones or said isolated, labeled and multimerized soluble T cell receptors is characterized by a nucleic acid sequence selected from SEQ ID NO 007 to SEQ ID NO 012 or SEQ ID NO 037 to SEQ ID NO 060 or SEQ ID NO 063 to SEQ ID NO 064 and/or an amino acid sequence selected from SEQ ID NO 001 to SEQ ID 006 or SEQ ID NO 013 to SEQ ID NO 036 or SEQ ID NO 061 to SEQ ID NO 062; or

-a T cell receptor alpha chain nucleic acid sequence selected from SEQ ID NO 007, 009 to 011 or SEQ ID NO 037 to SEQ ID NO 048 and/or an amino acid sequence selected from SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024; or an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity and the same biological activity as SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024, in particular an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity with SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024, comprising a CDR sequence selected from SEQ ID NO 065 to SEQ ID NO 079 and/or

-a T cell receptor beta chain nucleic acid sequence selected from SEQ ID NO 008, 010 to 012 or SEQ ID NO 049 to SEQ ID NO 060 and/or an amino acid sequence selected from SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036 or an amino acid sequence having at least 85% (≧ 90%, 95%, 98%) identity and the same biological activity as SEQ ID SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036, in particular an amino acid sequence having at least 85% (≧ 90%, 95%, 98%) identity with SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036 comprising a CDR sequence selected from SEQ ID NO 080 to SEQ ID NO 094;

or

-the T cell receptor gamma chain nucleic acid sequence SEQ ID NO 61 and/or the amino acid sequence SEQ ID NO 063 or a sequence having at least 85% (≧ 90%, 95%, 98%) identity therewith and having the same biological activity, in particular an amino acid sequence having at least 85% (≧ 90%, 95%, 98%) identity with SEQ ID NO 063 and comprising the CDR3 of SEQ ID NO 095; and/or

-T cell receptor delta chain nucleic acid sequence SEQ ID NO: 64 and/or the amino acid sequence SEQ ID NO 062, or an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity to SEQ ID NO 062 and comprising the CDR3 of SEQ ID NO 096;

or

Wherein each said T cell clone or said isolated, labeled and multimerized soluble T cell receptor is characterized by a pair of T cell receptor alpha and beta chain nucleic acid sequences selected from the group consisting of:

-SEQ ID NO 007 and SEQ ID NO 008; or SEQ ID NO 009 and SEQ ID NO 010; or SEQ ID NO 011 and SEQ ID NO 012,

-SEQ ID NO 037 and SEQ ID NO 049; or SEQ ID NO 038 and SEQ ID NO 050; or SEQ ID NO 039 and SEQ ID NO 051; or SEQ ID NO 040 and SEQ ID NO 052; or SEQ ID NO 041 and SEQ ID NO 053; or SEQ ID NO 042 and SEQ ID NO 054; or SEQ ID NO 043 and SEQ ID NO 055; or SEQ ID NO 044 and SEQ ID NO 056; or SEQ ID NO 045 and SEQ ID NO 057; or SEQ ID NO 046 and SEQ ID NO 058; or SEQ ID NO 047 and SEQ ID NO 059; or SEQ ID NO 048 and SEQ ID NO 060;

or

Wherein each said T cell clone or said isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor gamma and delta chain nucleic acid sequence pair selected from the group consisting of SEQ ID NO 063 and SEQ ID NO 064;

or

Wherein each said T cell clone or said isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor alpha chain and beta chain amino acid sequence pair selected from the group consisting of:

-SEQ ID NO 001 and SEQ ID NO 002; or SEQ ID NO 003 and SEQ ID NO 004; or SEQ ID NO 005 and SEQ ID NO 006,

-SEQ ID NO 013 and SEQ ID NO 025; or SEQ ID NOs 014 and 026; or SEQ ID NO 015 and SEQ ID NO 027; or SEQ ID NO 016 and SEQ ID NO 028; or SEQ ID NO 017 and SEQ ID NO 029; or SEQ ID NO 018 and SEQ ID NO 030; or SEQ ID NO 019 and SEQ ID NO 031; or SEQ ID NO 20 and SEQ ID NO 032; or SEQ ID NO 021 and SEQ ID NO 033; or SEQ ID NO 022 and SEQ ID NO 034; or SEQ ID NO 023 and SEQ ID NO 035; or SEQ ID NO 024 and SEQ ID NO 036;

or a pair selected from the pair given in the first two paragraphs, wherein each partner may have at least 85% (. gtoreq.90%, 95%, 98%) identity to the indicated SEQ ID NO and the pair has the same biological activity as the unmutated pair;

or

Wherein each of said T cell clones or said isolated, labeled and multimerized soluble T cell receptor are characterized by the T cell receptor gamma chain SEQ ID NO 061 and the delta chain amino acid sequence SEQ ID NO 062, or wherein each partner may have a pair with at least 85% (. gtoreq.90%, 95%, 98%) identity thereto and which pair has the same biological activity as the unmutated pair;

identifying a receptor of MR1T cells that specifically reacts with the tumor sample;

c. providing a preparation of T cells;

d. introducing a nucleic acid expression construct into said T cell preparation to produce a transgenic T cell preparation, said nucleic acid construct encoding an MR 1-reactive T cell receptor which is identified in step iii.

2. The method of claim 1, wherein the T cell preparation is obtained from the same patient (autologous adoptive T cell therapy).

3. The method according to claim 1, wherein the T cell preparation is obtained from another subject, in particular an HLA-matched subject (allogeneic adoptive T cell therapy).

4. The method of claims 1 to 3, wherein the T cell preparation is obtained from peripheral blood, in particular wherein the T cell preparation is obtained by expressing one or more T cell markers selected from the group consisting of CD4, CD8, CD27, CD45RA and CD57, in particular selected CD3, by selected PBMCs⁺CD4⁺Or CD3⁺CD8⁺Or CD3⁺CD27⁺CD45RA⁺Or CD3⁺CD27⁺CD45RA^-Or CD3⁺CD27^-CD45RA^-Or CD3⁺CD57^-Or CD3⁺CD57⁺T cells.

5. The method of claim 1 or 2, wherein the T cell preparation is obtained after tumor biopsy followed by in vitro expansion.

6. A preparation of MR 1-specific T cells obtained by the method of any one of claims 1 to 5 for use in a method of treatment or prevention of cancer, in particular a method of treatment or prevention of cancer characterised by MR1 expression.

7. An expression vector comprising a nucleic acid sequence encoding:

a. a functional T cell receptor heterodimer;

or

b.T a cell receptor alpha chain capable of forming a functional T cell receptor heterodimer with a T cell receptor beta chain; and/or

c.T a cell receptor beta chain capable of forming a functional T cell receptor heterodimer with a T cell receptor alpha chain;

wherein the T cell receptor heterodimer is capable of specifically binding to an MR1 molecule, wherein the MR1 molecule is expressed on tumor cells and presents a tumor associated antigen,

and wherein the nucleic acid sequence

Is or comprises a nucleic acid sequence selected from SEQ ID NO 007 to SEQ ID NO 012, and/or encodes an amino acid sequence selected from SEQ ID NO 001 to SEQ ID 006; or an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity and the same biological activity as SEQ ID NO 001, 003 to 005, in particular an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity to SEQ ID NO 001, 003 to 005, comprising a CDR sequence selected from SEQ ID NO 065 to SEQ ID NO 067;

or

Is or comprises a T cell receptor alpha chain nucleic acid sequence selected from SEQ ID NO 007, SEQ ID NO 009 or SEQ ID NO 011, and/or

Encoding an amino acid sequence selected from SEQ ID NO 001, SEQ ID NO 003 or SEQ ID NO 005;

or

Is or comprises a T cell receptor beta chain nucleic acid sequence selected from SEQ ID NO 008, SEQ ID NO 010 or SEQ ID NO 012, and/or

Encoding an amino acid sequence selected from SEQ ID NO 002, SEQ ID NO 004 or SEQ ID NO 006;

or

Is or comprises a pair of T cell receptor alpha and beta chain nucleic acid sequences selected from the group consisting of: SEQ ID NO 007 and SEQ ID NO 008; SEQ ID NO 009 and SEQ ID NO 010; or SEQ ID NO 011 and SEQ ID NO 012;

or a pair of T cell receptor alpha and beta chain amino acid sequences selected from the following pairs: SEQ ID NO 001 and SEQ ID NO 002; SEQ ID NO 003 and SEQ ID NO 004; or SEQ ID NO 005 and SEQ ID NO 006.

8. An isolated T cell receptor protein heterodimer comprising an amino acid sequence selected from SEQ ID NOs 001 to 006, or a sequence having at least 85% (≧ 90%, 95%, 98%) identity to the amino acid sequence selected from SEQ ID NOs 001 to 006 and having the same biological activity, particularly wherein the sequence comprises a CDR3 sequence selected from SEQ ID NOs 65, 66, 67, 80, 81 and 82.

9. The isolated T cell receptor protein heterodimer according to claim 8, wherein the isolated T cell receptor protein comprises a pair of amino acid sequences selected from the group consisting of:

SEQ ID NO 001 and SEQ ID NO 002,

SEQ ID NO 003 and SEQ ID NO 004,

SEQ ID NO 005 and SEQ ID NO 006

Or a pair selected from the pairs given above, wherein each partner may have a sequence with at least 85% (. gtoreq.90%, 95%, 98%) identity to the indicated SEQ ID NO and the pair has the same biological activity as the unmutated pair; in particular wherein each amino acid sequence comprises the same CDR3 sequence as the indicated SEQ ID NO.

10. The isolated T cell receptor protein heterodimer according to claim 8 or 9, which binds to an MR1 molecule, wherein the MR1 molecule presents a tumor associated antigen.

11. A recombinant cell comprising the expression vector of claim 7 or the T cell receptor protein heterodimer of any one of claims 8 to 10, wherein the recombinant cell is a T cell obtained from:

a. peripheral blood; or

b. Tumors infiltrate lymphocytes.

12. A recombinant cell according to claim 11 for use in a method of treatment or prevention of cancer, in particular a method of treatment or prevention of cancer characterized by MR1 expression.

13. The recombinant cell for use in a method of treating or preventing cancer according to claim 12, wherein the cell is administered by adoptive T cell immunotherapy.

14. A collection of nucleic acid sequences, wherein each sequence of the collection promotes the expression of a different T cell receptor alpha chain, T cell receptor beta chain, or combination of T cell receptor alpha and beta chains in a mammalian cell, wherein the combination is capable of specifically binding to a MR1 molecule presenting a cancer antigen, wherein the collection comprises a sequence selected from SEQ ID NO 007 to SEQ ID NO 012 and/or comprises a sequence encoding a T cell receptor molecule (or a T cell receptor constituting an alpha or beta chain) selected from SEQ ID NO 001 to SEQ ID NO 006.

15. A collection of recombinant T cells, wherein each cell of the collection expresses as a transgene a T cell receptor capable of specifically binding to the cancer antigen presenting MR1 molecule according to claims 15 to 18.

16. A nucleic acid expression vector expressing MR1 comprising a nucleic acid sequence encoding MR1 under the control of a promoter sequence operable in mammalian cells for use in cancer therapy, wherein the nucleic acid expression vector expressing MR1 is administered prior to, concurrently with, or subsequent to administration of:

a. a recombinant cell comprising the expression vector of claim 7 or the T cell receptor protein heterodimer of any one of claims 8 to 10, and/or

b. An MR 1-specific T cell preparation obtained by the method of any one of claims 1 to 5, and/or

c. An expression vector according to claim 7.

Background

T lymphocytes can detect a variety of different non-peptide antigens presented by non-polymorphic cell surface molecules, including lipids and phosphorylated isoprenoids. The diverse phenotype and functional properties of these T cells serve a special role in protecting the host from infection, autoimmunity, and cancer. The T cell pool for non-peptide antigens has recently increased and contains mucosa-associated constant T (mait) cells that respond to the small riboflavin precursors produced by numerous yeasts and bacteria and are presented by the MHC class I-associated protein MR 1. MAIT cells are common in human blood, kidney and intestine and contain most of the T cells residing in the liver. Upon activation, MAIT cells release a range of pro-inflammatory and immunoregulatory cytokines and can mediate direct killing of microbial infected cells. It is not clear whether MR1 has other effects besides presenting microbial metabolites to MAIT cells.

MR1 is a non-polymorphic MHC class I protein that is expressed at low levels on the surface of many cell types. MR1 is highly conserved across species, with > 90% sequence homology at the protein level between human and mouse MR 1.

The inventors propose the presence of human T cells that recognize a tumor associated antigen presented by MR 1. These novel T cells may be involved in tumor immune surveillance and therefore represent a new tool for cancer immunotherapy. Adoptive therapy with donor or patient-derived T cells, designed to express TCRs of selected tumor-associated antigens, represents a guaranteed and safe strategy for inducing clinically relevant anti-tumor immune responses in cancer patients. However, most tumor associated antigens identified to date are peptides presented by polymorphic MHC molecules. Extreme polymorphisms in the MHC gene limit the utility of this approach in patients expressing distinct MHC alleles. Targeting tumor antigens bound to non-polymorphic antigen presenting molecules such as MR1 can overcome this limitation and is in principle applicable to all patients with tumors expressing MR 1. The use of tumor reactive T cell receptors recognizing MR 1-presented antigens may also have the advantage of complementing the anti-tumor response mediated by MHC-presented peptide antigens, which precludes cross-competition of tumor antigens with binding of the same type of presenting molecule. Furthermore, this strategy may offer the possibility to target antigens of different nature on the same tumor cell, thereby minimizing the potential occurrence of tumor escape variants under selective immunological pressure. Thus, the identification of tumor associated antigens presented by MR1 and characterization of MR 1-restricted TCRs that recognize these antigens may be of great interest for cancer immunotherapy.

Based on the above state of the art, it was an object of the present invention to provide novel means and methods for the treatment of cancer. This object is achieved by the subject matter of the independent claims and further advantageous solutions are provided by the dependent claims, examples and figures disclosed herein.

Definition of

In the context of the present specification, the term MR1 refers to the MR1 gene (Entrez 3140) or the MR1 gene product (Uniprot Q95460).

MR1 presents bacterial riboflavin by-products (referred to above as "foreign microbe-derived antigens") in the physiological environment of non-neoplastic patients and to mucosal non-denatured T cells.

Cancer cells expressing MR1 present a specific cancer antigen or a number of specific cancer antigens on MR 1.

In the context of the present specification, the term MR1T cell refers to a T cell expressing a T cell receptor capable of specifically binding to an MR1 molecule presented by a cancer cell.

In the context of the present specification, the term MR1T cell receptor refers to a T cell receptor capable of specifically binding to an antigen presented by a cancer cell associated with the MR1 molecule.

The TCR sequences or TCR molecules described herein comprise fully functional TCR α and TCR β polypeptide chains, or TCR γ and TCR δ polypeptide chains. If reference is made to a TCR a or β polypeptide having a particular sequence, it is to be understood that the presence of a complementary (β or α, respectively) polypeptide chain is required for it to be fully functional in the methods and cells described herein. Likewise, the necessary modifications apply to the γ δ pair. Reference to a particular TCR α, β, γ or δ sequence means that it is likely to pair with the TCR sequence with which it is paired in the original clone described herein, or a sequence with some identity to the original paired sequence, as specified herein. Reference to a particular TCR α, β, γ or δ sequence also means that it may be paired with another paired TCR sequence.

Recognition of the cancer antigen presented by MR1 is primarily through the CDR3 sequence. Where reference is made herein to a TCR sequence which is characterised only by a particular CDR3 sequence, it is implied that the TCR sequence is the complete α, β, γ or δ TCR sequence provided herein, and that the resultant TCR molecule is paired with an appropriate second sequence.

In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter that represents the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods of sequence alignment for comparison are well known in the art. The alignment of sequences for comparison can be performed as follows: local homology algorithms of Smith and Waterman, adv.Appl.Math.2:482(1981), global alignment algorithms of Needleman and Wunsch, J.mol.biol.48:443(1970), methods of finding similarities of Pearson and Lipman, Proc.Nat.Acad.Sci.85:2444(1988), or by computerized implementation of these algorithms, including but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, for example, through the national center for Biotechnology information (http:// BLAST. ncbi. nlm. nih. gov. /).

One example of comparing amino acid sequences is the BLASTP algorithm using default settings: desired threshold value: 10; word length: 3; maximum match within query range: 0; matrix: BLOSUM 62; gap penalties: presence 11, extension 1; composition adjustment: the conditions constitute a scoring matrix adjustment. One such example for comparing nucleic acid sequences is the BLASTN algorithm using default settings: desired threshold value: 10; word length: 28; maximum match within query range: 0; match/no match score: 1. -2; gap penalties: and (4) linearity. Unless otherwise indicated, sequence identity values provided herein refer to values obtained using the BLAST suite of programs, Altschul et al, J.Mol.biol.215: 403-.

Reference to identical sequences without an assigned percentage value indicates 100% identical sequences (i.e., identical sequences).

In the present specification, the term positive when used in the context of marker expression refers to the expression of antigen as determined by a fluorescently labeled antibody wherein the median fluorescence intensity of the fluorescence is at least 30% (. gtoreq.30%) higher, in particular. gtoreq.50% or. gtoreq.80% higher, than the fluorescence intensity obtained after staining with a isotype-matched antibody which does not specifically bind to the same target cell. Such marker expression is indicated by the superscript "plus" (+) following the name of the marker, e.g. CD4⁺。

In the present specification, the term negative when used in the context of marker expression refers to the expression of antigen as determined by a fluorescently labeled antibody wherein the median fluorescence intensity of the fluorescence is not higher than 130%, in particular not higher than 115%, of the fluorescence intensity obtained after staining with an isotype-matched antibody that does not specifically bind to the same target cell. Such marker expression is indicated by a superscript minus (-) following the marker name, e.g., CD127^-。

In the context of the present specification, the term nucleic acid expression vector relates to a plasmid or a viral genome, which is used to transfect (in the case of a plasmid) or transduce (in the case of a viral genome) a target cell carrying a certain gene of interest in a certain way. The gene of interest is under the control of a promoter sequence, and the promoter sequence is operable within the target cell, so that the gene of interest can be constitutive or in response to stimulation of transcription, or depending on the state of the cell. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector capable of transducing a target cell.

Disclosure of Invention

In its broadest sense, the present invention relates to a method of treating cancer in which TCR sequences isolated from T cells reactive with cancer cells expressing MR1 (MR1T cells) are expressed following gene transfer into a T cell population of a patient. These exogenous, transgene-expressed TCR sequences are used to confer T cells with the ability to specifically recognize MR 1-expressing cancer cells as a treatment for patient tumors.

The invention similarly provides T cells and T cell preparations comprising a plurality of T cells transduced with an MR1T cell-specific TCR gene. In certain embodiments, T cells transduced with the MR1T cell TCR gene can be used in adoptive cellular immunotherapy in combination with other therapeutic interventions.

The invention also relates to a method for preparing tumor-infiltrating T-cells from the same cancer tissue biopsy according to our previously established protocol (De Libero, supra). Individual T cell clones were tested against a tumor cell line expressing MR1 protein. The MR1 restriction, tumor lethality, and release of inflammatory cytokines were studied for the most reactive T cell clones. The TCR genes of selected T cell clones were sequenced.

Drawings

FIG. 1 MR1T cells do not recognize microbial antigens. (A) Surface expression of MR1 by CCRFSB, THP-1 and A375-MR1 cells. Grey histograms indicate staining with isotype matched control mAb. (B) MR1T cell clone DGB129 or (C) MAIT cell clone SMC3 were stimulated in A by three cell lines in the absence (no Ag) or in the presence of E.coli lysate (E.coli) and/or anti-MR 1 blocking mAb (. alpha. -MR 1). MAIT clone SMC3 was previously isolated from PBMCs of healthy donors and expressed the classical MAIT phenotype and function. Bars represent IFN-. gamma.release (mean. + -. SD). THP-1 cells stimulated (D) DGB129 MR1T or (E) SMC3 MAIT cells, structurally expressed surface MR1, loaded with synthetic 6, 7-dimethyl-8-D-ribosyloxazine (RL-6, 7-dimer) and with or without anti-MR 1 mAb. Bars represent mean IFN-. gamma.release + SD. Data represent four (A, B and C), two (D and E). P <0.05 (unpaired student T-test).

FIG. 2. strategy for isolation of MR1T cell clones from peripheral T cells. (A) FACS analysis of purified T cells pre-expanded with irradiated A375-MR1 cells after overnight co-culture with A375-MR1 cells in the absence of foreign antigen. The left dot plot shows CD3 in live cells and the cells follow purple (CTV) staining. Right dot plots show CD69 and CD137 expression of CD3 positive CTV negative gated cells. Arrows indicate gating hierarchies. Number ofWords indicate the percentage of cells within the gate. Cells from donor a are shown as representative donors. (B, D) cumulative results of T cell clones screened from donors A and B. As shown in a, from CD3⁺CTV^-CD137⁺Sorted T cells produced T cell clones. The graph shows individual clones (x-axis) and their IFN- γ release (y-axis) as a ratio between the amounts of cytokines secreted in response to a375-MR1 cells versus a375 WT cells. Each dot represents a single T cell clone, tested simultaneously under the indicated experimental conditions. The vertical lines indicate the number of T cell clones showing MR 1-restricted reactivity (i.e. clones showing IFN- γ release rate above an arbitrary cut-off value of 2). Results are representative of two independent experiments. (C, E) IFN-. gamma.was released by donor A and 14 representative clones from 11 clones of donor B after stimulation with A375 WT, A375-MR1 and A375-MR1 in the presence of blocking anti-MR 1mAb (. alpha. -MR 1). Dots represent IFN-. gamma.release from each clone (mean. + -. SD of replicate cultures). Results are representative of three independent experiments. P<0.05 (unpaired Student T test).

FIG. 3 MR1T cells are common in the blood of healthy individuals. (A) Flow cytometry analysis of purified T cells from representative donors (donor C) after overnight co-culture with a375 WT or a375-MR1 cells. Dot plot live CD3⁺CD69 and CD137 expression on cells. Numbers indicate the percentage of cells in the gate. (B) CD69 from 5 different donors after overnight co-culture with A375 WT or A375-MR1 cells⁺CD137⁺Frequency of T cells. (C) Cumulative results of T cell clone stimulation assays from donor C. From CD3⁺CD69⁺CD137⁺Sorted T cells produced T cell clones as shown in the right dot plot. The figure shows the number of test clones (x-axis) and IFN- γ release (y-axis), expressed as the ratio between the amount of cytokines secreted in response to a375-MR1 cells versus a375 WT cells. Each dot represents a single T cell clone, tested simultaneously under the indicated experimental conditions. The vertical lines indicate the number of T cell clones showing MR 1-restricted reactivity (i.e. clones showing IFN- γ release rate above an arbitrary cut-off value of 2). Results are representative of two independent experiments. (D) 8 representative MR 1-restricted T cell clones from donor C in the absence of foreign antigenA375-MR1 was identified but A375 WT cells were not identified. The reactivity of T cell clones to A375-MR1 cells was inhibited by blocking anti-MR 1mAb (α -MR 1). Dots represent IFN- γ release (mean ± SD of replicate cultures) for each clone tested under three experimental conditions. Results are representative of three independent experiments. P<0.05 (unpaired Student T test).

FIG. 4 MR1T TCR gene transfer confers MR1 restriction recognition on A375 cells. A375 cells expressing (A375-MR1) and lacking (A375 WT) MR1 stimulated (A) SKW-3 cells expressing DGB129 TCR (SKW3-DGB129) and (B) J.RT3-T3.5 cells expressing MAIT MRC25 TCR (J.RT3-MAIT), in the presence or absence of E.coli lysate and anti-MR 1mAb, respectively. The TCR-expressing SKW-3 cells were stimulated with A375-MR1 or A375 WT cells by three separate MR1T cell clones (C) DGA4(SKW3-DGA4), (D) DGB70(SKW3-DGB70), and (E) JMA (SKW3-JMA), in the presence or absence of anti-MR 1 mAb. The Median Fluorescence Intensity (MFI) + SD for CD69 of replicate cultures of transduced T cells is shown. CD69 MFI of transduced T cells cultured in the absence of APC is also shown. Mock transduced T cells showed background levels of CD69 expression when incubated with a375-MR1 or a375 WT (not shown). Data are representative of three independent experiments. P <0.05 (unpaired Student T-test).

FIG. 5 differential identification of various types of tumor cells by MR1T cell clone. (A) Four human cell lines expressing constitutive surface levels of MR1 were identified by representative SMC3 MAIT cell clones in the absence (no Ag) or in the presence of e.coli lysate (e.coli) with or without anti-MR 1 blocking mAb (α -MR 1). (B) The same cell type as in a was recognized by 13 MR1T cell clones with or without anti-MR 1mAb (α -MR 1). The graph shows IFN-. gamma.release (mean. + -. SD of replicate cultures).

FIG. 6 MR1T cell clone did not react with microbial ligands or 6-FP. (A) Reaction of 7 MR1T cell clones and one control MAIT cell clone in coculture with cells expressing (A375-MR1) or without A375 (A375 WT) MR1 in the presence or absence of E.coli lysate. Blocking of T cell clonal reactivity by anti-MR 1mAb (α -MR1) is also shown. (B) Response of MR1T cell clones to A375 cells expressing WT MR1 molecules (A375-MR1) or K43A mutated MR1 molecules (A375-MR1K43A) in the presence of 6-formylpterin (6-FP). (C) Control MAIT cell clone MRC25 or control TCR V γ 9V δ 2 clone G2B9 was stimulated with A375-MR1 or A375-MR1K43A cells, which were previously incubated with E.coli lysate or zoledronate in the absence or presence of 6-FP, respectively. Results are expressed as mean + -SD of IFN- γ measured in duplicate cultures. Results are representative of three independent experiments. P <0.05 (unpaired Student T-test).

FIG. 7 MR1T clone did not recognize Ac-6-FP. (A) Three representative MR1T cell clones were stimulated by A375-MR1 cells in the absence or presence of acetyl-6-formylpterin (Ac-6-FP). (B) Two MAIT cell clones (MRC25 and SMC3) were stimulated with E.coli lysate pulsed A375-MR1 cells in the absence or presence of Ac-6-FP. (C) A375-MR1 cells were treated with zoledronate (Zol) in the absence or presence of Ac-6-FP (25. mu.g/ml) and used to stimulate a TCRV γ 9-V δ 2 cell clone (G2B 9). (D) Three MR1T cell clones shown in A were stimulated with A375 cells expressing a K43A mutant MR1 molecule (A375-MR1K43A) in the absence or presence of Ac-6-FP (25. mu.g/ml). (E) Two MAIT cell clones used in B were stimulated with E.coli lysate pulsed A375-MR1K43A cells in the absence or presence of Ac-6-FP (25. mu.g/ml). Results are expressed as mean ± SD of IFN- γ release assessed in duplicate cultures and are representative of three independent experiments. P <0.05 (unpaired Student T-test).

FIG. 8 MR1T cells recognized antigens present in tumor cells, but not derived from RPMI 1640 medium. Overexpression of (A) A375 cells (A375-MR1) and (B) THP-1 cells (THP1-MR1) by MR1 growth in RPMI 1640 or PBS for 4 days stimulated the DGB129 MR1T cell clone, both supplemented with 5% human serum. The inhibition of T cell clonal reactivity by the anti-MR 1 blocking mAb (α -MR1) is shown. DGB129 cells recognized a load of APC isolates loaded from (C) THP-1 cell lysate or from (D) mouse mammary tumor EMT6 grown in vivo. Isolates E1 and E2 contained hydrophobic molecules; isolates N1 to N4 contained hydrophilic molecules. (E) DGB70 MR1T cells were reacted with N3 isolate of THP-1 lysate. (F) DGB129 and DGB70T cells were stimulated by THP-1 derived isolates N3 and N4 loaded onto plastic bound recombinant MR 1. Mean values of IFN- γ or GM-CSF + -SD T cell release for replicate cultures are shown (representing three independent experiments). Total cytokine release is shown in group a, group B, group F; fold increase over background is shown in groups C, D, E. P <0.05 (unpaired Student T-test).

FIG. 9 MR1T cells showed different anti-tumor responses. Tumor cell lines THP-1 and a375 expressing MR1 and MR1T cell clones (a) DGB129 or (B) DGB70 were cloned with the indicated effectors: the targeting (E: T) ratio was incubated overnight. The graph shows the percentage of apoptotic target cells under various experimental conditions, assessed by flow cytometry using annexin V and propidium iodide staining. MR1T cells were identified by staining with anti-CD 3 mAb and excluded from the analysis. Inhibition of the killing ability of MR1T cell clones by anti-MR 1(α -MR1) mAb was also reported as 1: 1E: the T scale shows. (C) Identification of Mo-DCs isolated from healthy individuals by 13 MR1T cell clones with or without anti-MR 1mAb (. alpha. -MR 1). The graph shows IFN-. gamma.release (mean. + -. SD of replicate cultures). (D) Mo-DCs from three donors were recognized by representative DGB129 MR1T cell clones in the absence or presence of anti-MR 1(α -MR1) mAb. IFN- γ release in the supernatant is shown and expressed as mean. + -. SD. (E) Flow cytometry analysis of co-stimulatory molecules CD83 and CD86 on Mo-DC after co-culture with DGB129 MR1T cells with or without anti-MR 1mAb (α -MR 1). A control group consisting of Mo-DC stimulated with LPS (10ng/ml) in the absence of T cells is also shown. The numbers represent the percentage of cells in each quadrant. (F) JMAN MR1T cell clones were stimulated by LS174T and HCT116 gastrointestinal tumor cell lines and normal intestinal epithelial cells (GEC) in the presence or absence of anti-MR 1mAb (. alpha. -MR 1). The column shows IFN-. gamma.release (mean. + -. SD of replicate cultures). All results are representative of at least three independent experiments. P <0.05 (t-test of unpaired students).

FIG. 10 functional diversity of MR1T cell clones. (A) IFN- γ released from clones of 7 selected MR1T cells stimulated with A375-MR1 cells. ELISA results are expressed as mean + -SD of IFN-. gamma.release measured in duplicate cultures. (B) Additional 16 cytokines were analyzed by performing multiple cytokine assays on the same supernatant, with IFN- γ shown in a. Results are representative of two independent experiments.

FIG. 11 MR1T cell clones showed multiple chemokine receptor expression profiles. Surface expression of CXCR3, CCR4 and CCR6 was analyzed by flow cytometry through seven selected resting MR1T cell clones. The graph shows the relative fluorescence intensity calculated by dividing the Median Fluorescence Intensity (MFI) of a particular mAb staining by the MFI of the corresponding isotype control. Data are representative of two independent experiments.

Figure 12 MR1T cells reduced the number of human melanoma lung nodules in mice. Immunocompromised NSG mice were injected with human melanoma a375 cells expressing MR1(a375-MR1) and MR1T cells. On day 14, mice were sacrificed and lung nodules were counted after indian ink perfusion.

P <0.0001 (unpaired Student T test).

Examples of the invention

Method

A cell. The following human cell lines were obtained from the american type culture collection: a375 (melanoma), THP-1 (myelomonocytic leukemia), J.RT3-T3.5(TCR β deficient T cell leukemia), LS174T (colon adenocarcinoma), HCT116 (colon cancer), Huh7 (hepatocellular carcinoma), HEK 293 (human embryonic kidney), and CCRF-SB (acute B cell lymphoblastic leukemia). SKW-3 cells (TCR alpha, beta, gamma and delta gene deficient human T cell leukemia) were obtained from the Lei-Bl research institute DSMZ-German Collection of microorganisms and cell cultures. Two representative MAIT clones (MRC25 and SMC3) and one TCR γ δ clone (G2B9) ((Gober et al, The Journal of experimental media 197,163-^TMHuman T cell enrichment assayThe kit, StemCell), was stimulated once a week with radiation (80 grey scale) a375-MR1 cells (ratio 2:1) for three weeks. Human rIL-2 (5U/ml; Hoffmann-La Roche), rIL-7 and rIL-15 (both 5ng/ml, Peprotech) were added on days +2 and +5 after each stimulation. 12 days after the last stimulation, cells were washed and co-cultured overnight with A375-MR1 cells (ratio 2: 1). CD3+ CD69+ CD37+ cells were then plated on PHA (1. mu.g/ml, Wellcome Research Laboratories), human rIL-2(100U/ml, Hoffmann-La Roche) and irradiated PBMC (5X 10)⁵Cells/ml) were sorted and cloned by limiting dilution. In other experiments, CD3 was used with sorting⁺CD69⁺CD137⁺In the same protocol, a single overnight stimulation with A375-MR1 cells (ratio 2:1) yielded a clone of MR1T cells. T cell clones were restimulated periodically following the same protocol (Lepore et al, supra). Purification of monocytes and B cells from PBMCs of healthy donors using EasySep human CD14 and CD19 positive selection kit (Stemcell Technologies) according to the manufacturer's instructions (r) ((r))>90% purity). Mo-DCs were combined with purified CD14 by culturing in the presence of GM-CSF and IL-4 as described previously (Lepore et al, supra)⁺Monocyte differentiation. Human normal intestinal epithelial cells (GEC) were isolated from intestinal biopsies of tumor-free individuals according to published protocols (Graves et al, Journal of immunological methods 414,20-31 (2014)).

Production of cells expressing the MR1A gene covalently linked to. beta.2m. The human MR1A cDNA construct linked to β 2m via a flexible Gly-Ser linker was generated by PCR as described previously (Lepore et al, supra). The K43A substitution in MR1A cDNA was introduced into the fusion construct using the following primers: MR1K43A _ f5'-CTCGGCAGGCCGAGCCACGGGC (SEQ ID NO 097) and MR1K43A _ r5' GCCCGTGGCTCGGCCTGCCGAG (SEQ ID NO 098). The resulting WT and mutant constructs were cloned into a bidirectional Lentiviral Vector (LV) (Lepore et al, supra). HEK 293 cells were transfected with the LV-MR 1A-. beta.2m construct alone, together with lentiviral packaging plasmids pMD2.G, pMDLg/pRRE and pRSV-REV (Addgene), using Metactene Pro (Biontex) according to the manufacturer's instructions. A375 and THP-1 cells were transduced by spin infection of the supernatant containing the viral particles in the presence of 8. mu.g/ml protamine sulfate. Surface expression of MR1 was assessed by flow cytometry and positive cells were FACS sorted.

Soluble recombinant β 2m-MR1-Fc fusion protein. The β 2m-MR1-Fc fusion construct was obtained using the human MR1A- β 2m construct described above as a template. Using primers: β 2mXhoI _ f5'-CTCGAGATGTCTCGCTCCGTGGCCTTA (SEQ ID NO 099) and MR1-IgG1_ r5' -GTGTGAGTTTTGTCGCTAGCCTGGGGGACCTG (SEQ ID NO 100), DNA complementary to the β 2m-MR1A gene was amplified by PCR, thereby excluding the MR1 transmembrane and intracellular domains. DNA complementary to the hinge region and the CH2-CH3 domain of the heavy chain of human IgG1 was generated using the following primers: NheI-hinge-f5'-CAGGTCCCCCAGGCTAGCGACAAAACTCACAC (SEQ ID NO 101) and IgG1NotI _ r5' -GCGGCCGCTCATTTACCCGGAGACAGGGAGA (SEQ ID NO 102) pFUSE-hIgG1-Fc1 (InvivoGen). The β 2m-MR1A and IgG1 PCR products were ligated together using two-step splicing with overlap extension PCR, and the resulting construct was subcloned into the XhoI/NotI site of the BCMGSNeo expression vector. CHO-K1 cells were transfected with the final construct using metaflecene Pro (Biotex), cloned by limiting dilution and screened by ELISA to generate β 2m-MR1-Fc fusion protein. Selected clones suitable for EX-CELL acfcho serum-free medium (Sigma) were used for Protein production and β 2m-MR1-Fc was purified using Protein-a-sepharose (thermo Fisher scientific) according to the manufacturer's instructions. Protein integrity and purity was verified by SDS-PAGE and Western blotting using anti-MR 1mAb 25.6 (Biolegend).

Flow cytometry and antibodies. Cell surface labeling was performed using standard protocols. Use of True-Nuclear according to the manufacturer's instructions^TMThe transcription factor buffer set was labeled intracellularly. The following anti-human mabs were obtained from Biolegend: CD4-APC (OKT4), CD8 a-PE (TuGh4), CD161-Alexa Fluor 647(HP-3G10), CD69-PE (FN50), CD3-PE/Cy7, Leu Violet-711, or Alexa-700(UCHT1), CD 137-biotin (n4b4-1), CXCR 3-Leu Violet 421(G025H7), CD 83-biotin (HB15e), MR1-PE (26.5), and TRAV1-2-PE (10C 3). CD86-FITC (2331), CCR4-PECy7(1G1) and CCR6-PE (11A9) mAbs were from BD Pharmingen. All of these mAbs were used at 5. mu.g/ml. Biotinylated mAb was visualized with streptavidin-PE, -fluorophore 488, or-brilliant violet 421 (2. mu.g/ml, Biolegend). In LSR Fortessa flow cytometrySamples were obtained on a meter (Becton Dickinson). Cell sorting experiments were performed using an Influx instrument (Becton Dickinson). Dead cells and doublets were excluded based on forward scatter area and width/side scatter and DAPI staining. All data were analyzed using FlowJo software (TreeStar).

TCR gene analysis of MR1T cell clones. TCR α and β or TCR γ and δ expression of genes of MR1T cell clones were assessed by RT-PCR using total cDNA and specific primers, or by using monoclonal antibodies specific for the pan γ δ TCR (B1, Biolegend) according to the manufacturer's instructions or pan γ δ TCR (Biolegend)Flow cytometry with the BetaMark TCRV β Repeartoire kit (Beckman Coulter). For RT-PCR, RNA was prepared using the NucleoSpin RNA II kit (Macherey Nagel) and cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). TCR α, β, γ and δ cdnas were amplified using V α, V β, V γ and V δ set primers according to the manufacturer's instructions (TCR typing amplification kit, Clontech). Functional transcripts were identified by sequencing and then analyzed using the ImmunoGeneTiCs information System (http:// www.imgt.org).

TCR gene transfer. The TCR α and β functional cDNAs from MAIT cell clone MRC25 were cloned into the XhoI/NotI site of the BCMGSNeo expression vector (Karasuyama and Melchers Eur.J.Immunol.198818: 97-104) and the resulting constructs were used to co-transfect J.RT3-T3.5 cells by electroporation according to standard procedures. Transfectants expressing TRAV1-2 and CD3 were FACS sorted. TCR α and β or TCR γ and δ functional cdnas from the MR1T clone were cloned into XmaI/BamHI sites of a modified version of the plasmid 52962(Addgene) expression vector. SKW-3 cells were transduced with the virus particle-containing supernatant produced as described above. FACS sorting of cells based on CD3 expression.

Isolation of cells and whole tumor lysate. Disrupted in water by mild sonication, from 2.5X 10⁹A single pellet of THP-1 cells produced a total cell lysate. The sonicated mass was then centrifuged (15,000g, at 4 ℃ for 15 minutes), and the supernatant was collected (S1). Next, the precipitate was resuspended in methanolSonicated, centrifuged as before, and the resulting supernatant combined with the S1 supernatant. The final concentration of methanol was 10%. The total cell extract was then applied to a C18 Sep-Pak column (Waters Corporation) and the unbound material was collected and dried (E-FT isolate). Bound material was eluted in portions with 75% methanol (isolate E1) and 100% methanol (isolate E2). E-FT material was resuspended in acetonitrile/water (9: 1 vol/vol) and loaded into NH₂Sep-Pak column (Waters Corporation). The unbound material (isolate N-FT) and 4 additional isolates were eluted with increasing amounts of water. Isolate N1 was washed with 35% H₂O elution, isolate N2 with 60% H₂O elution, isolate N3 with 100% H₂O elution and isolate N4 with 100% H₂O and 50mM ammonium acetate (pH 7.0). All isolates were dried and then resuspended in 20% methanol (isolates E1, E2 and N-FT) or 100% H before storage at-70 deg.C₂O (all other isolates).

Mouse EMT6 breast tumors were prepared as described in this document (Zippelius et al, Cancer Immunol Res 3,236-244 (2015)). Freshly excised tumors were washed thoroughly in saline, weighed, and homogenized in 7ml HPLC grade water using a Dounce tissue grinder for 4g mass. Tumor homogenates were subjected to two freeze-thaw cycles, centrifuged (3,250g) at 4 ℃ for 10 minutes, and the supernatants collected and stored at-70 ℃. The precipitate was extracted a second time with 2ml HPLC grade water, centrifuged (5,100g) at 4 ℃ for 10 minutes, the supernatant collected and stored at-70 ℃. The precipitate was further extracted with 9ml of HPLC grade methanol at room temperature for 5 minutes by vortexing, centrifuged (5,100g) at 4 ℃ for 10 minutes, and the supernatant was collected. The three supernatants were combined, dried and resuspended in water: methanol (10: 1). C18 and NH as described above₂Sep-Pak column separates the material.

T cell activation assay. MR1 restricted T cells (5X 10)⁴Perwell, unless otherwise indicated) with the indicated target cells (5X 10)⁴Per well) were co-cultured in duplicate or triplicate in a total volume of 200 μ l. T cells were cultured with the indicated APCs for 24 hours. In some experiments, anti-MR 1mAb (clone 26.5) or mouse IgG2a isotype control mAb (both 30. mu.g/ml) were added) And incubated for 30 minutes before addition of T cells. Coli lysates were prepared from DH5 α strain (Invitrogen) grown in LB medium and collected during exponential growth. Bacterial cells were washed twice in PBS and then lysed by sonication. After centrifugation (15,000g for 15 minutes), the supernatant was collected, dried, and stored at-70 ℃. Before the addition of T cells, the amount of 10 is equivalent⁸CFU/ml (unless otherwise stated) of E.coli lysate APC pulse for 4 hours. In some experiments, APC were preincubated with 6-FP or Ac-6-FP (Schircks laboratories)) for 4 hours prior to co-culture with T cells. In a control experiment with TCR γ δ cells expressing TCR γ 9 and V δ 2 chains, the APC was first treated with zoledronate (10 μ g/ml) for 6 hours prior to addition of T cells. Activation experiments were performed by coating β 2m-MR1-Fc on 96-well plates (4 μ g/ml) and loading the column purified cell lysate with plate-bound recombinant human β 2m-MR1-Fc at 37 ℃ for 4 hours, followed by two washes and addition of T cells. Supernatants were collected after 24 hours and evaluated for IFN-. gamma.or GM-CSF by ELISA. The various cytokines and chemokines in the cell culture supernatants were used according to the manufacturer's instructions with Millipox MAP human cytokine/chemokine magnetic bead plate-premix 41plex (HCYTMAG-60K-PX 41; Merck Millipore). Samples were obtained on a Flexmap 3D system (Merck Millipore) and mean fluorescence intensity and analyte concentration were determined using Milliplex analysis software.

Killing tumor cells. The target cell line (2X 10) was used in the presence or absence of anti-MR 1mAb (30. mu.g/ml, clone 26.5)⁴Cells/ml) were subjected to killing assays, and the target cell lines were incubated for 24 hours, either alone or with T cells of different E/T ratios. Target cells were stained with PE-annexin v (bd) and Propidium Iodide (PI) (Sigma-Aldrich) as previously described (2). T cells were identified by staining with anti-CD 3 mAb and excluded from the assay. Apoptosis was assessed as follows: annexin V⁺PI⁺Late apoptosis and annexin V^-PI⁺Necrosis. The percentage of apoptotic + necrotic cells in the absence of T cells (spontaneous apoptosis; no T cells) is also shown.

And (5) counting the value. Data were analyzed using unpaired Student T-test (Prism 6, GraphPad software).

Identification and characterization of novel tumor-reactive MR 1-restricted T cells in healthy donors

The inventors detected atypical MR 1-restricted T cell clones that did not react with microbial ligands during early studies of the human MAIT cell bank. This T cell clone (DGB129) recognized a cell line constitutively displaying surface MR1 (CCRF-SB lymphocytic leukemia cells, or THP-1 monocytic leukemia cells; FIG. 1A) or transfected with the MR1 gene (A375 melanoma cells; A375-MR 1; FIG. 1A), in the absence of any exogenously added antigen (FIG. 1B). The sterile recognition of MR1+ target cells was completely inhibited by blocking with anti-MR 1 monoclonal antibody (mAb) (fig. 1B), thus resembling the response of MAIT cells to e.coli-derived antigens assessed in parallel (fig. 1C). Importantly, DGB129T cells also failed to recognize the synthetic MAIT cell agonist 6, 7-dimethyl-8-D-ribosyloxazine (RL-6, 7-diMe; FIG. 1D) as distinct from the control MAIT cell clone, which was stimulated by this compound in an MR 1-dependent manner (FIG. 1E). DGB129 cells do not express the classical semi-invariant TCR of classical MAIT cells (table 1).

The inventors investigated whether DGB129 clone represents a novel tumor-reactive MR 1-restricted T cell population distinct from microbial-reactive MAIT cells. Therefore, they established a method to isolate and study these unpredictable MR 1-restricted T cells. Purified T cells from two healthy donors were labeled with the proliferation marker CellTrace purple (CTV) and stimulated with irradiated a375-MR1 cells in the absence of exogenous antigen. Proliferating cells were again challenged with a375-MR1 cells and those expressing the high level activation marker CD137 were sorted and cloned by limiting dilution (fig. 2A). The ability of individual T cell clones to recognize A375-MR1 and A375 cells (A375-WT) lacking MR1 was then studied. Among the two donors, the inventors found that the majority of T cell clones (126/195 and 37/57, respectively) showed specific recognition of a375-MR1 cells (fig. 2B, panel D), which were inhibited by anti-MR 1 blocking mabs (fig. 2C, panel E). TCR V β specific mAb staining with 12 MR1 reactive T cell clones showed that they expressed 7 different TRBV chains (TRBV4-3, 6-5/6-6/6-9, 18, 25-1, 28, 29-1) some of which share the same TRBV gene. Furthermore, none of the expressed TRAV1-2 chains was classical for MAIT cells.

The lack of specific markers makes it difficult to identify these novel T cells individually in vitro by standard flow cytometry. Thus, their frequency was estimated by combining flow cytometry analysis after very short in vitro stimulation and single T cell cloning experiments. Purified blood T cells from five healthy donors were co-cultured overnight with MR 1-deficient or MR 1-sufficient a375 cells and analyzed for expression of the activation markers CD69 and CD137 (fig. 3A). Of all five donors screened, CD69 detected after stimulation with A375-MR1 cells (ranging from 0.034 to 0.072% of T cells)⁺CD137⁺The percentage of T cells was consistently higher than after co-culture with A375-WT cells (ranging from 0.015 to 0.032%) (FIGS. 3A, B). Since the two types of APCs express differently to MR1, MR1 reactive T cells resulted in an increase in the number of activated T cells after stimulation with MR1 positive APCs. Using this method, the inventors estimated that the circulating T cell pool of the analyzed individuals contained a375-MR1 reactive T cells, with a frequency range of 1: 2500(0.072-0.032 ═ 0.04%) and 1: 5000(0.034-0.015 ═ 0.019). This estimated frequency is higher than the peptide-specific CD4 after antigen exposure⁺Frequency of T cells (Lucas et al, J Virol 78: 7284-. These observations were supported by parallel experiments in which sorted CD69 from one of these donors (donor C, fig. 3A, right panel) was cloned⁺CD137⁺Overnight activated T cells. Indeed, 31 of the 96 screened T cell clones (32%) showed specific reactivity to a375-MR1 cells (fig. 3C), which were inhibited by anti-MR 1mAb (fig. 3D). Thus, the frequency of a375-MR 1-reactive T cells calculated in the donor's blood T cells was 1: 5000(0.065 × 0.32 ═ 0.02%), which agrees with the estimated range. Detailed analysis of representative T cell clones from three donors confirmed that they displayed different TCR α and β chains and indicated differential expression of CD4, CD8 and CD161 (table 1).

Taken together, these findings indicate that the identified tumor-reactive MR 1-restricted T cells are a new but common polyclonal population of lymphocytes in the blood of healthy human individuals (hereinafter referred to as MR1T cells).

MR1T cell TCR Gene transfer conferring MR1 restricted tumor cell recognition

The inventors next investigated whether the reactivity of MR1T cells to tumor cells was mediated by TCR. Paired TCR α and β genes from different MR1T cell clones were cloned and expressed in TCR-deficient SKW-3 cells, conferring recognition ability of MR1 on tumor cells, similar to MR1 recognition shown by the original MR1T cells, and were completely blocked by anti-MR 1-mAb (fig. 4A-panel C). In control experiments, the transfer of the TCR α and β genes of representative MAIT cell clones conferred the ability to recognize a375-MR1 cells in an MR 1-dependent manner only in the presence of e.coli antigen (fig. 4D). These data highlight the key role of TCRs in mediating MR1T cell recognition of tumor cells and indicate that MR1T cell TCR gene transfer can effectively redirect the reactivity of selected T cells to MR 1-expressing tumor cells.

Differential identification of MR1T cell clone on tumor cells

Having generated a large number of MR1T cell clones reactive with a375 melanoma cells expressing MR1, the inventors next investigated whether they could also recognize other types of tumor cells constitutively expressing surface MR1, including THP-1 myelomonocytic cells, Huh7 hepatoma cells, HCT116 colon cancer cells and LS174T goblet colon adenocarcinoma cells. All these cell types supported MAIT cell activation in an MR 1-dependent manner in the presence of microbial antigens (fig. 5A). The same cells were able to induce sterile activation of selected MR1T cell clones to varying degrees. Most of the tested MR1T cell clones identified THP-1 cells followed by Huh7 hepatoma cells, LS174T goblet cells and HCT116 colon cancer cells (fig. 5B). Importantly, all reactions were blocked by anti-MR 1 mAb.

These data further demonstrate that MR1T cells are novel and diverse cells limited to tumor-reactive T cells of the non-polymorphic antigen presenting molecule MR 1.

MR1T cell recognitionMR 1-binding antigens present in other tumor cells

The inventors next investigated the basis of the reactivity of MR1T cells to tumor cells. First, they sought to specifically exclude the possibility that the MR1T cell clone could recognize microbial antigens, similar to MAIT cells. Whereas the control MAIT cell clone reacted with a375-MR1 cells only in the presence of e.coli lysate, the activation of the different MR1T cell clone was not enhanced by e.coli lysate (fig. 6A). Consistent with these data, MR 1-negative a375-WT cells failed to stimulate any type of T cells, regardless of the addition of e.coli lysate (fig. 6A), and it is important that the anti-MR 1mAb effectively blocks both MR1T and MAIT cell responses (fig. 6A). These findings confirm that the microbial ligand present in E.coli and stimulating MAIT cells does not stimulate the tested MR1T cells.

The inventors then tested the response of MR1T cells to the known MR1 ligands 6-FP and Ac-6-FP, which have previously been reported to stimulate a rare subset of TRAV1-2 negative T cells and inhibit MAIT cell activation by microbial antigens. Stimulation of MR1T cells was impaired in the presence of 6-FP or Ac-6-FP ligands, which also impaired E.coli stimulation of control MAIT cells, but did not disrupt the control TCR γ δ cell response to the cognate antigen presented by the same APC, thus precluding compound toxicity (FIG. 6B, panel C, and FIGS. 7A-C). Notably, when the target A375 cells were transcribed to express the mutant MR1 molecule with defective ligand binding capacity, 6-FP or Ac-6-FP failed to inhibit the activation of MR1T cells or MAIT cells (A375-MR1K 34A with ligand blocking Schiff base formation by lysine 43 mutation to alanine; FIG. 6B, FIG. C and FIG. 7D, FIG. E). The specific inhibition observed with 6-FP or Ac-6-FP indicates that MR1T cells i) do not recognize 6-FP and Ac-6-FP; ii) reacting with a cellular antigen bound by MR 1; and iii) by ligands that do not require the formation of Schiff bases with MR 1.

To obtain further information on the origin of the identified antigens, the inventors investigated whether the stimulatory capacity of tumor target cells is dependent on the media composition, since some MR1 ligands, e.g. 6-FP, may be derived from folate present in RPMI 1640 media used for cell culture. THP-1 and A375-MR1 cells were washed thoroughly and cultured in Phosphate Buffered Saline (PBS) supplemented with 5% human serum only for 4 days. Cells were washed daily and then used to stimulate DGB129 MR1T cells and T cell activation assays were performed in PBS. THP-1 and A375-MR1 cells grown in RPMI 1640 or PBS showed the same stimulatory capacity (FIG. 8A, panel B), thus indicating that the media components did not activate MR1T cells. To directly investigate whether stimulatory antigens are present in the target tumor cells, the inventors then performed T cell activation assays using both types of tumor lysates as antigen sources. The first lysate was obtained from THP-1 cells cultured in vitro, while the second lysate was prepared from mouse mammary tumors immediately after resection. Two hydrophobic and four hydrophilic isolates were obtained and tested using THP-1 cells constitutively expressing low levels of MR1 as APCs. The DGB129 clone reacted only with N4 isolate, which contained highly hydrophilic compounds isolated from both freshly transplanted mouse tumors and in vitro cultured THP-1 cells (fig. 8C, fig. D). These results exclude the possibility of the stimulatory antigen being derived from the RPMI 1640 component and suggest a cellular source thereof. The inventors also tested the isolates produced by THP-1 lysate with another representative MR1T cell clone, DGB 70. DGB70 cells recognized isolate N3 but not isolate N4 (fig. 8E), indicating that at least two different complexes differentially stimulated two MR1T clones. The same isolate was loaded onto a plastic-bound MR1 molecule and showed alternative and specific stimulatory capacity, i.e. N3 stimulated only DGB70 cells, while N4 stimulated only DGB129 cells (fig. 8F). In the absence of N3 and N4 isolates, both clones did not react with MR1, further indicating the need for specific antigens.

Taken together, these data indicate that MR1T cells recognize MR1 complexed with ligands that are not derived from culture media, and are also present in tumor cells grown in vivo.

MR1T cells showed different antitumor responses

To evaluate the antitumor activity of MR1T cells, the inventors tested their ability to kill tumor cells directly in vitro. Two representative MR1T cell clones (DGB129 and DGB70) were identified with various effectors: the target ratio was effective to kill both THP-1 and a375 cells expressing MR1 (fig. 9A, panel B). The control MAIT cell clone failed to kill both cell types, although it was fully capable of killing when the target was infected with e. These results indicate that MR1T cells showed specific cytotoxic activity against MR1 expressing tumor cells.

It has been found that MR1T cells recognize and kill the myelomonocytic tumor cell line THP-1, and the inventors next addressed whether they could also recognize normal bone marrow cells, including monocytes and monocyte-derived dendritic cells (Mo-DC) from different donors. None of the tested MR1T cell clones identified monocytes (not shown). In contrast, some MR1T cell clones reacted with Mo-DC in an MR 1-dependent manner (fig. 9C). Interestingly, experiments with representative DGB129 MR1T cell clones showed that Mo-DC recognition did not result in Mo-DC killing (not shown), but rather promoted up-regulation of the CD83 and CD86 activation markers by Mo-DC (FIG. 9D). Notably, the anti-MR 1mAb completely inhibited the DGB129 cell-induced activation of Mo-DC (fig. 9D). These data indicate that some tumor-reactive MR1T cells elicit direct antitumor activity and also promote activation of innate immune cells, which is of great significance for establishing an effective antitumor immune response.

While the inventors observed that some of the MR1T cell clones reacted with HCT116 and LS174T intestinal tumor cells, they next investigated whether they could also recognize normal intestinal epithelial cells (GEC) prepared from intestinal biopsies. GEC cells did not stimulate any of the HCT116 or LS 174T-responsive MR1T cell clones tested (fig. 9F, panel G), thus indicating that MR1T cell clones may display specific recognition of gastrointestinal tumor cells without responding to normal intestinal epithelial cells.

To further assess the specificity of MR1T cells to recognize tumors, the inventors finally investigated whether they could react with other types of normal cells, including neutrophils, NK cells, B cells and T cells. None of these cells was identified by the MR1T cell tested (not shown).

Collectively, these data identified MR1T cells as novel and diverse cells of human MR 1-restricted T lymphocytes that i) respond differently to various types of tumor cells; ii) exhibits cytotoxic activity against tumor cells; iii) Mo-DCs that do not recognize normal cells-differentiated in vitro and iv) Mo-DCs are not killed but induced for their activity. These findings indicate that MR1T cells exhibit important anti-tumor properties and are worthy of development for their immunotherapeutic potential.

MR1T cells were functionally diverse

The inventors finally analyzed the cytokine secretion profile of a representative MR1T cell clone after stimulation with a375-MR1 tumor cells. All clones tested released IFN-. gamma. (FIG. 10A). However, the inventors also observed different expression profiles of Th1(IL-2, TNF-. alpha.and TNF-. beta.), Th2(IL-3, IL-4, IL-5, IL-6, IL-10, IL-13) and Th17 cytokines (IL-17A, G-CSF, GM-CSF) and other soluble factors (MIP-1. beta., soluble CD40L PDGF-AA and VEGF; FIG. 10B). The variable combination and amount of cytokines expressed by MR1T cells indicates that the cells have considerable functional plasticity. For example, clone DGA4 secreted large amounts of IL-17A, IL-6, TNF- α and GM-CSF, but failed to secrete the prototype Th2 cytokines IL-4, IL-5, IL-10 or IL-13, thus showing an 'atypical' Th 17-like phenotype. In contrast, clone TC5A87 released large amounts of VEGF and PGDF-AA, but only small amounts of either Th1 or Th2 cytokines, and no IL-17A. Notably, 4 of the 7 clones studied (DGB129, CH9a3, DGB70, JMA) showed Th 2-skewed features of cytokine release, a functional phenotype recently associated with protective anti-tumor immunity.

The inventors next investigated the expression of three selected chemokine receptors known to be differentially expressed by subsets of T cells with different functions, and their surrogate combinations express regulatory T cell recycling and migration to different homing sites. All MR1T cell clones showed high levels of CXCR3 except DGA4 (fig. 11). In addition, the inventors observed different expression patterns of CCR4 and CCR6 (fig. 11), further indicating that MR1T cells are diverse.

In the last series of studies, it was investigated whether MR1T cells maintained their tumor killing ability in vivo using a lung solid tumor model. Mice injected intravenously with a375 melanoma cells expressing MR1 received DGB129 cells or untreated. On day 14, mice were sacrificed and the number of tumor nodules in the lungs was counted. While untreated mice showed 200 to 250 nodules, mice treated with MR1T cells showed 1-6 nodules (fig. 12). These results demonstrate that tumor cells grown in vivo produce antigens that stimulate MR1T cells. Importantly, they provide strong evidence of the effective ability of MR1T cells to kill solid tumor cells in vivo.

Taken together, these data indicate that the tumor MR 1-reactive T clones tested here are phenotypically and functionally diverse, thus indicating that MR1T cells comprise multiple subgroups with different recirculation patterns and tissue homing capacity, and may have different roles in tumor immunity. Finally, these data identified MR1T cells as recognizing MR 1: tumor associated antigen complexes and may be involved in a new population of human T lymphocytes with anti-tumor immune responses with multiple effector functions.

TABLE 1 phenotype of selected MR1 reactive T cell clones.

Table 2. tumor cell lines recognized by human MR1T cells.

The following examples further illustrate the clinical workflow for applying the present invention:

screening for MR1 expressing cancers

Tissue fresh or fresh frozen tissue biopsies from cancer patients were analyzed for MR1 expression using PCR amplification of mAb specific for human MR1 and MR1 mRNA.

Cancer treatment, example 1:the optimal MRT1 TCR gene was selected for identifying cancer cells expressing primary MR 1.

I. Isolation of in vitro Primary MR1⁺Cancer cellUsed to stimulate a pool of previously characterized MR1T cell clones. Each clone expresses a different TCR gene and recognizes a different type of cancer cell.

Selecting the clone of MR1T cell that best responds to the patient's cancer cells and using its TCR gene for TCR gene therapy. The response is determined based on cytokine release and/or surface marker expression. Cells are assayed by staining with internal (cytokines) or surface markers using antibodies reactive to the assayed activation markers, such as, but not limited to, CD3, CD69, CD137, CD150, and/or ICOS (surface markers) and INF- γ and GM-CSF (cytokines).

When available soluble MR1T TCR will be multimerized and used to stain tumor cells isolated from tumor biopsies. The MR1T TCR multimer bound to tumor cells would allow for the rapid selection of MR1T TCRs suitable for gene therapy in this patient.

Several circulating patient T cell populations can be used as recipient T cells (naive, central, effector, CD 4)⁺、CD8⁺Or CD4, CD8 double negative T cells). Naive cells were selected that, when transduced with a TCR gene that recognizes MR1, a tumor antigen, allowed unlabeled T lymphocytes to mature in the presence of tumor cells. Central and effector memory cells were used because they provide immediate proliferation and effector functions (tumor killing) when recognizing MR1 expressing tumor cells. CD4 cells were selected to provide sufficient numbers of T helper cells that promote the growth and expansion of other cells with anti-tumor function. CD 8T cells were selected to promote killing of tumor cells. CD4 to CD8 double negative T cells were selected for their innate-like functions, such as immediate release of large amounts of killing effector molecules (TNF α, granzyme and granulysin).

V. expression of the transduced TCR gene and T cells with selected effector function for Adoptive Cell Therapy (ACT).

T cells from the patient's peripheral blood were stained with monoclonal antibodies specific for surface markers (CD4, CD8, CD27, CD45RA, CD57) and sorted. By usingHuman T-Activator CD3/CD28(ThermoFisher) activated each sorted population and was transfected 24 hours later with the TCR gene encoding the MR1T TCR selected for the individual patient. This results in a modified T cell preparation (recipient T cells). In some cases, recipient T cells are also modified by gene editing methods to inactivate the PD1, ILT2, and ILT4 suppressor genes or transduced with the CD137 and CD134 genes to promote cell survival, cell expansion, and enhance anti-cancer effector function.

Cancer patients with the receptor use a non-myeloablative chemotherapy preparation regimen (60mg/kg cyclophosphamide administration for 2 days; 2525mg/m²Fludarabine was administered for 5 days) for lymphocyte depletion, followed by transfer of T cells and IL-2 to tolerance 720,000 IU/kg. In some cases, 200 or 1200 centigray (cGy; 1Gy ═ 100 rads) total body irradiation is added to the preparation protocol. T cells expressing the exogenous TCR gene (modified T cell preparation) of MR1T were transferred into recipients.

The TCR genes were cloned into a safe recombinant lentiviral vector (see e.g. Provasi et al, Nat Med 18, 807-. In some cases, TCR genes are cloned into vectors containing suicide genes (see, e.g., Greco et al, Front Pharmacol 6,95(2015)), thereby reducing the risk caused by unwanted gene insertion. In some cases, RNA encoding the TCR MR1T gene is transfected in recipient cells (see, e.g., Zhao et al. molecular therapy 13,151,2006)).

Cancer therapy, example 2:MR1T cells were isolated from Tumor Infiltrating Lymphocytes (TILs) of the patient to be treated.

Autologous TIL was prepared from cancer tissue biopsies according to our previously established protocol (De Libero, supra).

Expanding T cells in vitro for 2 to 3 weeks using medium supplemented with IL-2, IL-7 and IL-15.

Testing of expanded T cells versus autologous MR1⁺Reactivity of cancer cells. T cells that increase the surface expression of activation markers (CD137, CD150, CD69, ICOS) are considered cancer specific if they are singly treated with anti-MR 1The presence of clonal antibodies was inhibited, they were considered MR1 dependent.

Cancer responsive T cells are sorted for expression of one of the activation markers described above, expanded, and used for ACT, as described above.

Sequence of

If there is a difference between the sequence contained in the page and the sequence scheme submitted in parallel in the form of a text file, the following sequence in this specification shall control.

Full-length TCR α and β protein sequences, including leader sequences.

CDR3 sequences are underlined

New clone 1 of SEQ ID 001 TCR alpha

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAQIYNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

New clone 2 of SEQ ID 003 TCR alpha

MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

New clone 3 of SEQ ID 005 TCR alpha

MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEEPSNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

New clone 1 of SEQ ID 002 TCR beta

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFSSGKQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

New clone 2 of SEQ ID 004 TCR beta

MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSDVGTGDTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

New clone 3 of SEQ ID 006 TCR beta

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSRLLAGGQNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Full-length TCR α and β protein sequences, including leader sequences.

Clone 4 of SEQ ID NO 013 TCR α

MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVMDSSYKLIFGSGTRLLVRPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 5 of SEQ ID NO 014 TCR alpha

MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAAAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 6 of SEQ ID NO 015 TCR α

MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAETWTDRGSTLGRLYFGRGTQLTVWPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 7 of SEQ ID NO 016 TCR α

MAMLLGASVLILWLQTDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASLYNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

SEQ ID NO 017 TCR alpha clone 8

MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASGDSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 9 of SEQ ID NO 018 TCR α

MNYSPGLVSLILLLLGRTRGNSVTQMEGPVTLSEEAFLTINCTYTATGYPSLFWYVQYPGEGLQLLLKATKADDKGSNKGFEATYRKETTSFHLEKGSVQVSDSAVYFCALTIWDYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 10 of SEQ ID NO 019 TCR α

MVLKFSVSILWIQLAWVSTQLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSLHITAAQPGDTGLYLCAGENSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 11 of SEQ ID NO 020 TCR alpha

MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSLSGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 12 of SEQ ID NO 021 TCR α

MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQLGGAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 13 of SEQ ID NO 022 TCR alpha

MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAANWSPQGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 14 of TCR α of SEQ ID NO 023

MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCASMDSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 15 of SEQ ID NO 024 TCR alpha

MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNRFTRDGNKLVFGAGTILRVKSYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Clone 4 of SEQ ID NO 025 TCR beta

MSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASSEVTGGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Clone 5 of TCR beta of SEQ ID NO 026

MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVGAGQGPYTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

SEQ ID NO 027 TCR beta clone 6

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLGATGANEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Clone 7 of TCR beta of SEQ ID NO 028

MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSYRGTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Clone 8 of SEQ ID NO 029 TCR β

MGPGLLCWVLLCLLGAGPVDAGVTQSPTHLIKTRGQHVTLRCSPISGHKSVSWYQQVLGQGPQFIFQYYEKEERGRGNFPDRFSARQFPNYSSELNVNALLLGDSALYLCASSFDVGLPPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Clone 9 of SEQ ID NO 030 TCR β

MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREWETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

SEQ ID NO 031 TCR beta clone 10

MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSQLYRDTSNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Clone 11 of TCR beta of SEQ ID NO 032

MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASGISGTASSYNSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

TCR beta clone 12 of SEQ ID NO 033

MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVGGGLADTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Clone 13 of SEQ ID NO 034 TCR β

MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEYIQYSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

SEQ ID NO 035 TCR beta clone 14

MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAKVTSGQHQGTTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Clone 15 of SEQ ID NO 036 TCR β

MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVEGRGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

Full-length TCR γ and δ protein sequences, including leader sequences.

Clone 1 of SEQ ID NO 061 TCR γ

MQWALAVLLAFLSPASQKSSNLEGRTKSVIRQTGSSAEITCDLAEGSTGYIHWYLHQEGKAPQRLLYYDSYTSSVVLESGISPGKYDTYGSTRKNLRMILRNLIENDSGVYYCATWETQELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS

SEQ ID NO 062 TCR delta clone 1

MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAVKSVALTISALQLEDSAKYFCALGVQALLPILGDTTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL

Table X assignment of sequence ID NO

Sequence listing

<110> university of Basel

<120> MR 1-restricted T cell receptor for cancer immunotherapy

<130> uz363wo

<160> 102

<170> PatentIn version 3.5

<210> 1

<211> 280

<212> PRT

<213> human

<400> 1

Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro

1 5 10 15

Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln

20 25 30

Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn

35 40 45

Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys

50 55 60

Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys

65 70 75 80

Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala

85 90 95

Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala

100 105 110

Val Tyr Phe Cys Ala Ala Gln Ile Tyr Asn Gln Gly Gly Lys Leu Ile

115 120 125

Phe Gly Gln Gly Thr Glu Leu Ser Val Lys Pro Asn Ile Gln Asn Pro

130 135 140

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

145 150 155 160

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

165 170 175

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg

180 185 190

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

195 200 205

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

210 215 220

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

225 230 235 240

Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val

245 250 255

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

260 265 270

Met Thr Leu Arg Leu Trp Ser Ser

275 280

<210> 2

<211> 309

<212> PRT

<213> human

<400> 2

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys

20 25 30

Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His

35 40 45

Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro

65 70 75 80

Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile

85 90 95

Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Ser Ser Gly Lys Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

115 120 125

Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

130 135 140

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

145 150 155 160

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

165 170 175

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro

180 185 190

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

225 230 235 240

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

290 295 300

Lys Asp Ser Arg Gly

305

<210> 3

<211> 269

<212> PRT

<213> human

<400> 3

Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser

1 5 10 15

Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe

20 25 30

Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn

35 40 45

Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu

50 55 60

Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg

65 70 75 80

Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile

85 90 95

Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Thr

100 105 110

Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val Ile Pro

115 120 125

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

130 135 140

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

145 150 155 160

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

165 170 175

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

180 185 190

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

195 200 205

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

210 215 220

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

225 230 235 240

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

245 250 255

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 4

<211> 313

<212> PRT

<213> human

<400> 4

Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr

1 5 10 15

Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr

20 25 30

Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His

35 40 45

Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser

65 70 75 80

Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser

85 90 95

Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr

100 105 110

Ser Asp Val Gly Thr Gly Asp Thr Gly Glu Leu Phe Phe Gly Glu Gly

115 120 125

Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 5

<211> 277

<212> PRT

<213> human

<400> 5

Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val

1 5 10 15

Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser

20 25 30

Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg

35 40 45

Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu

50 55 60

Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile

65 70 75 80

Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn

85 90 95

Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys

100 105 110

Ala Leu Ser Glu Glu Pro Ser Asn Thr Gly Lys Leu Ile Phe Gly Gln

115 120 125

Gly Thr Thr Leu Gln Val Lys Pro Asp Ile Gln Asn Pro Asp Pro Ala

130 135 140

Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu

145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser

165 170 175

Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp

180 185 190

Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala

195 200 205

Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe

210 215 220

Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe

225 230 235 240

Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe

245 250 255

Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu

260 265 270

Arg Leu Trp Ser Ser

275

<210> 6

<211> 313

<212> PRT

<213> human

<400> 6

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys

20 25 30

Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His

35 40 45

Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro

65 70 75 80

Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile

85 90 95

Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser

100 105 110

Ser Arg Leu Leu Ala Gly Gly Gln Asn Glu Gln Phe Phe Gly Pro Gly

115 120 125

Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 7

<211> 840

<212> DNA

<213> human

<400> 7

atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60

agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120

gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180

tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240

gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 300

ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcccagatt 360

tataaccagg gaggaaagct tatcttcgga cagggaacgg agttatctgt gaaacccaat 420

atccagaacc ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct 480

gtctgcctat tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat 540

gtgtatatca cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt 600

gctgtggcct ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt 660

attccagaag acaccttctt ccccagccca gaaagttcct gtgatgtcaa gctggtcgag 720

aaaagctttg aaacagatac gaacctaaac tttcaaaacc tgtcagtgat tgggttccga 780

atcctcctcc tgaaagtggc cgggtttaat ctgctcatga cgctgcggct gtggtccagc 840

<210> 8

<211> 927

<212> DNA

<213> human

<400> 8

atgggaatca ggctcctgtg tcgtgtggcc ttttgtttcc tggctgtagg cctcgtagat 60

gtgaaagtaa cccagagctc gagatatcta gtcaaaagga cgggagagaa agtttttctg 120

gaatgtgtcc aggatatgga ccatgaaaat atgttctggt atcgacaaga cccaggtctg 180

gggctacggc tgatctattt ctcatatgat gttaaaatga aagaaaaagg agatattcct 240

gaggggtaca gtgtctctag agagaagaag gagcgcttct ccctgattct ggagtccgcc 300

agcaccaacc agacatctat gtacctctgt gccagcagtt tttctagcgg aaagcagtac 360

ttcgggccgg gcaccaggct cacggtcaca gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210> 9

<211> 807

<212> DNA

<213> human

<400> 9

atgatatcct tgagagtttt actggtgatc ctgtggcttc agttaagctg ggtttggagc 60

caacggaagg aggtggagca ggatcctgga cccttcaatg ttccagaggg agccactgtc 120

gctttcaact gtacttacag caacagtgct tctcagtctt tcttctggta cagacaggat 180

tgcaggaaag aacctaagtt gctgatgtcg gtatactcca gtggtaatga agatggaagg 240

tttacagcac agctcaatag agccagccag tatatttccc tgctcatcag agactccaag 300

ctcagtgatt cagccaccta cctctgtgtg gtgaccggta accagttcta ttttgggaca 360

gggacaagtt tgacggtcat tccaaatatc cagaaccctg accctgccgt gtaccagctg 420

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 480

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 540

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 600

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 660

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 720

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 780

ctcatgacgc tgcggctgtg gtccagc 807

<210> 10

<211> 939

<212> DNA

<213> human

<400> 10

atggcctccc tgctcttctt ctgtggggcc ttttatctcc tgggaacagg gtccatggat 60

gctgatgtta cccagacccc aaggaatagg atcacaaaga caggaaagag gattatgctg 120

gaatgttctc agactaaggg tcatgataga atgtactggt atcgacaaga cccaggactg 180

ggcctacggt tgatctatta ctcctttgat gtcaaagata taaacaaagg agagatctct 240

gatggataca gtgtctctcg acaggcacag gctaaattct ccctgtccct agagtctgcc 300

atccccaacc agacagctct ttacttctgt gccaccagtg atgtcgggac aggggacacc 360

ggggagctgt tttttggaga aggctctagg ctgaccgtac tggaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939

<210> 11

<211> 831

<212> DNA

<213> human

<400> 11

atgctgactg ccagcctgtt gagggcagtc atagcctcca tctgtgttgt atccagcatg 60

gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 120

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 180

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 240

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 300

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgagtgagga acctagcaac 360

acaggcaaac taatctttgg gcaagggaca actttacaag taaaaccaga tatccagaac 420

cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480

ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540

acagacaaaa ctgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660

gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720

gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780

ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag c 831

<210> 12

<211> 939

<212> DNA

<213> human

<400> 12

atgggaatca ggctcctgtg tcgtgtggcc ttttgtttcc tggctgtagg cctcgtagat 60

gtgaaagtaa cccagagctc gagatatcta gtcaaaagga cgggagagaa agtttttctg 120

gaatgtgtcc aggatatgga ccatgaaaat atgttctggt atcgacaaga cccaggtctg 180

gggctacggc tgatctattt ctcatatgat gttaaaatga aagaaaaagg agatattcct 240

gaggggtaca gtgtctctag agagaagaag gagcgcttct ccctgattct ggagtccgcc 300

agcaccaacc agacatctat gtacctctgt gccagcagtc gactactagc gggggggcag 360

aatgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939

<210> 13

<211> 267

<212> PRT

<213> human

<400> 13

Met Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr

1 5 10 15

Thr Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly

20 25 30

Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly

35 40 45

Leu Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser

50 55 60

Tyr Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe

65 70 75 80

Leu Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln

85 90 95

Met Lys Asp Ser Ala Ser Tyr Leu Cys Ala Val Met Asp Ser Ser Tyr

100 105 110

Lys Leu Ile Phe Gly Ser Gly Thr Arg Leu Leu Val Arg Pro Asp Ile

115 120 125

Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser

130 135 140

Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val

145 150 155 160

Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu

165 170 175

Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser

180 185 190

Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile

195 200 205

Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys

210 215 220

Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn

225 230 235 240

Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

245 250 255

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 14

<211> 271

<212> PRT

<213> human

<400> 14

Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln

1 5 10 15

Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln

20 25 30

Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser

35 40 45

Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu

50 55 60

Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr

65 70 75 80

Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala

85 90 95

Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Ala Ala Gly Gly

100 105 110

Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val

115 120 125

His Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp

130 135 140

Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser

145 150 155 160

Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp

165 170 175

Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala

180 185 190

Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn

195 200 205

Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

210 215 220

Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu

225 230 235 240

Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys

245 250 255

Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265 270

<210> 15

<211> 277

<212> PRT

<213> human

<400> 15

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu

1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

20 25 30

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

35 40 45

Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu

50 55 60

Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln

65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg

85 90 95

Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu

100 105 110

Thr Trp Thr Asp Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe Gly Arg

115 120 125

Gly Thr Gln Leu Thr Val Trp Pro Asp Ile Gln Asn Pro Asp Pro Ala

130 135 140

Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu

145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser

165 170 175

Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp

180 185 190

Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala

195 200 205

Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe

210 215 220

Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe

225 230 235 240

Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe

245 250 255

Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu

260 265 270

Arg Leu Trp Ser Ser

275

<210> 16

<211> 280

<212> PRT

<213> human

<400> 16

Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Thr

1 5 10 15

Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln

20 25 30

Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn

35 40 45

Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys

50 55 60

Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys

65 70 75 80

Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala

85 90 95

Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala

100 105 110

Val Tyr Phe Cys Ala Ala Ser Leu Tyr Asn Gln Gly Gly Lys Leu Ile

115 120 125

Phe Gly Gln Gly Thr Glu Leu Ser Val Lys Pro Asn Ile Gln Asn Pro

130 135 140

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

145 150 155 160

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

165 170 175

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg

180 185 190

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

195 200 205

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

210 215 220

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

225 230 235 240

Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val

245 250 255

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

260 265 270

Met Thr Leu Arg Leu Trp Ser Ser

275 280

<210> 17

<211> 274

<212> PRT

<213> human

<400> 17

Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His

1 5 10 15

Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser

20 25 30

Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro

35 40 45

Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys

50 55 60

Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys

65 70 75 80

Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr

85 90 95

Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys

100 105 110

Ala Ser Gly Asp Ser Gly Tyr Ala Leu Asn Phe Gly Lys Gly Thr Ser

115 120 125

Leu Leu Val Thr Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser

<210> 18

<211> 276

<212> PRT

<213> human

<400> 18

Met Asn Tyr Ser Pro Gly Leu Val Ser Leu Ile Leu Leu Leu Leu Gly

1 5 10 15

Arg Thr Arg Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu

20 25 30

Ser Glu Glu Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly

35 40 45

Tyr Pro Ser Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln

50 55 60

Leu Leu Leu Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly

65 70 75 80

Phe Glu Ala Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys

85 90 95

Gly Ser Val Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr

100 105 110

Ile Trp Asp Tyr Gly Gly Ser Gln Gly Asn Leu Ile Phe Gly Lys Gly

115 120 125

Thr Lys Leu Ser Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val

130 135 140

Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe

145 150 155 160

Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp

165 170 175

Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe

180 185 190

Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys

195 200 205

Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro

210 215 220

Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu

225 230 235 240

Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg

245 250 255

Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg

260 265 270

Leu Trp Ser Ser

275

<210> 19

<211> 269

<212> PRT

<213> human

<400> 19

Met Val Leu Lys Phe Ser Val Ser Ile Leu Trp Ile Gln Leu Ala Trp

1 5 10 15

Val Ser Thr Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln

20 25 30

Glu Gly Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser

35 40 45

Ser Leu Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu

50 55 60

Val Thr Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr

65 70 75 80

Phe Gln Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His Ile Thr Ala

85 90 95

Ala Gln Pro Gly Asp Thr Gly Leu Tyr Leu Cys Ala Gly Glu Asn Ser

100 105 110

Gly Tyr Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro

115 120 125

His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

130 135 140

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

145 150 155 160

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

165 170 175

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

180 185 190

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

195 200 205

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

210 215 220

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

225 230 235 240

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

245 250 255

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 20

<211> 275

<212> PRT

<213> human

<400> 20

Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro

20 25 30

Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser

35 40 45

Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys

50 55 60

Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp

65 70 75 80

Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu

85 90 95

Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala

100 105 110

Met Ser Leu Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr

115 120 125

Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr

130 135 140

Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr

145 150 155 160

Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val

165 170 175

Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys

180 185 190

Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala

195 200 205

Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser

210 215 220

Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr

225 230 235 240

Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile

245 250 255

Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu

260 265 270

Trp Ser Ser

275

<210> 21

<211> 275

<212> PRT

<213> human

<400> 21

Met Leu Leu Glu His Leu Leu Ile Ile Leu Trp Met Gln Leu Thr Trp

1 5 10 15

Val Ser Gly Gln Gln Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln

20 25 30

Glu Gly Glu Asp Val Ser Met Asn Cys Thr Ser Ser Ser Ile Phe Asn

35 40 45

Thr Trp Leu Trp Tyr Lys Gln Asp Pro Gly Glu Gly Pro Val Leu Leu

50 55 60

Ile Ala Leu Tyr Lys Ala Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr

65 70 75 80

Ala Gln Phe Gly Ile Thr Arg Lys Asp Ser Phe Leu Asn Ile Ser Ala

85 90 95

Ser Ile Pro Ser Asp Val Gly Ile Tyr Phe Cys Ala Gly Gln Leu Gly

100 105 110

Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr

115 120 125

Ile Leu Thr Val His Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr

130 135 140

Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr

145 150 155 160

Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val

165 170 175

Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys

180 185 190

Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala

195 200 205

Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser

210 215 220

Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr

225 230 235 240

Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile

245 250 255

Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu

260 265 270

Trp Ser Ser

275

<210> 22

<211> 274

<212> PRT

<213> human

<400> 22

Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp

1 5 10 15

Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val

20 25 30

Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala

35 40 45

Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Gly Pro Gln

50 55 60

Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg

65 70 75 80

Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile

85 90 95

Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Asn

100 105 110

Trp Ser Pro Gln Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg

115 120 125

Leu Thr Ile Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser

<210> 23

<211> 267

<212> PRT

<213> human

<400> 23

Met Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr

1 5 10 15

Thr Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly

20 25 30

Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly

35 40 45

Leu Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser

50 55 60

Tyr Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe

65 70 75 80

Leu Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln

85 90 95

Met Lys Asp Ser Ala Ser Tyr Leu Cys Ala Ser Met Asp Ser Asn Tyr

100 105 110

Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys Pro Asp Ile

115 120 125

Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser

130 135 140

Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val

145 150 155 160

Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu

165 170 175

Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser

180 185 190

Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile

195 200 205

Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys

210 215 220

Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn

225 230 235 240

Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

245 250 255

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 24

<211> 274

<212> PRT

<213> human

<400> 24

Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser

1 5 10 15

Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe

20 25 30

Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn

35 40 45

Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu

50 55 60

Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg

65 70 75 80

Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile

85 90 95

Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Asn

100 105 110

Arg Phe Thr Arg Asp Gly Asn Lys Leu Val Phe Gly Ala Gly Thr Ile

115 120 125

Leu Arg Val Lys Ser Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser

<210> 25

<211> 312

<212> PRT

<213> human

<400> 25

Met Ser Ile Gly Leu Leu Cys Cys Val Ala Phe Ser Leu Leu Trp Ala

1 5 10 15

Ser Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Asn Ser Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile Tyr Tyr Ser Ala Ser Glu Gly Thr Thr Asp Lys Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Leu Asn Lys Arg Glu Phe Ser Leu Arg

85 90 95

Leu Glu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Ser Glu Val Thr Gly Gly Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 26

<211> 311

<212> PRT

<213> human

<400> 26

Met Leu Ser Leu Leu Leu Leu Leu Leu Gly Leu Gly Ser Val Phe Ser

1 5 10 15

Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Cys Gln Arg Gly Thr

20 25 30

Ser Leu Thr Ile Gln Cys Gln Val Asp Ser Gln Val Thr Met Met Phe

35 40 45

Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr Ala

50 55 60

Asn Gln Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp Lys

65 70 75 80

Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val Ser

85 90 95

Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Gly Ala

100 105 110

Gly Gln Gly Pro Tyr Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

115 120 125

Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala

130 135 140

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

195 200 205

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

210 215 220

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

225 230 235 240

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

245 250 255

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln

260 265 270

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

275 280 285

Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

290 295 300

Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 27

<211> 311

<212> PRT

<213> human

<400> 27

Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val

1 5 10 15

Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys

20 25 30

Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His

35 40 45

Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu

50 55 60

Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro

65 70 75 80

Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile

85 90 95

Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser

100 105 110

Ser Leu Gly Ala Thr Gly Ala Asn Glu Lys Leu Phe Phe Gly Ser Gly

115 120 125

Thr Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Phe

305 310

<210> 28

<211> 308

<212> PRT

<213> human

<400> 28

Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Tyr Arg Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu

115 120 125

Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val

130 135 140

Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser

195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His

210 215 220

Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp

225 230 235 240

Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala

245 250 255

Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly

260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr

275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys

290 295 300

Arg Lys Asp Phe

305

<210> 29

<211> 309

<212> PRT

<213> human

<400> 29

Met Gly Pro Gly Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Pro Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln His Val Thr Leu Arg Cys Ser Pro Ile Ser Gly His

35 40 45

Lys Ser Val Ser Trp Tyr Gln Gln Val Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Glu Lys Glu Glu Arg Gly Arg Gly Asn Phe Pro

65 70 75 80

Asp Arg Phe Ser Ala Arg Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Asp Val Gly Leu Pro Pro Leu His Phe Gly Asn Gly Thr Arg

115 120 125

Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala

130 135 140

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

195 200 205

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

210 215 220

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

225 230 235 240

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

245 250 255

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln

260 265 270

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

275 280 285

Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

290 295 300

Lys Arg Lys Asp Phe

305

<210> 30

<211> 309

<212> PRT

<213> human

<400> 30

Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr

1 5 10 15

Gly His Gly Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr

20 25 30

Gln Phe Gly Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His

35 40 45

Asn Val Met Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu

50 55 60

Leu Phe His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro

65 70 75 80

Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp

85 90 95

Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr

100 105 110

Ser Arg Glu Trp Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu

115 120 125

Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

130 135 140

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

145 150 155 160

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

165 170 175

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro

180 185 190

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

225 230 235 240

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

290 295 300

Lys Asp Ser Arg Gly

305

<210> 31

<211> 315

<212> PRT

<213> human

<400> 31

Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe Tyr Phe Leu Gly Ala

1 5 10 15

Gly Leu Met Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile

20 25 30

Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His

35 40 45

Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu

50 55 60

Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser

65 70 75 80

Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr

85 90 95

Leu Glu Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser

100 105 110

Ser Gln Leu Tyr Arg Asp Thr Ser Asn Thr Gly Glu Leu Phe Phe Gly

115 120 125

Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro

130 135 140

Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr

145 150 155 160

Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His

165 170 175

Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val

180 185 190

Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser

195 200 205

Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln

210 215 220

Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser

225 230 235 240

Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile

245 250 255

Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu

260 265 270

Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu

275 280 285

Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu

290 295 300

Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly

305 310 315

<210> 32

<211> 313

<212> PRT

<213> human

<400> 32

Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala

1 5 10 15

Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

85 90 95

Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Gly Ile Ser Gly Thr Ala Ser Ser Tyr Asn Ser Pro Leu His Phe Gly

115 120 125

Asn Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro

130 135 140

Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr

145 150 155 160

Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His

165 170 175

Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val

180 185 190

Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser

195 200 205

Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln

210 215 220

Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser

225 230 235 240

Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile

245 250 255

Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val

260 265 270

Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu

275 280 285

Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu

290 295 300

Met Ala Met Val Lys Arg Lys Asp Phe

305 310

<210> 33

<211> 312

<212> PRT

<213> human

<400> 33

Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala

1 5 10 15

Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr

20 25 30

Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp

35 40 45

Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe

50 55 60

Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu

65 70 75 80

Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn

85 90 95

Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser

100 105 110

Ser Val Gly Gly Gly Leu Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 34

<211> 311

<212> PRT

<213> human

<400> 34

Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe Tyr Phe Leu Gly Ala

1 5 10 15

Gly Leu Met Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile

20 25 30

Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His

35 40 45

Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu

50 55 60

Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser

65 70 75 80

Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr

85 90 95

Leu Glu Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser

100 105 110

Ser Glu Tyr Ile Gln Tyr Ser Gly Asn Thr Ile Tyr Phe Gly Glu Gly

115 120 125

Ser Trp Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Phe

305 310

<210> 35

<211> 314

<212> PRT

<213> human

<400> 35

Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly Leu Gly Ala

1 5 10 15

Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser

20 25 30

Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe

35 40 45

Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser

50 55 60

Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys

65 70 75 80

Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr

85 90 95

Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Lys Val

100 105 110

Thr Ser Gly Gln His Gln Gly Thr Thr Asp Thr Gln Tyr Phe Gly Pro

115 120 125

Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro

130 135 140

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln

145 150 155 160

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val

165 170 175

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser

180 185 190

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg

195 200 205

Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn

210 215 220

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu

225 230 235 240

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val

245 250 255

Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser

260 265 270

Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu

275 280 285

Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met

290 295 300

Ala Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 36

<211> 307

<212> PRT

<213> human

<400> 36

Met Leu Ser Leu Leu Leu Leu Leu Leu Gly Leu Gly Ser Val Phe Ser

1 5 10 15

Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Cys Gln Arg Gly Thr

20 25 30

Ser Leu Thr Ile Gln Cys Gln Val Asp Ser Gln Val Thr Met Met Phe

35 40 45

Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr Ala

50 55 60

Asn Gln Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp Lys

65 70 75 80

Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val Ser

85 90 95

Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Val Glu Gly

100 105 110

Arg Gly Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

115 120 125

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

130 135 140

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

145 150 155 160

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

165 170 175

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

180 185 190

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

225 230 235 240

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

245 250 255

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

290 295 300

Ser Arg Gly

305

<210> 37

<211> 801

<212> DNA

<213> human

<400> 37

atgtggggag ttttccttct ttatgtttcc atgaagatgg gaggcactac aggacaaaac 60

attgaccagc ccactgagat gacagctacg gaaggtgcca ttgtccagat caactgcacg 120

taccagacat ctgggttcaa cgggctgttc tggtaccagc aacatgctgg cgaagcaccc 180

acatttctgt cttacaatgt tctggatggt ttggaggaga aaggtcgttt ttcttcattc 240

cttagtcggt ctaaagggta cagttacctc cttttgaagg agctccagat gaaagactct 300

gcctcttacc tctgtgctgt gatggatagc agctataaat tgatcttcgg gagtgggacc 360

agactgctgg tcaggcctga tatccagaac cctgaccctg ccgtgtacca gctgagagac 420

tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca aacaaatgtg 480

tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga catgaggtct 540

atggacttca agagcaacag tgctgtggcc tggagcaaca aatctgactt tgcatgtgca 600

aacgccttca acaacagcat tattccagaa gacaccttct tccccagccc agaaagttcc 660

tgtgatgtca agctggtcga gaaaagcttt gaaacagata cgaacctaaa ctttcaaaac 720

ctgtcagtga ttgggttccg aatcctcctc ctgaaagtgg ccgggtttaa tctgctcatg 780

acgctgcggc tgtggtccag c 801

<210> 38

<211> 813

<212> DNA

<213> human

<400> 38

atgctactca tcacatcaat gttggtctta tggatgcaat tgtcacaggt gaatggacaa 60

caggtaatgc aaattcctca gtaccagcat gtacaagaag gagaggactt caccacgtac 120

tgcaattcct caactacttt aagcaatata cagtggtata agcaaaggcc tggtggacat 180

cccgtttttt tgatacagtt agtgaagagt ggagaagtga agaagcagaa aagactgaca 240

tttcagtttg gagaagcaaa aaagaacagc tccctgcaca tcacagccac ccagactaca 300

gatgtaggaa cctacttctg tgcggctgct ggtggtacta gctatggaaa gctgacattt 360

ggacaaggga ccatcttgac tgtccatcca aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agc 813

<210> 39

<211> 831

<212> DNA

<213> human

<400> 39

atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60

agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120

ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180

ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240

agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300

cagactgggg actcagctat ctacttctgt gcagagacct ggaccgacag aggctcaacc 360

ctggggaggc tatactttgg aagaggaact cagttgactg tctggcctga tatccagaac 420

cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480

ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540

acagacaaaa ctgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660

gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720

gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780

ctgaaagtgg ccgggtttaa tctgctcatg acgttgcggc tgtggtccag c 831

<210> 40

<211> 840

<212> DNA

<213> human

<400> 40

atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagacaga ctgggtaaac 60

agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120

gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180

tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240

gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 300

ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagtctt 360

tataaccagg gaggaaagct tatcttcgga cagggaacgg agttatctgt gaaacccaat 420

atccagaacc ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct 480

gtctgcctat tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat 540

gtgtatatca cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt 600

gctgtggcct ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt 660

attccagaag acaccttctt ccccagccca gaaagttcct gtgatgtcaa gctggtcgag 720

aaaagctttg aaacagatac gaacctaaac tttcaaaacc tgtcagtgat tgggttccga 780

atcctcctcc tgaaagtggc cgggtttaat ctgctcatga cgctgcggct gtggtccagc 840

<210> 41

<211> 822

<212> DNA

<213> human

<400> 41

atggagaaga atcctttggc agccccatta ctaatcctct ggtttcatct tgactgcgtg 60

agcagcatac tgaacgtgga acaaagtcct cagtcactgc atgttcagga gggagacagc 120

accaatttca cctgcagctt cccttccagc aatttttatg ccttacactg gtacagatgg 180

gaaactgcaa aaagccccga ggccttgttt gtaatgactt taaatgggga tgaaaagaag 240

aaaggacgaa taagtgccac tcttaatacc aaggagggtt acagctattt gtacatcaaa 300

ggatcccagc ctgaagactc agccacatac ctctgtgcct cgggggattc cgggtatgca 360

ctcaacttcg gcaaaggcac ctcgctgttg gtcacacccc atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 822

<210> 42

<211> 828

<212> DNA

<213> human

<400> 42

atgaactatt ctccaggctt agtatctctg atactcttac tgcttggaag aacccgtgga 60

aattcagtga cccagatgga agggccagtg actctctcag aagaggcctt cctgactata 120

aactgcacgt acacagccac aggataccct tcccttttct ggtatgtcca atatcctgga 180

gaaggtctac agctcctcct gaaagccacg aaggctgatg acaagggaag caacaaaggt 240

tttgaagcca cataccgtaa agaaaccact tctttccact tggagaaagg ctcagttcaa 300

gtgtcagact cagcggtgta cttctgtgct ctgacaatat gggattatgg aggaagccaa 360

ggaaatctca tctttggaaa aggcactaaa ctctctgtta aaccaaatat ccagaaccct 420

gaccctgccg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc 480

accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca 540

gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg 600

agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 660

accttcttcc ccagcccaga aagttcctgt gatgtcaagc tggtcgagaa aagctttgaa 720

acagatacga acctaaactt tcaaaacctg tcagtgattg ggttccgaat cctcctcctg 780

aaagtggccg ggtttaatct gctcatgacg ctgcggctgt ggtccagc 828

<210> 43

<211> 807

<212> DNA

<213> human

<400> 43

atggtcctga aattctccgt gtccattctt tggattcagt tggcatgggt gagcacccag 60

ctgctggagc agagccctca gtttctaagc atccaagagg gagaaaatct cactgtgtac 120

tgcaactcct caagtgtttt ttccagctta caatggtaca gacaggagcc tggggaaggt 180

cctgtcctcc tggtgacagt agttacgggt ggagaagtga agaagctgaa gagactaacc 240

tttcagtttg gtgatgcaag aaaggacagt tctctccaca tcactgcagc ccagcctggt 300

gatacaggcc tctacctctg tgcaggagaa aattccgggt atgcactcaa cttcggcaaa 360

ggcacctcgc tgttggtcac accccatatc cagaaccctg accctgccgt gtaccagctg 420

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 480

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 540

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 600

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 660

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 720

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 780

ctcatgacgc tgcggctgtg gtccagc 807

<210> 44

<211> 825

<212> DNA

<213> human

<400> 44

atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60

agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120

gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180

tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240

ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300

tcacagccca gtgattcagc cacctacctc tgtgcaatga gcctatcagg aggaagctac 360

atacctacat ttggaagagg aaccagcctt attgttcatc cgtatatcca gaaccctgac 420

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660

ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720

gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780

gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825

<210> 45

<211> 825

<212> DNA

<213> human

<400> 45

atgctccttg aacatttatt aataatcttg tggatgcagc tgacatgggt cagtggtcaa 60

cagctgaatc agagtcctca atctatgttt atccaggaag gagaagatgt ctccatgaac 120

tgcacttctt caagcatatt taacacctgg ctatggtaca agcaggaccc tggggaaggt 180

cctgtcctct tgatagcctt atataaggct ggtgaattga cctcaaatgg aagactgact 240

gctcagtttg gtataaccag aaaggacagc ttcctgaata tctcagcatc catacctagt 300

gatgtaggca tctacttctg tgctgggcag ctaggagggg ctggtggtac tagctatgga 360

aagctgacat ttggacaagg gaccatcttg actgtccatc caaatatcca gaaccctgac 420

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660

ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720

gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780

gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825

<210> 46

<211> 822

<212> DNA

<213> human

<400> 46

atgacatcca ttcgagctgt atttatattc ctgtggctgc agctggactt ggtgaatgga 60

gagaatgtgg agcagcatcc ttcaaccctg agtgtccagg agggagacag cgctgttatc 120

aagtgtactt attcagacag tgcctcaaac tacttccctt ggtataagca agaacttgga 180

aaaggacctc agcttattat agacattcgt tcaaatgtgg gcgaaaagaa agaccaacga 240

attgctgtta cattgaacaa gacagccaaa catttctccc tgcacatcac agagacccaa 300

cctgaagact cggctgtcta cttctgtgca gcaaactgga gcccgcaagg aaatgagaaa 360

ttaacctttg ggactggaac aagactcacc atcataccca atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 822

<210> 47

<211> 801

<212> DNA

<213> human

<400> 47

atgtggggag ttttccttct ttatgtttcc atgaagatgg gaggcactac aggacaaaac 60

attgaccagc ccactgagat gacagctacg gaaggtgcca ttgtccagat caactgcacg 120

taccagacat ctgggttcaa cgggctgttc tggtaccagc aacatgctgg cgaagcaccc 180

acatttctgt cttacaatgt tctggatggt ttggaggaga aaggtcgttt ttcttcattc 240

cttagtcggt ctaaagggta cagttacctc cttttgaagg agctccagat gaaagactct 300

gcctcttacc tctgtgcttc catggatagc aactatcagt taatctgggg cgctgggacc 360

aagctaatta taaagccaga tatccagaac cctgaccctg ccgtgtacca gctgagagac 420

tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca aacaaatgtg 480

tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga catgaggtct 540

atggacttca agagcaacag tgctgtggcc tggagcaaca aatctgactt tgcatgtgca 600

aacgccttca acaacagcat tattccagaa gacaccttct tccccagccc agaaagttcc 660

tgtgatgtca agctggtcga gaaaagcttt gaaacagata cgaacctaaa ctttcaaaac 720

ctgtcagtga ttgggttccg aatcctcctc ctgaaagtgg ccgggtttaa tctgctcatg 780

acgctgcggc tgtggtccag c 801

<210> 48

<211> 822

<212> DNA

<213> human

<400> 48

atgatatcct tgagagtttt actggtgatc ctgtggcttc agttaagctg ggtttggagc 60

caacggaagg aggtggagca ggatcctgga cccttcaatg ttccagaggg agccactgtc 120

gctttcaact gtacttacag caacagtgct tctcagtctt tcttctggta cagacaggat 180

tgcaggaaag aacctaagtt gctgatgtcc gtatactcca gtggtaatga agatggaagg 240

tttacagcac agctcaatag agccagccag tatatttccc tgctcatcag agactccaag 300

ctcagtgatt cagccaccta cctctgtgtg gtgaacagat tcacaaggga tggaaacaaa 360

ctggtctttg gcgcaggaac cattctgaga gtcaagtcct atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 822

<210> 49

<211> 936

<212> DNA

<213> human

<400> 49

atgagcatcg gcctcctgtg ctgtgtggcc ttttctctcc tgtgggcaag tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccataactcc atgtactggt atcgacaaga cccaggcatg 180

ggactgaggc tgatttatta ctcagcttct gagggtacca ctgacaaagg agaagtcccc 240

aatggctaca atgtctccag attaaacaaa cgggagttct cgctcaggct ggagtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcagtg aggtgacagg gggatacaat 360

gagcagttct tcgggccagg gacacggctc accgtgctag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210> 50

<211> 933

<212> DNA

<213> human

<400> 50

atgctgagtc ttctgctcct tctcctggga ctaggctctg tgttcagtgc tgtcatctct 60

caaaagccaa gcagggatat ctgtcaacgt ggaacctccc tgacgatcca gtgtcaagtc 120

gatagccaag tcaccatgat gttctggtac cgtcagcaac ctggacagag cctgacactg 180

atcgcaactg caaatcaggg ctctgaggcc acatatgaga gtggatttgt cattgacaag 240

tttcccatca gccgcccaaa cctaacattc tcaactctga ctgtgagcaa catgagccct 300

gaagacagca gcatatatct ctgcagcgtt ggggcggggc aaggacctta cacagatacg 360

cagtattttg gcccaggcac ccggctgaca gtgctcgagg acctgaaaaa cgtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 900

atggccatgg tcaagagaaa ggattccaga ggc 933

<210> 51

<211> 933

<212> DNA

<213> human

<400> 51

atgggaatca ggctcctgtg tcgtgtggcc ttttgtttcc tggctgtagg cctcgtagat 60

gtgaaagtaa cccagagctc gagatatcta gtcaaaagga cgggagagaa agtttttctg 120

gaatgtgtcc aggatatgga ccatgaaaat atgttctggt atcgacaaga cccaggtctg 180

gggctacggc tgatctattt ctcatatgat gttaaaatga aagaaaaagg agatattcct 240

gaggggtaca gtgtctctag agagaagaag gagcgcttct ccctgattct ggagtccgcc 300

agcaccaacc agacatctat gtacctctgt gccagcagct taggggcgac aggggctaat 360

gaaaaactgt tttttggcag tggaacccag ctctctgtct tggaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccctgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacggacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttc 933

<210> 52

<211> 924

<212> DNA

<213> human

<400> 52

atgggctcct ggaccctctg ctgtgtgtcc ctttgcatcc tggtagcaaa gcacacagat 60

gctggagtta tccagtcacc ccggcacgag gtgacagaga tgggacaaga agtgactctg 120

agatgtaaac caatttcagg acacgactac cttttctggt acagacagac catgatgcgg 180

ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240

gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300

tcagaaccca gggactcagc tgtgtacttc tgtgccagca gctacagggg cactgaagct 360

ttctttggac aaggcaccag actcacagtt gtagaggacc tgaacaaggt gttcccaccc 420

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480

gtgtgcctgg ccacaggctt cttccctgac cacgtggagc tgagctggtg ggtgaatggg 540

aaggaggtgc acagtggggt cagcacggac ccgcagcccc tcaaggagca gcccgccctc 600

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720

acccaggata gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780

gactgtggct ttacctcggt gtcctaccag caaggggtcc tgtctgccac catcctctat 840

gagatcctgc tagggaaggc caccctgtat gctgtgctgg tcagcgccct tgtgttgatg 900

gccatggtca agagaaagga tttc 924

<210> 53

<211> 927

<212> DNA

<213> human

<400> 53

atgggccctg ggctcctctg ctgggtgctg ctttgtctcc tgggagcagg cccagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca cgtgactctg 120

agatgctctc ctatctctgg gcacaagagt gtgtcctggt accaacaggt cctgggtcag 180

gggccccagt ttatctttca gtattatgag aaagaagaga gaggaagagg aaacttccct 240

gatcgattct cagctcgcca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct gtatctctgt gccagcagct ttgacgttgg tttgccaccc 360

ctccactttg ggaacgggac caggctcact gtgacagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttccct gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcacg gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttc 927

<210> 54

<211> 927

<212> DNA

<213> human

<400> 54

atgggtcctg ggcttctcca ctggatggcc ctttgtctcc ttggaacagg tcatggggat 60

gccatggtca tccagaaccc aagataccag gttacccagt ttggaaagcc agtgaccctg 120

agttgttctc agactttgaa ccataacgtc atgtactggt accagcagaa gtcaagtcag 180

gccccaaagc tgctgttcca ctactatgac aaagatttta acaatgaagc agacacccct 240

gataacttcc aatccaggag gccgaacact tctttctgct ttcttgacat ccgctcacca 300

ggcctggggg acgcagccat gtacctgtgt gccaccagca gagagtggga gacccagtac 360

ttcgggccag gcacgcggct cctggtgctc gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210> 55

<211> 945

<212> DNA

<213> human

<400> 55

atgactatca ggctcctctg ctacatgggc ttttattttc tgggggcagg cctcatggaa 60

gctgacatct accagacccc aagatacctt gttataggga caggaaagaa gatcactctg 120

gaatgttctc aaaccatggg ccatgacaaa atgtactggt atcaacaaga tccaggaatg 180

gaactacacc tcatccacta ttcctatgga gttaattcca cagagaaggg agatctttcc 240

tctgagtcaa cagtctccag aataaggacg gagcattttc ccctgaccct ggagtctgcc 300

aggccctcac atacctctca gtacctctgt gccagcagcc aactttaccg ggacacctcg 360

aacaccgggg agctgttttt tggagaaggc tctaggctga ccgtactgga ggacctgaaa 420

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 480

caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 540

tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 600

gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 660

accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 720

gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 780

gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 840

gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 900

gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 945

<210> 56

<211> 939

<212> DNA

<213> human

<400> 56

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcggaa tcagcgggac agcgagctcc 360

tataattcac ccctccactt tgggaacggg accaggctca ctgtgacaga ggacctgaac 420

aaggtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 480

caaaaggcca cactggtgtg cctggccaca ggcttcttcc ctgaccacgt ggagctgagc 540

tggtgggtga atgggaagga ggtgcacagt ggggtcagca cggacccgca gcccctcaag 600

gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 660

accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 720

gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 780

gcctggggta gagcagactg tggctttacc tcggtgtcct accagcaagg ggtcctgtct 840

gccaccatcc tctatgagat cctgctaggg aaggccaccc tgtatgctgt gctggtcagc 900

gcccttgtgt tgatggccat ggtcaagaga aaggatttc 939

<210> 57

<211> 936

<212> DNA

<213> human

<400> 57

atgggcttca ggctcctctg ctgtgtggcc ttttgtctcc tgggagcagg cccagtggat 60

tctggagtca cacaaacccc aaagcacctg atcacagcaa ctggacagcg agtgacgctg 120

agatgctccc ctaggtctgg agacctctct gtgtactggt accaacagag cctggaccag 180

ggcctccagt tcctcattca gtattataat ggagaagaga gagcaaaagg aaacattctt 240

gaacgattct ccgcacaaca gttccctgac ttgcactctg aactaaacct gagctctctg 300

gagctggggg actcagcttt gtatttctgt gccagcagcg tcggaggggg attggcagat 360

acgcagtatt ttggcccagg cacccggctg acagtgctcg aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210> 58

<211> 933

<212> DNA

<213> human

<400> 58

atgactatca ggctcctctg ctacatgggc ttttattttc tgggggcagg cctcatggaa 60

gctgacatct accagacccc aagatacctt gttataggga caggaaagaa gatcactctg 120

gaatgttctc aaaccatggg ccatgacaaa atgtactggt atcaacaaga tccaggaatg 180

gaactacacc tcatccacta ttcctatgga gttaattcca cagagaaggg agatctttcc 240

tctgagtcaa cagtctccag aataaggacg gagcattttc ccctgaccct ggagtctgcc 300

aggccctcac atacctctca gtacctctgt gccagcagtg aatatatcca gtactctgga 360

aacaccatat attttggaga gggaagttgg ctcactgttg tagaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc ttccctgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacggacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 900

gtgttgatgg ccatggtcaa gagaaaggat ttc 933

<210> 59

<211> 942

<212> DNA

<213> human

<400> 59

atgctgctgc ttctgctgct tctggggcca ggctccgggc ttggtgctgt cgtctctcaa 60

catccgagct gggttatctg taagagtgga acctctgtga agatcgagtg ccgttccctg 120

gactttcagg ccacaactat gttttggtat cgtcagttcc cgaaacagag tctcatgctg 180

atggcaactt ccaatgaggg ctccaaggcc acatacgagc aaggcgtcga gaaggacaag 240

tttctcatca accatgcaag cctgaccttg tccactctga cagtgaccag tgcccatcct 300

gaagacagca gcttctacat ctgcagtgcg aaggtgacta gcgggcaaca ccaagggacc 360

acagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga cctgaaaaac 420

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480

aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 540

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 600

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720

aatgacgagt ggacccagga tagggccaaa cctgtcaccc agatcgtcag cgccgaggcc 780

tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 840

accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 900

ctcgtgctga tggccatggt caagagaaag gattccagag gc 942

<210> 60

<211> 921

<212> DNA

<213> human

<400> 60

atgctgagtc ttctgctcct tctcctggga ctaggctctg tgttcagtgc tgtcatctct 60

caaaagccaa gcagggatat ctgtcaacgt ggaacctccc tgacgatcca gtgtcaagtc 120

gatagccaag tcaccatgat gttctggtac cgtcagcaac ctggacagag cctgacactg 180

atcgcaactg caaatcaggg ctctgaggcc acatatgaga gtggatttgt cattgacaag 240

tttcccatca gccgcccaaa cctaacattc tcaactctga ctgtgagcaa catgagccct 300

gaagacagca gcatatatct ctgcagcgtt gaaggcaggg gttacgagca gtacttcggg 360

ccgggcacca ggctcacggt cacagaggac ctgaaaaacg tgttcccacc cgaggtcgct 420

gtgtttgagc catcagaagc agagatctcc cacacccaaa aggccacact ggtgtgcctg 480

gccacaggct tctaccccga ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg 540

cacagtgggg tcagcacaga cccgcagccc ctcaaggagc agcccgccct caatgactcc 600

agatactgcc tgagcagccg cctgagggtc tcggccacct tctggcagaa cccccgcaac 660

cacttccgct gtcaagtcca gttctacggg ctctcggaga atgacgagtg gacccaggat 720

agggccaaac ctgtcaccca gatcgtcagc gccgaggcct ggggtagagc agactgtggc 780

ttcacctccg agtcttacca gcaaggggtc ctgtctgcca ccatcctcta tgagatcttg 840

ctagggaagg ccaccttgta tgccgtgctg gtcagtgccc tcgtgctgat ggccatggtc 900

aagagaaagg attccagagg c 921

<210> 61

<211> 310

<212> PRT

<213> human

<400> 61

Met Gln Trp Ala Leu Ala Val Leu Leu Ala Phe Leu Ser Pro Ala Ser

1 5 10 15

Gln Lys Ser Ser Asn Leu Glu Gly Arg Thr Lys Ser Val Ile Arg Gln

20 25 30

Thr Gly Ser Ser Ala Glu Ile Thr Cys Asp Leu Ala Glu Gly Ser Thr

35 40 45

Gly Tyr Ile His Trp Tyr Leu His Gln Glu Gly Lys Ala Pro Gln Arg

50 55 60

Leu Leu Tyr Tyr Asp Ser Tyr Thr Ser Ser Val Val Leu Glu Ser Gly

65 70 75 80

Ile Ser Pro Gly Lys Tyr Asp Thr Tyr Gly Ser Thr Arg Lys Asn Leu

85 90 95

Arg Met Ile Leu Arg Asn Leu Ile Glu Asn Asp Ser Gly Val Tyr Tyr

100 105 110

Cys Ala Thr Trp Glu Thr Gln Glu Leu Gly Lys Lys Ile Lys Val Phe

115 120 125

Gly Pro Gly Thr Lys Leu Ile Ile Thr Asp Lys Gln Leu Asp Ala Asp

130 135 140

Val Ser Pro Lys Pro Thr Ile Phe Leu Pro Ser Ile Ala Glu Thr Lys

145 150 155 160

Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu Leu Glu Lys Phe Phe Pro

165 170 175

Asp Val Ile Lys Ile His Trp Gln Glu Lys Lys Ser Asn Thr Ile Leu

180 185 190

Gly Ser Gln Glu Gly Asn Thr Met Lys Thr Asn Asp Thr Tyr Met Lys

195 200 205

Phe Ser Trp Leu Thr Val Pro Glu Lys Ser Leu Asp Lys Glu His Arg

210 215 220

Cys Ile Val Arg His Glu Asn Asn Lys Asn Gly Val Asp Gln Glu Ile

225 230 235 240

Ile Phe Pro Pro Ile Lys Thr Asp Val Ile Thr Met Asp Pro Lys Asp

245 250 255

Asn Cys Ser Lys Asp Ala Asn Asp Thr Leu Leu Leu Gln Leu Thr Asn

260 265 270

Thr Ser Ala Tyr Tyr Met Tyr Leu Leu Leu Leu Leu Lys Ser Val Val

275 280 285

Tyr Phe Ala Ile Ile Thr Cys Cys Leu Leu Arg Arg Thr Ala Phe Cys

290 295 300

Cys Asn Gly Glu Lys Ser

305 310

<210> 62

<211> 295

<212> PRT

<213> human

<400> 62

Met Leu Phe Ser Ser Leu Leu Cys Val Phe Val Ala Phe Ser Tyr Ser

1 5 10 15

Gly Ser Ser Val Ala Gln Lys Val Thr Gln Ala Gln Ser Ser Val Ser

20 25 30

Met Pro Val Arg Lys Ala Val Thr Leu Asn Cys Leu Tyr Glu Thr Ser

35 40 45

Trp Trp Ser Tyr Tyr Ile Phe Trp Tyr Lys Gln Leu Pro Ser Lys Glu

50 55 60

Met Ile Phe Leu Ile Arg Gln Gly Ser Asp Glu Gln Asn Ala Lys Ser

65 70 75 80

Gly Arg Tyr Ser Val Asn Phe Lys Lys Ala Val Lys Ser Val Ala Leu

85 90 95

Thr Ile Ser Ala Leu Gln Leu Glu Asp Ser Ala Lys Tyr Phe Cys Ala

100 105 110

Leu Gly Val Gln Ala Leu Leu Pro Ile Leu Gly Asp Thr Thr Asp Lys

115 120 125

Leu Ile Phe Gly Lys Gly Thr Arg Val Thr Val Glu Pro Arg Ser Gln

130 135 140

Pro His Thr Lys Pro Ser Val Phe Val Met Lys Asn Gly Thr Asn Val

145 150 155 160

Ala Cys Leu Val Lys Glu Phe Tyr Pro Lys Asp Ile Arg Ile Asn Leu

165 170 175

Val Ser Ser Lys Lys Ile Thr Glu Phe Asp Pro Ala Ile Val Ile Ser

180 185 190

Pro Ser Gly Lys Tyr Asn Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser

195 200 205

Asn Ser Val Thr Cys Ser Val Gln His Asp Asn Lys Thr Val His Ser

210 215 220

Thr Asp Phe Glu Val Lys Thr Asp Ser Thr Asp His Val Lys Pro Lys

225 230 235 240

Glu Thr Glu Asn Thr Lys Gln Pro Ser Lys Ser Cys His Lys Pro Lys

245 250 255

Ala Ile Val His Thr Glu Lys Val Asn Met Met Ser Leu Thr Val Leu

260 265 270

Gly Leu Arg Met Leu Phe Ala Lys Thr Val Ala Val Asn Phe Leu Leu

275 280 285

Thr Ala Lys Leu Phe Phe Leu

290 295

<210> 63

<211> 930

<212> DNA

<213> human

<400> 63

atgcagtggg ccctagcggt gcttctagct ttcctgtctc ctgccagtca gaaatcttcc 60

aacttggaag ggagaacgaa gtcagtcatc aggcagactg ggtcatctgc tgaaatcact 120

tgtgatcttg ctgaaggaag taccggctac atccactggt acctacacca ggaggggaag 180

gccccacagc gtcttctgta ctatgactcc tacacctcca gcgttgtgtt ggaatcagga 240

atcagcccag ggaagtatga tacttatgga agcacaagga agaacttgag aatgatactg 300

cgaaatctta ttgaaaatga ctctggagtc tattactgtg ccacctggga aactcaagag 360

ttgggcaaaa aaatcaaggt atttggtccc ggaacaaagc ttatcattac agataaacaa 420

cttgatgcag atgtttcccc caagcccact atttttcttc cttcaattgc tgaaacaaag 480

ctccagaagg ctggaacata cctttgtctt cttgagaaat ttttccctga tgttattaag 540

atacattggc aagaaaagaa gagcaacacg attctgggat cccaggaggg gaacaccatg 600

aagactaacg acacatacat gaaatttagc tggttaacgg tgccagaaaa gtcactggac 660

aaagaacaca gatgtatcgt cagacatgag aataataaaa acggagttga tcaagaaatt 720

atctttcctc caataaagac agatgtcatc acaatggatc ccaaagacaa ttgttcaaaa 780

gatgcaaatg atacactact gctgcagctc acaaacacct ctgcatatta catgtacctc 840

ctcctgctcc tcaagagtgt ggtctatttt gccatcatca cctgctgtct gcttagaaga 900

acggctttct gctgcaatgg agagaaatca 930

<210> 64

<211> 885

<212> DNA

<213> human

<400> 64

atgctgttct ccagcctgct gtgtgtattt gtggccttca gctactctgg atcaagtgtg 60

gcccagaagg ttactcaagc ccagtcatca gtatccatgc cagtgaggaa agcagtcacc 120

ctgaactgcc tgtatgaaac aagttggtgg tcatattata ttttttggta caagcaactt 180

cccagcaaag agatgatttt ccttattcgc cagggttctg atgaacagaa tgcaaaaagt 240

ggtcgctatt ctgtcaactt caagaaagca gtgaaatccg tcgccttaac catttcagcc 300

ttacagctag aagattcagc aaagtacttt tgtgctcttg gggtccaagc cctcctaccc 360

atactggggg ataccaccga taaactcatc tttggaaaag gaacccgtgt gactgtggaa 420

ccaagaagtc agcctcatac caaaccatcc gtttttgtca tgaaaaatgg aacaaatgtc 480

gcttgtctgg tgaaggaatt ctaccccaag gatataagaa taaatctcgt gtcatccaag 540

aagataacag agtttgatcc tgctattgtc atctctccca gtgggaagta caatgctgtc 600

aagcttggta aatatgaaga ttcaaattca gtgacatgtt cagttcaaca cgacaataaa 660

actgtgcact ccactgactt tgaagtgaag acagattcta cagatcacgt aaaaccaaag 720

gaaactgaaa acacaaagca accttcaaag agctgccata aacccaaagc catagttcat 780

accgagaagg tgaacatgat gtccctcaca gtgcttgggc tacgaatgct gtttgcaaag 840

actgttgccg tcaattttct cttgactgcc aagttatttt tcttg 885

<210> 65

<211> 17

<212> PRT

<213> human

<400> 65

Cys Ala Ala Gln Ile Tyr Asn Gln Gly Gly Lys Leu Ile Phe Gly Gln

1 5 10 15

Gly

<210> 66

<211> 13

<212> PRT

<213> human

<400> 66

Cys Val Val Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly

1 5 10

<210> 67

<211> 18

<212> PRT

<213> human

<400> 67

Cys Ala Leu Ser Glu Glu Pro Ser Asn Thr Gly Lys Leu Ile Phe Gly

1 5 10 15

Gln Gly

<210> 68

<211> 15

<212> PRT

<213> human

<400> 68

Cys Ala Val Met Asp Ser Ser Tyr Lys Leu Ile Phe Gly Ser Gly

1 5 10 15

<210> 69

<211> 17

<212> PRT

<213> human

<400> 69

Cys Ala Ala Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln

1 5 10 15

Gly

<210> 70

<211> 20

<212> PRT

<213> human

<400> 70

Cys Ala Glu Thr Trp Thr Asp Arg Gly Ser Thr Leu Gly Arg Leu Tyr

1 5 10 15

Phe Gly Arg Gly

20

<210> 71

<211> 17

<212> PRT

<213> human

<400> 71

Cys Ala Ala Ser Leu Tyr Asn Gln Gly Gly Lys Leu Ile Phe Gly Gln

1 5 10 15

Gly

<210> 72

<211> 15

<212> PRT

<213> human

<400> 72

Cys Ala Ser Gly Asp Ser Gly Tyr Ala Leu Asn Phe Gly Lys Gly

1 5 10 15

<210> 73

<211> 20

<212> PRT

<213> human

<400> 73

Cys Ala Leu Thr Ile Trp Asp Tyr Gly Gly Ser Gln Gly Asn Leu Ile

1 5 10 15

Phe Gly Lys Gly

20

<210> 74

<211> 15

<212> PRT

<213> human

<400> 74

Cys Ala Gly Glu Asn Ser Gly Tyr Ala Leu Asn Phe Gly Lys Gly

1 5 10 15

<210> 75

<211> 17

<212> PRT

<213> human

<400> 75

Cys Ala Met Ser Leu Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg

1 5 10 15

Gly

<210> 76

<211> 21

<212> PRT

<213> human

<400> 76

Cys Ala Gly Gln Leu Gly Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu

1 5 10 15

Thr Phe Gly Gln Gly

20

<210> 77

<211> 18

<212> PRT

<213> human

<400> 77

Cys Ala Ala Asn Trp Ser Pro Gln Gly Asn Glu Lys Leu Thr Phe Gly

1 5 10 15

Thr Gly

<210> 78

<211> 15

<212> PRT

<213> human

<400> 78

Cys Ala Ser Met Asp Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly

1 5 10 15

<210> 79

<211> 18

<212> PRT

<213> human

<400> 79

Cys Val Val Asn Arg Phe Thr Arg Asp Gly Asn Lys Leu Val Phe Gly

1 5 10 15

Ala Gly

<210> 80

<211> 15

<212> PRT

<213> human

<400> 80

Cys Ala Ser Ser Phe Ser Ser Gly Lys Gln Tyr Phe Gly Pro Gly

1 5 10 15

<210> 81

<211> 19

<212> PRT

<213> human

<400> 81

Cys Ala Thr Ser Asp Val Gly Thr Gly Asp Thr Gly Glu Leu Phe Phe

1 5 10 15

Gly Glu Gly

<210> 82

<211> 19

<212> PRT

<213> human

<400> 82

Cys Ala Ser Ser Arg Leu Leu Ala Gly Gly Gln Asn Glu Gln Phe Phe

1 5 10 15

Gly Pro Gly

<210> 83

<211> 18

<212> PRT

<213> human

<400> 83

Cys Ala Ser Ser Glu Val Thr Gly Gly Tyr Asn Glu Gln Phe Phe Gly

1 5 10 15

Pro Gly

<210> 84

<211> 19

<212> PRT

<213> human

<400> 84

Cys Ser Val Gly Ala Gly Gln Gly Pro Tyr Thr Asp Thr Gln Tyr Phe

1 5 10 15

Gly Pro Gly

<210> 85

<211> 19

<212> PRT

<213> human

<400> 85

Cys Ala Ser Ser Leu Gly Ala Thr Gly Ala Asn Glu Lys Leu Phe Phe

1 5 10 15

Gly Ser Gly

<210> 86

<211> 15

<212> PRT

<213> human

<400> 86

Cys Ala Ser Ser Tyr Arg Gly Thr Glu Ala Phe Phe Gly Gln Gly

1 5 10 15

<210> 87

<211> 17

<212> PRT

<213> human

<400> 87

Cys Ala Ser Ser Phe Asp Val Gly Leu Pro Pro Leu His Phe Gly Asn

1 5 10 15

Gly

<210> 88

<211> 15

<212> PRT

<213> human

<400> 88

Cys Ala Thr Ser Arg Glu Trp Glu Thr Gln Tyr Phe Gly Pro Gly

1 5 10 15

<210> 89

<211> 21

<212> PRT

<213> human

<400> 89

Cys Ala Ser Ser Gln Leu Tyr Arg Asp Thr Ser Asn Thr Gly Glu Leu

1 5 10 15

Phe Phe Gly Glu Gly

20

<210> 90

<211> 21

<212> PRT

<213> human

<400> 90

Cys Ala Ser Gly Ile Ser Gly Thr Ala Ser Ser Tyr Asn Ser Pro Leu

1 5 10 15

His Phe Gly Asn Gly

20

<210> 91

<211> 18

<212> PRT

<213> human

<400> 91

Cys Ala Ser Ser Val Gly Gly Gly Leu Ala Asp Thr Gln Tyr Phe Gly

1 5 10 15

Pro Gly

<210> 92

<211> 19

<212> PRT

<213> human

<400> 92

Cys Ala Ser Ser Glu Tyr Ile Gln Tyr Ser Gly Asn Thr Ile Tyr Phe

1 5 10 15

Gly Glu Gly

<210> 93

<211> 22

<212> PRT

<213> human

<400> 93

Cys Ser Ala Lys Val Thr Ser Gly Gln His Gln Gly Thr Thr Asp Thr

1 5 10 15

Gln Tyr Phe Gly Pro Gly

20

<210> 94

<211> 15

<212> PRT

<213> human

<400> 94

Cys Ser Val Glu Gly Arg Gly Tyr Glu Gln Tyr Phe Gly Pro Gly

1 5 10 15

<210> 95

<211> 19

<212> PRT

<213> human

<400> 95

Cys Ala Thr Trp Glu Thr Gln Glu Leu Gly Lys Lys Ile Lys Val Phe

1 5 10 15

Gly Pro Gly

<210> 96

<211> 24

<212> PRT

<213> human

<400> 96

Cys Ala Leu Gly Val Gln Ala Leu Leu Pro Ile Leu Gly Asp Thr Thr

1 5 10 15

Asp Lys Leu Ile Phe Gly Lys Gly

20

<210> 97

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 97

ctcggcaggc cgagccacgg gc 22

<210> 98

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 98

gcccgtggct cggcctgccg ag 22

<210> 99

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 99

ctcgagatgt ctcgctccgt ggcctta 27

<210> 100

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 100

gtgtgagttt tgtcgctagc ctgggggacc tg 32

<210> 101

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 101

caggtccccc aggctagcga caaaactcac ac 32

<210> 102

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 102

gcggccgctc atttacccgg agacagggag a 31

Detailed Description

Responsive cells respond to contact with MR 1-expressing cancer cells presenting cancer antigens in a MR 1-restricted manner, they upregulate activation markers (particularly those described in the preceding paragraph), release cytokines and begin to proliferate.

In other words, T cells showing MR 1-restricted activity are T cells that can be activated by tumor-associated antigens shown by MR 1.

These cells can be sorted by Fluorescence Activated Cell Sorting (FACS) after staining with an appropriate fluorescently labeled antibody specific for the marker, or by sorting with magnetic beads labeled with an appropriate antibody (this is a commonly used sorting method in clinical settings).

The first aspect of the invention relates to a method for producing a preparation of transgenic MR1T cells reactive with MR1 in the absence of exogenous antigen. The method first involves determining which T cell receptors are most likely to be responsive to a particular MR 1-expressing cancer in the patient, then preparing a population of T cells expressing these specific T cell receptor genes from the expression constructs transferred into the cells, and administering these designed T cells to the patient.

The method comprises the following steps:

a. providing a tumor sample obtained from a patient;

b. contacting the tumor sample with a plurality of MR1T cell receptor molecules that are responsive to MR1, and

-presentation on a plurality of T cell clones, wherein each T cell clone is characterized by having an MR1T cell receptor molecule reactive with MR 1; or

-as a labelled soluble MR1T cellular receptor molecule, recognized in a cell-independent manner;

c. identifying a plurality of T cell clones that are specifically reactive to the tumor sample;

d. providing a T cell preparation, in particular a T cell preparation obtained from the same patient;

e. introducing into said T cell preparation a nucleic acid expression construct encoding an MR 1-reactive T cell receptor molecule expressed on said T cell clone which is identified in step c as being specifically reactive with said tumor sample, thereby producing a transgenic T cell preparation.

The patient's transgenic T cell preparation can thus be administered to the patient.

In certain embodiments, each MR 1-specific T cell clone or isolated, labeled and multimerized soluble T cell receptor is characterized by a CDR3 sequence fragment selected from any one of SEQ ID NO 065 to SEQ ID NO 096. Likewise, a fragment of the CDR3 sequence is characterized by a sequence identical to a sequence selected from any one of SEQ ID NO 065 to SEQ ID NO 096 but with one or two amino acid substitutions. In particular embodiments, the CDR3 sequence substitutions are selected according to the following substitution rules:

-glycine (G) and alanine (a) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable, a and V are interchangeable;

tryptophan (W) and phenylalanine (F) are interchangeable, tyrosine (Y) and F are interchangeable;

-serine (S) and threonine (T) are interchangeable;

aspartic acid (D) and glutamic acid (E) are interchangeable

-asparagine (N) and glutamine (Q) are interchangeable; n and S are interchangeable; n and D are interchangeable; e and Q are interchangeable;

methionine (M) and Q are interchangeable;

cysteine (C), a and S are interchangeable;

-proline (P), G and a are interchangeable;

arginine (R) and lysine (K) are interchangeable.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a CDR3 sequence selected from any one of SEQ ID NO 065 to SEQ ID NO 096, wherein the sequence comprises a total of maximum 0, 1, or 2 substitutions in the three N-and/or C-terminal positions according to the above substitution rules in the three N-and/or C-terminal positions, while the central amino acid of the indicated CDR3 sequence is not altered. The central portion of the CDR3 sequence is known to contribute the most to antigen binding or recognition specificity.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a nucleic acid sequence selected from SEQ ID NO 007 to SEQ ID NO 012 or SEQ ID NO 037 to SEQ ID NO 060 or SEQ ID NO 063 to SEQ ID NO 064 and/or an amino acid sequence selected from SEQ ID NO 001 to SEQ ID 006 or SEQ ID NO 013 to SEQ ID NO 036 or SEQ ID NO 061 to SEQ ID NO 062.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by

Herein, "the same biological activity" refers to the ability of the recombinant TCR sequence to recognize (or facilitate recognition of) the MR1 molecule presenting the cancer antigen on cancer cells. Assays and methods for determining such interactions are described herein.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor alpha chain nucleic acid sequence selected from SEQ ID NO 007, 009 to 011 or SEQ ID NO 037 to SEQ ID NO 048 and/or an amino acid sequence selected from SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity and the same biological activity as SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024, in particular an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity with SEQ ID NO 001, 003 to 005 or SEQ ID NO 013 to SEQ ID NO 024 comprising a CDR sequence selected from SEQ ID NO 065 to SEQ ID NO 079

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor beta chain nucleic acid sequence selected from SEQ ID NO 008, 010 to 012 or SEQ ID NO 049 to SEQ ID NO 060 and/or an amino acid sequence selected from SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by an amino acid sequence with at least 85% (. gtoreq.90%, 95%, 98%) identity to SEQ ID SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036 and having the same biological activity, in particular an amino acid sequence with at least 85% (. gtoreq.90%, 95%, 98%) identity to SEQ ID NO 002, 004 to 006 or SEQ ID NO 025 to SEQ ID NO 036 comprising a CDR sequence selected from SEQ ID NO 080 to SEQ ID NO 094.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by the T cell receptor gamma chain nucleic acid sequence SEQ ID NO 61 and/or the amino acid sequence SEQ ID NO 063 or a sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity thereto and having the same biological activity, in particular a sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity to SEQ ID NO 063 and comprising the amino acid sequence CDR3 of SEQ ID NO 095

In certain embodiments, each MR 1-specific T cell clone or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor delta chain nucleic acid sequence SEQ ID NO: 64 and/or the amino acid sequence SEQ ID NO 062, or an amino acid sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity with SEQ ID NO 062 and comprising the CDR3 of SEQ ID NO 096.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor α chain and β chain nucleic acid sequence pair selected from the following pairs: SEQ ID NO 007 and SEQ ID NO 008; or SEQ ID NO 009 and SEQ ID NO 010; or SEQ ID NO 011 and SEQ ID NO 012, SEQ ID NO 037 and SEQ ID NO 049; or SEQ ID NO 038 and SEQ ID NO 050; or SEQ ID NO 039 and SEQ ID NO 051; or SEQ ID NO 040 and SEQ ID NO 052; or SEQ ID NO 041 and SEQ ID NO 053; or SEQ ID NO 042 and SEQ ID NO 054; or SEQ ID NO 043 and SEQ ID NO 055; or SEQ ID NO 044 and SEQ ID NO 056; or SEQ ID NO 045 and SEQ ID NO 057; or SEQ ID NO 046 and SEQ ID NO 058; or SEQ ID NO 047 and SEQ ID NO 059; or SEQ ID NO 048 and SEQ ID NO 060.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor gamma and delta chain nucleic acid sequence pair selected from the group consisting of SEQ ID NO 063 and SEQ ID NO 064, or sequences at least 85% (. gtoreq.90%, 95%, 98%) identical thereto, having the same biological activity as the unmutated pair.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor alpha chain and beta chain amino acid sequence pair selected from the group consisting of: SEQ ID NO 001 and SEQ ID NO 002; or SEQ ID NO 003 and SEQ ID NO 004; or SEQ ID NO 005 and SEQ ID NO 006, SEQ ID NO 013 and SEQ ID NO 025; or SEQ ID NOs 014 and 026; or SEQ ID NO 015 and SEQ ID NO 027; or SEQ ID NO 016 and SEQ ID NO 028; or SEQ ID NO 017 and SEQ ID NO 029; or SEQ ID NO 018 and SEQ ID NO 030; or SEQ ID NO 019 and SEQ ID NO 031; or SEQ ID NO 20 and SEQ ID NO 032; or SEQ ID NO 021 and SEQ ID NO 033; or SEQ ID NO 022 and SEQ ID NO 034; or SEQ ID NO 023 and SEQ ID NO 035; or SEQ ID NO 024 and SEQ ID NO 036; or a pair selected from the pair given in the first two paragraphs, wherein each partner may have at least 85% (. gtoreq.90%, 95%, 98%) identity to the indicated SEQ ID NO and the pair has the same biological activity as the unmutated pair.

In certain embodiments, each MR 1-specific T cell cloned or isolated, labeled and multimerized soluble T cell receptor is characterized by a T cell receptor gamma chain and delta chain amino acid sequence selected from the group consisting of SEQ ID NO 061 and SEQ ID NO 062.

In certain embodiments, the T cell preparation according to the invention is obtained from the same patient (autologous adoptive T cell therapy). This approach has the advantage of avoiding the risk of adverse reactions, particularly alloimmune reactions driven by the endogenous T cell receptors of the designed T cell preparation.

In certain embodiments, the T cell preparation according to the invention is obtained from another subject, in particular an HLA-matched subject (allogeneic adoptive T cell therapy). Depending on the quality of HLA-matching, the risk of alloimmunization may be great, but the logistical and procedural advantages of a large selection of pre-prepared TC formulations may help the therapy to be performed in a larger patient population compared to the much higher cost and regulatory hurdles of a customized individual patient therapy.

Introduction of the MR1T cell receptor expression construct into the T cell preparation can be achieved by lentiviral transduction, which the inventors frequently used in the work with MR1T cells, or by standard methods of DNA expression vector (plasmid) or RNA transfection. The person skilled in the art is aware of the relevant schemes and procedures.

Optionally, the transgenic T cell preparation may be maintained in culture for a period of time prior to administration to a patient to expand its number and, again optionally, further stimulate its differentiation into a particularly desired T cell subset.

In certain embodiments, the T cell preparation obtained from the patient is obtained from peripheral blood of the patient, particularly wherein the T cell preparation is obtained by selecting Peripheral Blood Mononuclear Cells (PBMCs) to express one or more T cell markers of CD4, CD8, CD27, CD45RA, and CD 57.

In certain embodiments, the T cell preparation obtained from the patient is obtained from a tumor biopsy, followed by subsequent in vitro expansion. In certain embodiments, the T cells are expanded in the presence of phytohemagglutinin, IL-2, IL-7, and IL-15. Proliferating T cells were isolated by magnetic sorting and used for T cell receptor design or for cloning and isolating tumor specific MR1 restricted T cells. Isolated MR1T cells were used for TCR gene cloning.

Multiple MR 1-specific T cell clones can be prepared prior to the above procedure and stored in a library or panel for temporary use when rapid characterization of emerging tumors is required. This step essentially identifies MR 1-specific T cell receptor molecules that recognize specific tumor entities.

Alternatively, soluble MR1T TCRs can be generated and multimerized (see subabramanian et al, nature Biotechnology,22,1429, (2004)). The TCR multimers will be labeled with fluorescent dyes and used to stain tumor cells isolated from tumor biopsies. Binding of the soluble MR1T TCR multimer would indicate the ability of the MR1T TCR to recognize tumor cells and would therefore contribute to the selection of an MR1T TCR suitable for gene therapy in this patient.

Another aspect of the invention relates to an expression vector comprising and causing transcription of a nucleic acid sequence encoding a functional T cell receptor heterodimer, or a T cell receptor alpha chain capable of forming a functional T cell receptor heterodimer with a T cell receptor beta chain, and/or a T cell receptor beta chain capable of forming a functional T cell receptor heterodimer with a T cell receptor alpha chain. Notably, the inventors have also found MR1 specific γ - δ heterodimers, and thus the above definitions apply equally to these chains.

In embodiments where the expression vector comprises a nucleic acid sequence encoding a T cell receptor alpha chain or a T cell receptor beta chain (or gamma or delta chain), two different expression vectors, one encoding an alpha chain (gamma chain) and one encoding a beta chain (delta chain), must be introduced into the cell to enable the cell to express a functional T cell receptor heterodimer. The T cell receptor heterodimer specifically binds to the MR1 molecule, wherein the MR1 molecule is expressed on tumor cells and presents a tumor associated antigen.

Expression of the above nucleic acid sequences is controlled by promoter sequences operable in mammalian cells, particularly human T cells. In certain embodiments, the promoter is a constitutively active promoter, such as the CMV immediate early promoter commonly used in molecular biology. In certain other embodiments, the promoter is an inducible promoter.

In certain embodiments of this aspect of the invention, the nucleic acid sequence comprised in the expression vector is or comprises a nucleic acid sequence selected from SEQ ID NO 007, SEQ ID NO 009 or SEQ ID NO 011 and/or encodes an amino acid sequence selected from SEQ ID NO 001, SEQ ID NO 003 or SEQ ID NO 005 (alpha chain).

In certain embodiments of this aspect of the invention, the nucleic acid sequence comprised in the expression vector is or comprises a nucleic acid sequence selected from SEQ ID NO 008, SEQ ID NO 010 or SEQ ID NO 012, and/or encodes an amino acid sequence selected from SEQ ID NO 002, SEQ ID NO 004 or SEQ ID NO 006 (beta chain).

Another aspect of the invention relates to a nucleic acid sequence encoding a functional T cell receptor heterodimer. T cell receptor heterodimers specifically bind to a non-polymorphic MHC I associated (MR1) antigen presenting molecule expressed on tumor cells presenting tumor associated antigens.

In certain embodiments, the nucleic acid sequence encodes a T cell receptor alpha chain and is selected from SEQ ID NO 007, SEQ ID NO 009, or SEQ ID NO 011, or encodes a T cell receptor alpha chain corresponding to an amino acid sequence selected from SEQ ID NO 001, SEQ ID NO 003, or SEQ ID NO 005.

In certain embodiments, the nucleic acid sequence encodes a T cell receptor beta chain and is selected from SEQ ID NO 008, SEQ ID NO 010 or SEQ ID NO 012 or encodes a T cell receptor beta chain specified by an amino acid sequence selected from SEQ ID NO 002, SEQ ID NO 004 or SEQ ID NO 006, or a sequence having at least 85% (. gtoreq.90%, 95%, 98%) identity and the same biological activity as an amino acid sequence selected from SEQ ID NO 001 to 006. In a particular embodiment, each amino acid sequence comprises a CDR3 sequence selected from SEQ ID 65, 66, 67, 80, 81 and 82.

In certain embodiments, the MR1T cell receptor consists of one alpha chain and one beta chain as disclosed herein. The inventors have surprisingly found that the α and β chains can be combined to produce a functional TCR molecule capable of recognising MR 1.

In certain embodiments, the MR1T cell receptor consists of one α chain and one β chain, corresponding to the sequences in the following list:

a.SEQ ID NO 001 and 002,

b.SEQ ID NO 003 and 004,

c. SEQ ID NO 005 and 006,

or a pair selected from the pairs given above, wherein each partner may have a sequence with at least 85% (. gtoreq.90%, 95%, 98%) identity to the indicated SEQ ID NO and the pair has the same biological activity as the unmutated pair. In particular embodiments, each amino acid sequence comprises the same CDR3 sequence as the indicated SEQ ID NO, as can be inferred from the following table.

Another aspect of the invention relates to a T cell receptor protein that binds to a non-polymorphic MHC I associated MR1 antigen presenting molecule. The MR1 molecule is expressed on tumor cells and presents tumor-associated antigens. In certain embodiments, a T cell receptor protein that binds to a non-polymorphic MHC I-associated MR1 antigen presenting molecule is identified by a method according to the first aspect of the invention.

In certain embodiments, the T cell receptor protein comprises a T cell receptor alpha chain characterized by an amino acid sequence selected from SEQ ID NO 001, SEQ ID NO 003, or SEQ ID NO 005, and a T cell receptor beta chain characterized by an amino acid sequence selected from SEQ ID NO 002, SEQ ID NO 004, or SEQ ID NO 006.

Another aspect of the invention relates to a recombinant cell comprising an expression vector according to the invention as described in the preceding paragraph and/or a T cell receptor polypeptide according to the invention. One skilled in the art will appreciate that where an expression vector comprises only a nucleic acid sequence encoding a T cell receptor alpha chain or a T cell receptor beta chain, but not both, two different expression vectors (one encoding an alpha chain and one encoding a beta chain) must be introduced into a recombinant cell in order to enable the cell to express a functional T cell receptor heterodimer. In certain embodiments, the recombinant cells are derived from peripheral blood T cells. In certain embodiments, the recombinant cells are derived from tumor infiltrating lymphocytes.

Another aspect of the invention relates to the use of a recombinant cell according to the previous aspect of the invention in a method for the treatment or prevention of cancer. The method comprises administering the recombinant cell.

In certain embodiments, the cancer is characterized by expression of MR 1.

In certain embodiments, the administration is achieved by adoptive T cell immunotherapy.

The invention further relates to a method of treating or preventing cancer recurrence comprising administering a recombinant cell according to the invention. In certain embodiments, the cancer is characterized by expression of MR 1.

In certain embodiments, the administration is achieved by adoptive T cell immunotherapy.

The invention also relates to a collection of nucleic acid sequences, wherein each sequence of the collection encodes a different T cell receptor alpha chain, T cell receptor beta chain, T cell receptor gamma chain, T cell receptor delta chain, or a combination of T cell receptor alpha and beta chains, or a combination of T cell receptor gamma and delta chains, wherein the combination is capable of specifically binding to an MR1 molecule presenting a cancer antigen. The nucleic acid sequence is capable of promoting expression of a T cell receptor alpha chain, beta chain, or a combination of alpha and beta chains in a mammalian cell.

This set will be used to select transgenic constructs for transfer into T cells collected from a patient. After identifying the TCR sequences that are best suited to initiate a response to a particular set of tumor antigens presented by a tumor in the first stage of the treatment method, the physician will need to be able to select pre-generated expression vectors from this collection manufactured according to GMP in order to achieve rapid gene transfer into the patient's T cells.

In certain embodiments, the collection comprises a sequence selected from SEQ ID NO 007 to SEQ ID NO 012, and/or the collection comprises a sequence encoding a T cell receptor molecule (or a T cell receptor constituting alpha or beta) selected from SEQ ID NO 001 to SEQ ID NO 006.

Another aspect of the invention relates to a collection of recombinant T cells, wherein each cell of the collection expresses a T cell receptor capable of specifically binding to a MR1 molecule presenting a cancer antigen as a transgene. In certain embodiments, the collection comprises a recombinant T cell comprising a T cell receptor protein heterodimer according to aspects of the invention.

The inventors identified and isolated a novel population of human MR 1-restricted T cells that responded to a variety of tumor cells in an MR 1-dependent manner. The MR1T cell clone was typically found in the blood of different healthy individuals, expressed different TCR genes, and did not recognize the previously identified MR1 microorganism or folate-derived ligands. Instead, they recognized a diverse set of unknown antigens isolated from tumor cells and presented by MR 1. The identification and characterization of stimulatory antigens associated with tumor cells is currently ongoing. The MR1T cell clone recognized and killed different types of tumor cells, thus showing labeled anti-tumor activity in vitro. In addition, they released different combinations of Th1, Th2 and Th17 cytokines and showed multiple chemokine receptor expression profiles, indicating phenotypic and functional diversity. Importantly, when paired TCR α and β genes or TCR γ and δ genes isolated from individual MR1T cell clones were transferred into TCR-deficient T cells, the recipient T cells acquired the ability to recognize MR 1-expressing tumor cells, indicating that MR1T cells TCR gene transfer was sufficient for this type of tumor recognition and could be used to indicate that selected T cells recognize MR 1-expressing tumor cells.

In summary, these findings reveal a novel functionally diverse population of tumor-reactive human T cells, limited to the non-polymorphic MR1 molecule, with diverse potential roles in tumor immunity, providing a new conceptual framework for cancer immune monitoring and immunotherapy.

In the present specification, the following abbreviations are used: APC: an antigen presenting cell; β 2 m: beta 2 microglobulin; dc: a dendritic cell; GM-CSF: granulocyte-macrophage colony stimulating factor; HPLC: high pressure liquid chromatography; IFN-gamma: interferon-gamma; mAb: a monoclonal antibody; MAIT cells: mucosal associated invariant T cells; MHC: a major histocompatibility complex; MR 1: MHC class I-related molecules; MR1T cells: MR1 restricted T cells; PBMC: peripheral blood mononuclear cells; TCR: a T cell receptor; TIL: tumors infiltrate lymphocytes.

Further embodiments and advantages can be derived from the following examples and the figures, which further illustrate the invention. These examples are intended to illustrate the invention, but not to limit its scope.