RU2782276C2 - Anti-hla-a2 antibodies and their application methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: humanized anti-HLA-A2 antibodies are proposed. CAR are also proposed, which include humanized anti-HLA-A2 antibodies. Modified cells including CAR, and methods for use of such modified cells for stimulation of immune tolerance in a subject are also proposed.

EFFECT: invention provides obtaining CAR with reduced cross reactivity to several allelic variants of HLA-A.

18 cl, 13 dwg, 9 tbl

Description

Cross-references to related applications

The application claims priority over U.S. provisional application serial number 62/560574, dated September 19, 2017, and U.S. provisional application serial number 62/692386, dated June 29, 2018, which are expressly incorporated herein by reference in in full.

The technical field to which the invention belongs

The present invention relates in some aspects to molecules that bind HLA-A2, in particular to humanized anti-HLA-A2 antibodies. The present invention also relates to recombinant receptors containing such antibodies, including chimeric antigen receptors (CAR), which include such antibodies. The disclosure further relates to engineered cells expressing such receptors and antibodies and their use in cell therapy.

Prior Art

Class I HLA antigens are polymorphic proteins expressed on all nucleated cells and are critical targets for immune recognition in the context of transplantation. Indeed, the development of T cells and/or antibodies specific for HLA class I is a major risk factor for acute and chronic allograft rejection, and the presence of preformed antibodies against donor HLA class I can lead to hyperacute rejection (Konvalinka et al., 2015) . Thus, finding ways to control the immune response to HLA class I proteins would be a major breakthrough in transplantation.

The classic HLA class I molecules are polymorphic and encoded by many different alleles that have evolved in response to evolutionary pressure from infections. There are three loci that code for the classic HLA class I proteins, which are called loci A, B, and C. Within the HLA-A locus, the HLA-A2 allele family is the largest and most diverse family, with at least 31 different HLA- A2 known to exist in humans. Interestingly, unlike many other families of HLA alleles, HLA-A2 is common in all ethnic groups, occurring in 50% of Caucasians and 35% of African Americans (Ellis et al., 2000). Many HLA-A2 alleles differ by only 1 to 9 amino acids, with most of the polymorphism centered around the peptide-binding groove (Hilton et al., 2013). The HLA-A2 alleles are split into two main branches: those obtained through interallelic gene conversion events from A*0201 or A*0205 (Ellis et al., 2000).

Adoptive immunotherapy with T-regulatory (Treg) cells as a way to control unwanted immunity to HLA proteins and other antigens that drive transplant rejection is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). The use of polyclonal Treg cell transfer in the prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) has been reported (Brunstein et al., 2011; Di Ianni et al., 2011; Trzonkowski et al., 2009 ). The use of Treg cell transfer to maintain c-peptide levels in type 1 diabetes has also been reported (Bluestone et al., 2015; Marek-Trzonkowska et al., 2012). Notably, it has been reported that there may be a temporary risk of generalized immunosuppression associated with the use of polyclonal Treg cells for such cell therapy (Brunstein et al., 2013).

Animal data show that the efficacy and specificity of Treg cell therapy can be significantly enhanced by the use of antigen-specific cells. For example, in models of autoimmunity, antigen-specific Treg cells are superior to polyclonal Treg cells in reducing morbidity: Treg cells isolated from pancreatic lymph nodes or activated with islet antigen are significantly better at preventing or curing type 1 diabetes than polyclonal Treg cells ( Green et al., 2002; Masteller et al., 2005; Tang et al., 2004; Tarbell et al., 2007; Tarbell et al., 2004), and Treg cells expressing the autoantigen-specific transgenic T-cell receptor (TCR ), are superior to polyclonal Treg cells in suppressing central nervous system inflammation in an experimental autoimmune encephalomyelitis (EAE) model (Stephens et al., 2009). Similarly, alloantigen-specific Treg cells enriched with stimulated alloantigen in vitro expansion or engineered to express the TCR transgene are more effective than polyclonal Treg cells in preventing organ and tissue transplant rejection (Golshayan et al., 2007; Joffre et al. ., 2008; Nishimura et al., 2004; Sanchez-Fueyo et al., 2006; Tsang et al., 2008). There is some evidence that Treg cells expanded with alloantigens effectively prevent GVHD (Trenado et al., 2006) and that in vivo induction of antigen-specific Treg cells promotes acceptance of hematopoietic allografts without GVHD (Verginis et al., 2008) . Humanized mouse models have shown similar results: alloantigen-expanded human Treg cells are stronger suppressors of skin graft rejection than polyclonal Treg cells (Putnam et al., 2013; Sagoo et al., 2011).

An alternative approach to overexpressing transgenic TCRs or antigen-stimulated expansion for enrichment in antigen-specific T cells is the use of chimeric antigen receptors (CARs). In cell-based adoptive immunotherapy, immune cells isolated from a patient can be modified to express synthetic proteins that allow the cells to perform new therapeutic functions after they are subsequently transferred back to the patient. An example of such a synthetic protein is CAR. An example of a currently used CAR is the fusion of an extracellular recognition domain (eg, an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon incorporation of the antigen, the intracellular signaling portion of CAR can initiate an activation-related response in the immune cell. For example, T cells can be genetically engineered to express extracellular single chain antibody antigen-binding domains (scFv) fused to intracellular signaling domains (Gill and June, 2015; June et al., 2015). In particular, Treg cells expressing CARs specific for model antigens have been reported (Blat et al., 2014; Elinav et al., 2009; Elinav et al., 2008; Fransson et al., 2012; Hombach et al., 2009, Boardman et al., 2016; MacDonald et al., 2016; Noyan et al., 2016).

The essence of the invention

Aspects of the present disclosure include anti-HLA-A2 antibodies. Also provided are chimeric antigen receptors (CARs) comprising an extracellular domain comprising any of the anti-HLA-A2 antibodies of the present invention. Also provided are nucleic acids encoding anti-HLA-A2 antibodies and CARs of the present invention, expression vectors containing them, and host cells containing such expression vectors. Aspects of the present disclosure also include humanized anti-HLA-A2 antibodies. Also provided are chimeric antigen receptors (CARs) comprising an extracellular domain comprising any of the humanized anti-HLA-A2 antibodies of the present invention. Also provided are nucleic acids encoding humanized anti-HLA-A2 antibodies and CARs of the present invention, expression vectors containing them, and host cells comprising such expression vectors. Also provided are immune cells, for example, immunoregulatory cells, which include CARs and/or expression vectors of the present invention, compositions and pharmaceutical compositions, including such immune cells, kits of parts, including such immune cells and/or reagents (for example, nucleic acid or vector encoding an anti-HLA-A or CAR antibody of the present invention) to obtain such immune cells, and methods for producing such immune cells. Also provided are methods of using anti-HLA-A2 antibodies, CARs, immune cells, and pharmaceutical compositions of the present invention. For example, contemplated anti-HLA-A2 antibodies, CARs, immune cells (e.g., immunoregulatory cells), and pharmaceutical compositions find use, for example, in promoting immune tolerance in a subject, preventing or treating graft-versus-host disease (GVHD) in a subject, preventing or treatment of organ or tissue transplant rejection in a subject, and the like.

In some embodiments, a humanized anti-HLA-A2 antibody is provided, wherein the antibody is capable of constituting an antigen-binding domain of a chimeric antigen receptor (CAR), wherein the CAR is capable of being expressed in a human cell (e.g., a human immune cell, such as a human immune regulatory cell), such that CAR binds specifically to HLA-A2. In certain aspects, an antibody of the present invention competes for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190.

In certain aspects, a humanized anti-HLA-A2 antibody is provided, wherein the antibody competes for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190.

In certain aspects, the humanized anti-HLA-A2 antibody set forth above binds to the same HLA-A2 epitope, wherein the antibody comprises: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190.

In some embodiments, the humanized anti-HLA-A2 antibody of the present invention is less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A* 29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, and any combination thereof compared to the BB7.2 antibody. For example, in some embodiments, the humanized anti-HLA-A2 antibody of the present invention is less reactive to at least one HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29, HLA -A*30 and any combination thereof compared to the BB7.2 antibody.

In certain aspects, a humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SYHIQ (SEQ ID NO: 1) and GYTFTSY (SEQ ID NO: 2).

In certain embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: YPGDGS (SEQ ID NO: 4) and WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G ( SEQ ID NO: 10), where X ¹⁰ is Q or K, X ¹² is N or S, X ¹³ is E or Q, and X ¹⁶ is K or Q. Such an antibody may include, for example, a heavy a chain comprising an amino acid sequence selected from the group consisting of: WIYPGDGSTQYNEKFKG (SEQ ID NO: 3) and YPGDGS (SEQ ID NO: 4). Also by way of example, such an antibody may include, for example, a heavy chain variable region comprising the amino acid sequence WIYPGDGSTKYSQKFQG (SEQ ID NO: 5). In certain aspects, a humanized antibody of the present invention comprises a heavy chain variable region comprising the amino acid sequence EGTYYAMDY (SEQ ID NO: 6).

In some embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 7). In certain aspects, a humanized antibody of the present invention includes a light chain variable region comprising the amino acid sequence KVSNRFS (SEQ ID NO: 8). In some embodiments, a humanized antibody of the present invention comprises a light chain variable region comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 9).

In certain aspects, the humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising framework region 1 (VH FR1) comprising an amino acid sequence selected from the group consisting of: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 11) and QVQLVQSGAEVKKPGASVKVSCKASGYTFT ( SEQ ID NO: 12).

In certain embodiments, a humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising framework region 2 (VH FR2) comprising an amino acid sequence selected from the group consisting of:

WVRQAPGQX ⁹ LEWMGX ¹⁵ (SEQ ID NO: 13),

WVRQAPGQX ⁹ LEWMGX ¹⁵ WI (SEQ ID NO: 17),

HIQWVRQAPGQX ¹² LEWMGX ¹⁸ WI (SEQ ID NO: 21), and

HIQWVRQAPGQX ¹² LEWMGX ¹⁸ (SEQ ID NO: 25), where:

X ⁹ is R or G and X ¹⁵ is I or not present in SEQ ID NO: 13;

X ⁹ is R or G and X ¹⁵ is I or not present in SEQ ID NO: 17;

X ¹² is R or G and X ¹⁸ is I or not present in SEQ ID NO: 21; and

X ¹² is R or G and X ¹⁸ is I or not present in SEQ ID NO: 25.

In some embodiments, a humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising framework region 3 (VH FR3) comprising an amino acid sequence selected from the group consisting of:

X ¹ VTX4TX ⁶ DTSX ¹⁰ STAYMX ¹⁶ LSX ¹⁹ LRSX ²³ DX ²⁵ AVYYCAR (SEQ ID NO: 29),

TX ² YX ⁴ X ⁵ KFX ⁸ GX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴

AVYYCAR (SEQ ID NO: 35),

TQYNEKFKGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴

AVYYCAR (SEQ ID NO: 36) and

TKYSQKFQGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR (SEQ ID NO: 37), where:

X ¹ is R or absent, X ⁴ is I or M, X ⁶ is R or A, X ¹⁰ is A, T or I, X ¹⁶ is E or L, X ¹⁹ is S or R, X ²³ is E or D and X ²⁵ is T or M in SEQ ID NO: 29;

X ² is Q or K, X ⁴ is N or S, X ⁵ is E or Q, X ⁸ is K or Q, X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO: 35;

X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO: 36; and

X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO: 37.

In certain aspects, the humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising framework region 4 (VH FR4) comprising the amino acid sequence WGQGTTVTVSS (SEQ ID NO: 44). In some embodiments, a humanized anti-HLA-A2 antibody of the present invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-66.

In certain embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising a framework region 1 (VL FR1) comprising the amino acid sequence DX ² VMTQX ⁷ PLSX ¹¹ X ¹² VTX ¹⁵ GQPASISX ²³ (SEQ ID NO: 46) where X ² is V or I, X ⁷ is S or T, X ¹¹ is L or S, X ¹² is P or S, X ¹⁵ is L or P and X ²³ is C or F.

In some embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising framework region 2 (VL FR2) comprising the amino acid sequence WX ² X ³ QX ⁵ PGQX ⁹ PX ¹¹ X ¹² LIY (SEQ ID NO: 51 ), where X ² is F or Y, X ³ is Q or L, X ⁵ is R or K, X ⁹ is S or P, X ¹¹ is R or Q, and X ¹² is R or L.

In certain aspects, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising a framework region 3 (VL FR3) comprising the amino acid sequence GVPDRFSGSGX ¹¹ GTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 56), where X ¹¹ is S or A In some embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising framework region 4 (VL FR4) comprising the amino acid sequence FGGGTKVEIK (SEQ ID NO: 59). In some embodiments, the humanized anti-HLA-A2 antibody of the present invention comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-71. In some embodiments, the humanized anti-HLA-A2 antibody of the present invention is a full length antibody, single chain antibody, dimeric single chain antibody, Fv, scFv, Fab, F(ab)'2, defucosylated antibody, bispecific antibody, diabody, triatebody, tetrabody, fragment an antibody selected from the group consisting of a unitel, a domain antibody, and a nanobody or antibody mimetic selected from the group consisting of an affitel, an alpha body, an armadillo repeat protein scaffold, knottin, a Kunitz domain peptide, affilin, affitin, adnectin , atrimer, evasine, DARPin, anticalin, avimer, finomer, versatel, or duocalin.

In certain embodiments, the humanized anti-HLA-A2 antibody of the present invention is a scFv. For example, a humanized anti-HLA-A2 antibody of the present invention may be an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 72-91.

In some embodiments, a humanized anti-HLA-A2 antibody as defined above is provided, wherein the antibody is capable of constituting an antigen-binding domain of a chimeric antigen receptor (CAR), wherein the CAR is capable of being expressed in an immune cell (e.g., a regulatory T cell (Treg)), such that CAR binds specifically to HLA-A2. In certain aspects, a humanized anti-HLA-A2 antibody as defined above is provided, wherein the antibody is capable of constituting an antigen-binding domain of a chimeric antigen receptor (CAR), wherein the CAR is capable of being expressed in an immune cell (e.g., a regulatory T cell (Treg)), such that the immune cell is activated by HLA-A2.

In certain aspects, a nucleic acid is provided that encodes any of the humanized anti-HLA-A2 antibodies identified above. Also provided are an expression vector and gene therapy vectors that include such a nucleic acid. Also provided is a host cell comprising such an expression or gene therapy vector.

Aspects of the present disclosure further include chimeric antigen receptors (CARs). For example, a CAR is provided that includes: (i) an extracellular domain comprising any of the humanized anti-HLA-A2 antibodies above; (ii) a transmembrane domain; and (iii) a cytoplasmic domain including an intracellular signaling domain; where CAR is capable of being expressed in an immune cell such that CAR specifically binds to HLA-A2. Such a CAR may be less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA -A*31, HLA-A*33, HLA-A*36, HLA-A*68, and any combination thereof compared to CAR including BB7.2 antibody. For example, in some embodiments, such a CAR has less reactivity to at least one HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29, HLA-A*30, and any combination thereof, compared to CAR containing the BB7.2 antibody. The CAR of the present invention may be capable of being expressed in an immune cell (eg, a regulatory T cell (Treg)), such that the immune cell is activated by HLA-A2. The CAR of the present invention may include a hinge region. In certain aspects, the hinge region includes the CD8α stem region.

The CAR of the present invention may include a transmembrane domain that includes the transmembrane domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor gamma chain, T cell receptor delta chain, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154 and any of them combinations. In some embodiments, the transmembrane domain includes the CD28 transmembrane domain.

According to some embodiments, a CAR of the present invention includes an intracellular signaling domain that includes a functional signaling domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, FcR gamma, FcR alpha, FcR epsilon, CD5, CD22, CD79a, CD79b and CD66d and any combination thereof. In some embodiments, the intracellular signaling domain includes a functional CD3 zeta signaling domain. In certain aspects, the intracellular signaling domain further includes a costimulatory domain. Such a co-stimulatory domain may include a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, Lymphocyte Function-Associated Antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 ( CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, ligand that specifically binds to CD83, IL2ra (CD25), IL6Ra (CD126), IL-7Ra (CD127), IL-13RA1, IL-13RA2, IL-33R (IL1RL1), IL-10RA, IL-10RB, IL- 4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1, TGFbR2, TGFbR3, common gamma chain, and any combination thereof. In some embodiments, the costimulatory domain includes a functional signaling domain of a protein selected from CD28 and 4-1BB. For example, the co-stimulatory domain may include a functional CD28 signaling domain.

Also provided are modified immune cells comprising any of the CARs of the present invention. In some embodiments, the modified immune cell is a regulatory T cell (Treg).

The present disclosure relates to nucleic acids encoding any of the CARs of the present disclosure. Also provided are expression vectors comprising such nucleic acids, as well as immune cells (eg, regulatory T cells (Treg)) comprising such expression vectors.

Compositions (eg, pharmaceutical compositions) are also provided. In certain aspects, a pharmaceutical composition is provided comprising a plurality of modified immune cells or immune cells of the present invention. Kits are also offered. In some embodiments, these kits contain in the first part the immune cells of the present invention and in the second part another therapeutic agent, such as, for example, an immunosuppressive agent. In some embodiments, said kits include one or more reagents (eg, a nucleic acid or an expression vector encoding an anti-HLA-A or CAR antibody of the present invention) for producing cells of the present invention. Methods for producing modified immune cells of the present invention are also provided. In some embodiments, such methods include transducing an immune cell with an expression vector of the present invention, resulting in a modified immune cell.

Also provided are methods of using antibodies, CARs, immune cells, modified immune cells, and pharmaceutical compositions of the present invention. In certain aspects, methods are provided for inducing immune tolerance in a subject, the methods comprising administering to the subject a pharmaceutical composition of the present invention, for example, a pharmaceutical composition comprising a plurality of modified immune cells or immune cells of the present invention. In some embodiments, immune tolerance is tolerance to a transplanted organ or tissue. According to certain embodiments, methods are provided for preventing or treating graft-versus-host disease (GVHD) in a subject, the methods comprising administering to the subject a pharmaceutical composition of the present invention, e.g., a pharmaceutical composition comprising a plurality of modified immune cells or immune cells of the present invention. In certain aspects, the subject is undergoing or has undergone hematopoietic stem cell transplantation. Also provided are methods for preventing or treating organ or tissue rejection in a subject, the methods comprising administering to the subject a pharmaceutical composition of the present invention, for example, a pharmaceutical composition comprising a plurality of modified immune cells or immune cells of the present disclosure. In some embodiments, the subject additionally receives an immunosuppressive agent. In certain embodiments, methods are provided for preventing or treating organ or tissue transplant rejection or graft-versus-host disease (GVHD) in a subject, the methods comprising administering to the subject an immune cell combination of the present invention with at least one immunosuppressive agent to induce immune tolerance. In any of the uses of antibodies, CARs, immune cells, modified immune cells, and pharmaceutical compositions of the present invention, the subject may be a human.

Brief description of the drawings

Figure 1. Construction of humanized anti-HLA-A2 CARs. Schematic representation of lentiviral constructs. Top: Shortened NGFR control design (no CAR); Bottom: Construction of a humanized anti-HLA-A2 CAR. "SP": signal peptide; "GS": glycine-serine linker; "TM": transmembrane region; "Hs": humanized.

Figure 2. Cell surface expression and specificity of humanized anti-HLA-A2 CARs. 293T cells were transiently transfected with the construct and antigen expression and specificity were measured 48 hours later by staining and flow cytometry with anti-ΔNGFR mAb and HLA-A2 tetramers. A & B show dot plots for constructs that retain or do not retain their ability to bind to HLA-A2, respectively. Data are representative of two independent experiments.

Figure 3. Comparison of binding strength of humanized anti-HLA-A2 CARs. 293T cells were transfected with the indicated humanized anti-HLA-A2 CAR constructs and stained with the indicated dilutions of the HLA-A2 tetramer. Figures A, B and C are graphs depicting the geometric mean fluorescence intensity of HLA-A2 tetramer binding in gated ΔNGFR+ cells, with constructs grouped according to light chain use.

Figure 4. Expression and specificity of humanized anti-HLA-A2 CARs for Treg. CD4+CD25hiCD127lo Tregs were activated, transduced every other day with the indicated lentivirus, and then allowed to propagate. Seven days after activation, ΔNGFR expressing cells were selected by separation on magnetic beads. Transduction efficiency and HLA-A2 binding were determined by flow cytometry before and after separation of ΔNGFR+ cells (A, B, C and D). The numbers represent the proportion of ΔNGFR+ tetramer+ cells. Data are representative of independent experiments. (E) summary of percent or mean fluorescence intensity of A*02:01-tetramer binding.

Figure 5. HLA-A2 CAR-mediated Treg activation. CD4+CD25hiCD127lo Tregs were activated, transduced with the indicated lentivirus and allowed to propagate. After 7 days, Tregs were left with 100 U/mL IL-2 overnight, then left unstimulated or stimulated by 2:1 (Treg cells: K562) co-cultivation of anti-CD3/28-loaded CD64-K562 cells (TCR ) or HLA-A2-K562 (CAR) cells. After 24 hours, the expression of CD69, CD154, CTLA-4 and LAP was measured by flow cytometry on live CD4+ cells. (A and B) show representative histograms and (C and D) show averaged data from two independent experiments (E). Control ΔNGFR/CAR Tregs were co-cultured at a 2:1 ratio (Treg: K562) with HLA-A2 expressing K562 cells. After 16 hours, the expression of CD69, CD71, CTLA-4 and LAP was measured by flow cytometry. Percentage positive and fold increase from baseline (without K562) in CD69 and CD71 expression. (F) Percent positive and fold increase from baseline (without K562) in CTLA-4 and LAP expression. Data are n=2-4 for each construct from at least two independent experiments. One-way analysis of variance and Holm-Sidak post hoc test comparing all constructs to mA2-CAR Treg. Mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. Determination of cross-reactivity of humanized anti-HLA-A2 CARs with common HLA-A and HLA-B allelic variants. (A) shows a schematic diagram of experimental setups and gating strategies for FlowPRT cell assay. ΔNGFR+ Tregs expressing these humanized CARs were incubated with Flow Panel Reactive Single Antigen beads and fixed viability dye for 30 minutes at room temperature. The samples were then washed, fixed and analyzed by flow cytometry. (B) Binding to HLA-A*02:01 coated beads for each m/hA2-CAR Treg relative to binding of the control ΔNGFR Treg. (C and D) Correlation between mean HLA-A*02:01 binding measured by FlowPRT cell assay and either (C) HLA-A*02:01-tetramer MFI estimated by flow cytometry or (D ) an increase in the proportion of CD69+ cells 16 hours after co-culture with HLA-A*02:01 compared to the negative control HLA-A*24:01 K562 cells. Data in E, F, G, and H show percent binding relative to control Tregs expressing truncated NGFR only, normalized by the number of HLA-negative beads collected by the cytometer. Data points above the shaded gray area (E, F, and G) or the horizontal dotted line (H) represent values that differed by more than two standard deviations from the mean of the bead-only control and are thus statistically significant (p<0, 05). Data are averages of three independent experiments. (I) ΔNGFR or m/hA2-CAR Tregs were co-cultured with these K562 cells transduced to express selected HLA-A alleles. After 16 hours, the expression of CD69, CD71, LAP and CTLA-4 was measured on live CD4+ T cells. n = 2-6 from at least 2 independent experiments. Statistical significance is determined by one-way analysis of variance and Holm-Sidak post hoc test compared to mA2-CAR. mean ± SEM, **p<0.01.

Figure 7 Tregs expressing humanized HLA-A2 CAR potently suppress T cell proliferation stimulated by HLA-A2+ dendritic cells. (A) Schematic representation of the experimental setups. Mature HLA-A2+ dendritic cells were used to stimulate HLA-A2neg CD4+ responder T cells labeled with cell proliferation dye (CPD)-e450. CPD-e660 labeled Tregs that were either not transduced or transduced with a ΔNGFR-encoding control lentivirus or humanized A2 CAR-expressing Tregs. (B, C, D and E) The indicated ratios of cells were co-cultured for six days, then the amount of proliferation of CD4+ responder T cells labeled with CPD-e450 was measured by flow cytometry. B shows representative scatter plots, and C, D, and E show graphical data for several cell ratios. C, D and E show average data for n = 3-7 from at least 3 independent experiments. Statistical data were performed using two-way analysis of variance with Holm-Sidak post hoc test versus control ΔNGFR Treg. * p<0.05, mean ± standard error of the mean.

Figure 8. Tregs expressing humanized HLA-A2 CAR potently suppress xenogeneic graft versus host disease. Irradiated NSG mice were injected with PBS (n = 3), 8 x 10 ⁶ HLA-A2+ PBMC (n = 5) or 4 x 10 ⁶ H1k2-CAR-expressing Tregs (n = 6). Engraftment of human cells in the blood was monitored every 7 days. (A) Survival curve and (B) percent change in weight from start of experiment. (C) Proportion of total mononuclear cells (live singlets) expressing human CD45 in blood. (D) Gating strategy to differentiate overall engraftment of CD45+ and CAR Treg (hCD45+hCD4+HLA-A2-) human cells. (E) In vivo cell engraftment after adoptive transfer in a mouse model of xenogeneic GVHD, as shown by the absolute engraftment of PBMC and CAR Treg per µl of blood over time. PBMC counts were calculated as hCD45+ minus total CAR Tregs as indicated in D.

Figure 9. m/hA2 CAR expression provides Treg with rapid and persistent homing to HLA-A2:01+ skin allografts. Tregs were co-transduced with lentivirus encoding luciferase and either control HER2-CAR, mA2-CAR or hA2-CAR (H1k2). The transduced double cells were sorted into FACS, expanded for 5 days, and then injected into NSG mice previously transplanted with adjacent skin grafts from both NSG and NSG-HLA-A*02:01 transgenic mice. (A) Schematic representation of the experimental conditions. (B) Representative luciferase imaging of skin grafts (left) at 72 hours or (right) 21 days after Treg injection. The amount of luciferase radiation was determined using the average number of photons/sec/cm ² /steradian and plotted as the ratio between (C) HLA-A*02:01-NSG skin grafts and NSG at 72 hours after Treg injection or (D) in depending on time. n = 2-3 per group of three independent experiments, mean ± SEM. Repeated measurements of ANOVA with Bonferroni correction.

Figure 10. Flow cytometry tracking m/hA2 CAR Treg with fast and persistent homing to HLA-A2:01+ skin allografts. Tregs were co-transduced with lentivirus containing luciferase and HER2-CAR, mA2-CAR or hA2-CAR constructs, expanded and injected into transplanted NSG mice as shown in Figure 9. (A) Pre-gating for flow cytometry plots was based on control HER2 spleen cells -CAR. (B) hCD4/hCD8 flow cytometry profile for the indicated constructs. The graphs were pre-gated to FvD-hCD45+ as in (A). (C) Flow cytometry plots showing staining for m/hA2-CAR Treg in the spleen and draining lymph node at the end of the experiment n = 1 per group from one independent experiment. * p < 0.05.

Figure 11. hA2-CAR-Treg reduce human skin allograft rejection. NSG mice were transplanted with HLA-A*02:01+ human skin and injected three weeks later with: PBS (n=3); HLA-A*02:01neg PBMC alone (n=4) or with a 2:1 ratio of autologous H1k2 CAR Treg (n=6). The PBMC/hA2-CAR Tregs were from two separate donors tested in one experiment. (A) Body weight was monitored thrice a week, and (B) the proportion of human CD45+ cells in blood (left) and spleen (right) was measured by experimental end point. (C) Cumulative histological score of skin grafts determined by hematoxylin-eosin staining. (D) Transplanted skin grafts were immunostained at the end of the experiment to quantify the degree of expression of involucrin and the proportion of Ki-67+ cells in the epidermis. (E) mRNA expression of the indicated genes in the skin grafts was determined by qRT-PCR. (F) Transplanted skin grafts were immunostained at the end of the experiment to quantify the proportion of FOXP3+ cells in human CD45+ cells. (G) Transplanted skin grafts, intestinal, lung and liver sections were immunostained at the end of the experiment to show the proportion of FOXP3+ cells in human CD45+ cells in each tissue. Each data point represents one mouse. Box-and-whisker charts show the mean ± range. Statistical significance was determined using a two-tailed Mann-Whitney test comparing PBMC to H1k2. * p < 0.05.

Figure 12: Flow cytometry tracking of hA2 CAR Treg in blood in a human skin graft model. NSG mice were grafted with human HLA-A*02+ skin and injected with cells as described in Figure 11. (A) Gating strategy to differentiate total engraftment of human CD45+ (PBMC) and CAR Treg (hCD45+ hCD4+ NGFR+). (B) Absolute engraftment of PBMCs and CAR Treg per µl of blood over time. The number of PBMC was calculated as hCD45 + minus the total number of Treg CAR, as indicated in (A).

Figure 13: Activation of hA2 CAR Treg using artificial antigen presenting cells. Control ΔNGFR/CAR Tregs were co-cultured for 16 hours without stimulation or with a 2:1 ratio (Treg : K562) of CD64-expressing K562 cells loaded with anti-CD3 and anti-CD28 monoclonal antibodies (TCR stimulation). (A) An example of a gating strategy. (B) Expression of CD69, CD71, CTLA-4 and LAP was measured by flow cytometry (left). The fold increase of each activation marker from baseline (no stimulation) was calculated (right). Data are n=2-4 for each construct from at least two independent experiments. One-way analysis of variance and Holm-Sidak multiple comparison test comparing all constructs to the mA2-CAR Treg. * p < 0.05, ** p < 0.01, **** p < 0.0001.

DETAILED DESCRIPTION

I. General Methods

In the practice of the present invention, unless otherwise indicated, standard methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are known to the person skilled in the art, will be used. Such methods are fully described in the literature, such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

Before the present invention is further described, it should be understood that this invention is not limited to the specific embodiments described, and therefore may, of course, vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which this invention belongs. While any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials will now be described. A person skilled in the art knows many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. Indeed, the present invention is in no way limited to the described methods and materials. For the purposes of the present invention, the following terms are further defined. All publications cited in this document are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. In the event that any of the following definitions conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

11. DEFINITIONS

Terms given in the singular also cover their plural. For example, "elementt" means one element or more than one element.

The term "about" when referring to a measurable value such as magnitude, time duration, etc., implies that it spans ±20% fluctuations, or in some cases ±10%, and in some cases ±5%, or in some cases ±1% or in some cases ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

Where a range of values is suggested, it is to be understood that every intermediate value, up to a tenth of the value of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range, and any other specified or intermediate value within that specified range, is covered by the invention. . The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also covered by the invention, subject to any particular exclusion limit within the specified range. If a given range includes one or both of the limits, ranges excluding either one or both of these included limits are also included in the invention.

As used herein, the terms "HLA-A2" and "A2" refer to human leukocyte antigen (HLA) proteins, including cell surface proteins encoded by the HLA-A*02 allele family at the HLA-A locus of the HLA gene complex. HLA proteins encompassed by the terms "HLA-A2" and "A2" include HLA proteins identified as belonging to the HLA-A*02 antigen type by serological testing or genotyping. Additional names for the HLA-A*02 antigen type include "HLA-A2", HLA-A02" and "HLA-A*2". Various naming systems have been developed that identify the HLA proteins encoded by this allele family, including the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors. The term "HLA-A2" and "A2" refers to HLA proteins encoded by alleles having designations in accordance with this naming system that begin with "HLA-A*02", including, but not limited to, designations beginning with " HLA-A*02:01", "HLA-A*02:02", "HLA-A*02:03", "HLA-A*02:04", "HLA-A*02:05", " HLA-A*02:06", "HLA-A*02:07", "HLA-A*02:08", "HLA-A*02:09", "HLA-A*02:10" and " HLA-A*02:11". In addition to the numbers that follow "HLA-A*02:", allele designators may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*02:01P or HLA-A*02:01:01G). Allele designations beginning with "HLA-A*02:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be italicized. The terms "HLA-A2" and "A2" also refer to HLA proteins identified with designations that begin with "HLA-A*02" in accordance with this naming system, including, but not limited to, the designations "HLA-A* 02:01", "HLA-A*02:02", "HLA-A*02:03", "HLA-A*02:04", "HLA-A*02:05", "HLA-A* 02:06", "HLA-A*02:07", "HLA-A*02:08", "HLA-A*02:09", "HLA-A*02:10", and "HLA-A *02:11".

As used herein, the term "HLA-A subtype" refers to a protein encoded by an allele of the HLA-A gene.

As used herein, the term "HLA-A*03" refers to HLA proteins, including cell surface proteins, encoded by the HLA-A*03 allele family at the HLA-A locus of the HLA gene complex. HLA proteins encompassed by the term "HLA-A*03" include HLA proteins identified as belonging to the HLA-A*03 antigen type by serological testing or genotyping. Additional names for the HLA-A*03 antigen type include "HLA-A03" and "HLA-A3". The term "HLA-A*03" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors that begin with "HLA-A*03", including, without limitation, designations starting with "HLA-A*03:01", "HLA-A*03:02", "HLA-A*03:04", "HLA-A*03:05", "HLA -A*03:06", "HLA-A*03:07", "HLA-A*03:08", "HLA-A*03:09", "HLA-A*03:10" and "HLA -A*03:12". In addition to the numbers that follow "HLA-A*03:", allele designators may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*03:01P or HLA-A*03:01:01G). Allele designations beginning with "HLA-A*03:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be italicized. The term "HLA-A*03" also refers to HLA proteins identified with designations that begin with "HLA-A*03:" in accordance with this naming system, including, but not limited to, the designations "HLA-A*03 :01", "HLA-A*03:02", "HLA-A*03:04", "HLA-A*03:05", "HLA-A*03:06", "HLA-A*03 :07", "HLA-A*03:08", "HLA-A*03:09", "HLA-A*03:10" and "HLA-A*03:12".

The terms "HLA-A*25", "HLA-A25", and "A25" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*25 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*25", "HLA-A25", and "A25" include HLA proteins identified as belonging to the HLA-A*25 antigen type by serological testing or genotyping. Additional names for the type of HLA-A*25 antigen include "HLA-A25". The term "HLA-A*25", "HLA-A25", and "A25" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*25:" including, but not limited to, designations starting with "HLA-A*25:01", "HLA-A*25:02", "HLA-A*25:03", " HLA-A*25:04", "HLA-A*25:05", "HLA-A*25:06", "HLA-A*25:07", "HLA-A*25:08", " HLA-A*25:09", "HLA-A*25:10" and "HLA-A*25:11". In addition to the numbers that follow "HLA-A*25:", allele designations may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*25:01P or HLA-A*25:01:01G). Allele designations beginning with "HLA-A*25:" followed by 2, 3, or 4 additional digits may constitute the full designation or the initial part of the designation. Allele designations may be in italics. The term "HLA-A*25", "HLA-A25", and "A25" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations “HLA-A*25:01”, “HLA-A*25:02”, “HLA-A*25:03”, “HLA-A*25:04”, “HLA-A*25 :05", "HLA-A*25:06", "HLA-A*25:07", "HLA-A*25:08", "HLA-A*25:09", "HLA-A*25 :10" and "HLA-A*25:11".

The terms "HLA-A*29", "HLA-A29", and "A29" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*29 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*29", "HLA-A29", and "A29" include HLA proteins identified as belonging to the HLA-A*29 antigen type by serological testing or genotyping. Additional names for the HLA-A*29 antigen type include "HLA-A29". The term "HLA-A*29", "HLA-A29", and "A29" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*29:" including, but not limited to, symbols beginning with "HLA-A*29:01", "HLA-A*29:02", "HLA-A*29:03", " HLA-A*29:04", "HLA-A*29:05", "HLA-A*29:06", "HLA-A*29:07", HLA-A*29:09", "HLA -A*29:10" and "HLA-A*29:11". In addition to the numbers that follow "HLA-A*29:", allele designators may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*29:01P or HLA-A*29:02:01G). Allele designations beginning with "HLA-A*29:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be in italics. The term "HLA-A*29", "HLA-A29", and "A29" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations "HLA-A*29:01", "HLA-A*29:02", "HLA-A*29:03", "HLA-A*29:04", "HLA-A*29 :05", "HLA-A*29:06", "HLA-A*29:07", "HLA-A*29:09", "HLA-A*29:10" and "HLA-A*29" :eleven".

The terms "HLA-A*30", "HLA-A30", and "A30" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*30 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*30", "HLA-A30", and "A30" include HLA proteins identified as belonging to the HLA-A*30 antigen type by serological testing or genotyping. Additional names for the type of HLA-A*30 antigen include "HLA-A30". The term "HLA-A*30", "HLA-A30", and "A30" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*30:" including, but not limited to, symbols beginning with "HLA-A*30:01", "HLA-A*30:02", "HLA-A*30:03", " HLA-A*30:04", "HLA-A*30:06", "HLA-A*30:07", "HLA-A*30:08", HLA-A*30:09", "HLA -A*30:10" and "HLA-A*30:11". In addition to the numbers that follow "HLA-A*30:", allele designations may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*30:01P , HLA-A*30:02P, HLA-A*30:04P, HLA-A*30:01:01G, HLA-A*30:02:01G or HLA-A*30:04:01G). Allele designations beginning with "HLA-A*30:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be in italics. The term "HLA-A*30", "HLA-A30", and "A30" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations “HLA-A*30:01”, “HLA-A*30:02”, “HLA-A*30:03”, “HLA-A*30:04”, “HLA-A*30 :05", "HLA-A*30:06", "HLA-A*30:07", "HLA-A*30:09", "HLA-A*30:10" and "HLA-A*30" :eleven".

The terms "HLA-A*31", "HLA-A31", and "A31" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*31 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*31", "HLA-A31", and "A31" include HLA proteins identified as belonging to the HLA-A*31 antigen type by serological testing or genotyping. Additional names for the HLA-A*31 antigen type include "HLA-A31". The term "HLA-A*31", "HLA-A31", and "A31" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*31:" including, but not limited to, symbols beginning with "HLA-A*31:01", "HLA-A*31:02", "HLA-A*31:03", " HLA-A*31:04", "HLA-A*31:05", "HLA-A*31:06", "HLA-A*31:07", "HLA-A*31:08", " HLA-A*31:09", "HLA-A*31:10" and "HLA-A*31:11". In addition to the numbers that follow "HLA-A*31:", allele designators may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*31:01P or HLA-A*31:01:01G). Allele designations beginning with "HLA-A*31:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be italicized. The term "HLA-A*31", "HLA-A31", and "A31" also refers to HLA proteins identified with designations that begin with "HLA-A*31:" under this naming system, including, without restrictions specified, designations “HLA-A*31:01”, “HLA-A*31:02”, “HLA-A*31:03”, “HLA-A*31:04”, “HLA-A*31 :05", "HLA-A*31:06", "HLA-A*31:07", "HLA-A*31:08", "HLA-A*31:09", "HLA-A*31 :10" and "HLA-A*31:11".

The terms "HLA-A*33", "HLA-A33", and "A33" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*33 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*33", "HLA-A33", and "A33" include HLA proteins identified as belonging to the HLA-A*33 antigen type by serological testing or genotyping. Additional names for the type of HLA-A*33 antigen include "HLA-A33". The terms "HLA-A*33", "HLA-A30", and "A30" refer to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*33:" including, but not limited to, symbols beginning with "HLA-A*33:01", "HLA-A*33:03", "HLA-A*33:04", " HLA-A*33:05", "HLA-A*33:06", "HLA-A*33:07", "HLA-A*33:08", HLA-A*33:09", "HLA -A*33:10" and "HLA-A*33:11". In addition to the numbers that follow "HLA-A*33:", allele designations may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*33:01P or HLA-A*33:01:01G). Allele designations beginning with "HLA-A*33:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be italicized. The term "HLA-A*33", "HLA-A33", and "A33" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations “HLA-A*33:01”, “HLA-A*33:02”, “HLA-A*33:03”, “HLA-A*33:04”, “HLA-A*33 :05", "HLA-A*33:06", "HLA-A*33:07", "HLA-A*33:09", "HLA-A*33:10" and "HLA-A*33" :eleven".

The terms "HLA-A*36", "HLA-A36", and "A36" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*36 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*36", "HLA-A36", and "A36" include HLA proteins identified as belonging to the HLA-A*36 antigen type by serological testing or genotyping. Additional names for the type of HLA-A*36 antigen include "HLA-A36". The term "HLA-A*36", "HLA-A36", and "A36" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*36:" including, but not limited to, symbols beginning with "HLA-A*36:01", "HLA-A*36:02", "HLA-A*36:03", " HLA-A*36:04" and "HLA-A*36:05". In addition to the numbers that follow "HLA-A*36:", allele designations may also contain capital letters, including, but not limited to, the capital letters "P" and "G". Allele designations beginning with "HLA-A*36:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be italicized. The term "HLA-A*36", "HLA-A36", and "A36" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations “HLA-A*36:01”, “HLA-A*36:02”, “HLA-A*36:03”, “HLA-A*36:04”, “HLA-A*36 :05" and "HLA-A*36:06".

The terms "HLA-A*68", "HLA-A68", and "A68" as used herein refer to HLA proteins, including cell surface proteins encoded by the HLA-A*68 allele family at the HLA-A locus of the HLA gene complex. . HLA proteins encompassed by the terms "HLA-A*68", "HLA-A68", and "A68" include HLA proteins identified as belonging to the HLA-A*68 antigen type by serological testing or genotyping. Additional names for the type of HLA-A*68 antigen include "HLA-A68". The term "HLA-A*68", "HLA-A68", and "A68" refers to HLA proteins encoded by alleles designated according to this naming system developed in 2010 by the WHO Committee on HLA System Factors beginning with "HLA-A*68:" including, but not limited to, symbols beginning with "HLA-A*68:01", "HLA-A*68:02", "HLA-A*68:03", " HLA-A*68:04", "HLA-A*68:05", "HLA-A*68:06", "HLA-A*68:07", "HLA-A*68:08", " HLA-A*68:09" and "HLA-A*68:10". In addition to the numbers that follow "HLA-A*68:", allele designators may also contain capital letters, including, but not limited to, capital letters "P" and "G" (e.g., HLA-A*68:01P , HLA-A*68:01:01G or HLA-A*68:01:02G). Allele designations beginning with "HLA-A*68:" followed by 2, 3, or 4 additional numbers may constitute the full designation or the initial part of the designation. Allele designations may be in italics. The term "HLA-A*68", "HLA-A68", and "A68" also refers to HLA proteins identified with designations that begin with "HLA-A*25:" under this naming system, including, without restrictions specified, designations "HLA-A*68:01", "HLA-A*68:02", "HLA-A*68:03", "HLA-A*68:04", "HLA-A*68 :05", "HLA-A*68:06", "HLA-A*68:07", "HLA-A*68:08", "HLA-A*68:09" and "HLA-A*68 :ten".

Specific HLA proteins may be referred to in this document using protein designations in accordance with the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors. For example, the term "HLA-A*02:01" as used herein refers to the HLA protein designated "HLA-A*02:01" according to this naming system. Similarly, the terms "HLA-A*03:01", "HLA-A*25:01", "HLA-A*29:02", "HLA-A*30:01", "HLA-A*31: 01", "HLA-A*33:01", "HLA-A*36:01" and "HLA-A*68:01" refer to HLA proteins with designation "HLA-A*03:01", "HLA -A*25:01", "HLA-A*29:02", "HLA-A*30:01", "HLA-A*31:01", "HLA-A*33:01", "HLA -A*36:01" and "HLA-A*68:01", respectively.

As used herein, the term "anti-HLA-A2 antibody" refers to an antibody that preferentially or specifically binds to HLA-A2.

As used herein, the term "BB7.2" refers to a mouse hybridoma identified as an ATCC deposit of HB-82. BB7.2 hybridoma cells secrete a mouse monoclonal antibody of the kappa IgG2b isotype, which was characterized by Parham P. et al. and Hilton et al. (Parham, P. et al., 1981; Hilton et al., 2013). The amino acid sequences of the six complementarity determining regions (CDRs) of the monoclonal antibody secreted by BB7.2 are as follows:

Heavy chain CDR1 (HCDR1): SYHIQ (SEQ ID NO: 183);

Heavy chain CDR2 (HCDR2): WIYPGDGSTQYNEKFKG (SEQ ID NO: 185);

Heavy chain CDR3 (HCDR3): EGTYYAMDY (SEQ ID NO: 187);

Light chain CDR1 (LCDR1): RSSQSIVHSNGNTYLE (SEQ ID NO: 188)

Light chain CDR2 (LCDR2): KVSNRFS (SEQ ID NO: 189);

Light chain CDR3 (LCDR3): FQGSHVPRT (SEQ ID NO: 190).

The term "BB7.2 antibody" as used herein is an antibody having the VH (SEQ ID NO: 191) and VL (SEQ ID NO: 192) of a monoclonal antibody secreted by BB7.2. The BB7.2 antibody may be a full length antibody or fragment thereof having the VH and VL of a monoclonal antibody secreted by BB7.2, such as a scFv having a monoclonal antibody VH and VL secreted by BB7.2.

The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies , single-domain antibodies (sdAbs), linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins containing an antigen-binding portion of an antibody and a non-antibody protein.

Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins and can be obtained from natural sources or from recombinant sources. The antibodies may be tetramers of immunoglobulin molecules. The basic 4-chain antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, a 4-chain unit typically has a mass of about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. Each H- and L-chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. VL is aligned with VH and CL is aligned with the first heavy chain constant domain (CH1). It is believed that specific amino acid residues form the interface between the variable domains of the light chain and the heavy chain. The pairing of VH and VL together forms one antigen-binding site. For the structure and properties of various classes of antibodies see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. ^Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, 71 and chapter 6. An L chain from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains referred to as α, δ, ε, γ, and μ, respectively. The γ and α classes are further subclassified based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The "variable region" or "variable domain" of an antibody refers to the N-terminal domains of the heavy or light chain of an antibody. The heavy chain variable domain may be referred to as "VH", "VH", or " _H ". The light chain variable domain may be referred to as "VL", "VL", or " _L ". These domains are usually the most variable parts of an antibody and include antigen-binding sites.

The term "variable" refers to the fact that certain segments of the V domains vary greatly in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is unevenly distributed over the 110 to 130 amino acid range of the variable domains. Meanwhile, the V regions are composed of relatively invariant regions called framework regions (FRs) of 15-30 amino acids, separated by shorter regions of excessive variability called "hypervariable regions", each of which is 9-12 amino acids long. Each of the native heavy and light chain variable domains comprises four FRs that largely adopt the β-sheet configuration, connected by three hypervariable regions that form loops connecting and in some cases are part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity to the FR and, together with the hypervariable regions from the other chain, contribute to the formation of an antibody antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ^Ed . Public Health Service, National Institutes of Health, Bethesda, M.D. (1991)).

An "intact" antibody is an antibody that includes an antigen binding site as well as CL and at least the heavy chain constant domains, CH1, CH2, and CH3. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. In one embodiment, an intact antibody may have one or more effector functions.

The term "antibody fragment" refers to at least one part of an intact antibody or recombinant variants thereof and refers to an antigen-binding domain, for example, to a region that determines the antigenic variability of an intact antibody, which is sufficient to confer recognition and specificity of binding of an antibody fragment to a target, such as an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, scFv fragments; dimers; single domain antibodies (sdAb); linear antibodies (see US Pat. No. 5641870, Example 2; Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); single chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of an intact antibody and thus retains the ability to bind the antigen. Also included within the anti-HLA-A2 antibody fragments are anti-HLA-A2 antibody portions (and combinations of anti-HLA-A2 antibody portions, e.g. scFv) that can be used as HLA-A2 antigen targeting arms in chimeric antigen receptors of CAR-modified immune cells. Such fragments are not necessarily proteolytic fragments, but rather are parts of polypeptide sequences that can confer affinity for the target. In addition, single-domain antibodies (sdAb) are included among anti-HLA-A2 antibody fragments (see, for example, Li et al. (2017); Jamnani et al. (2014)). Such single domain antibodies can be used as targeting arms in the CAR-modified immune cells of the present invention.

Cleavage of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to easily crystallize. The Fab fragment consists of the entire L chain along with the H chain variable region domain (VH) and the first constant domain of one heavy chain (CH1). Each Fab fragment is antigen-binding monovalent, that is, it has one antigen-binding site. Treating the antibody with pepsin produces one large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments having bivalent antigen-binding activity while retaining antigen cross-linking ability. Fab' fragments differ from Fab fragments by the presence of several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is referred to herein as Fab', in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments that contain hinge cysteines between them. Other chemical combinations of antibody fragments are also known.

The Fc fragment of an antibody includes the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibodies were determined by sequences in the Fc region, this region being also the part recognized by the Fc receptors (FcRs) found in certain cell types.

"Fv" is the minimum antibody fragment that includes a complete antigen recognition and antigen binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. In a single chain type Fv (scFv), one heavy chain variable domain and one light chain variable domain can be covalently linked to a flexible peptide linker such that the light and heavy chains can associate into a "dimeric" structure similar to that of the double chain type Fv. The folding of these two domains results in six hypervariable loops (3 loops each of the H and L chains) that contribute amino acid residues for antigen binding and confer antigen binding specificity on the antibody. However, even one variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, albeit at a lower affinity than the entire binding site.

"Single chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that contain the VH and VL antibody domains linked in a single polypeptide chain. The scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. Unless otherwise indicated, the scFv used herein may have the VL and VH variable regions in any order, e.g., relative to the N-terminal and C-terminal ends of the polypeptide, the scFv may contain a VL-linker-VH, or may contain a VH-linker-VL . For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Antibody Engineering, ed. Borrebaeck, Oxford University Press, New York (1995). In one embodiment, a scFv derived from an anti-HLA-A2 antibody can be used as the targeting arm of the CAR-modified immune cell disclosed herein.

The term "Adnectin", also known as a monobody, is well known in the art and refers to proteins designed to bind with high affinity and specificity to antigens. They belong to a class of molecules collectively referred to as "antibody mimetics".

The term alphabody, which may also be referred to as cell-penetrating alphabody, refers to a type of antibody mimetic composed of small 10 kDa proteins designed to bind to various antigens. Alfatels are able to reach and bind to intracellular protein targets.

The term "affitelo" is well known in the art and refers to affinity proteins based on a protein domain of 58 amino acid residues derived from one of the IgG-binding domains of staphylococcal protein A.

The term "Anticalin" is well known in the art and refers to antibody mimetic technology in which the binding specificity is derived from lipocalin. Anticalin can also be formatted as a dual targeting protein called duocalin.

The term "armadillo repeat protein scaffold" refers to a type of antibody mimetic corresponding to artificial peptide-binding armadillo protein-based scaffolds. Proteins with armadillo repeats are characterized by an armadillo domain consisting of tandem armadillo repeats of about 42 amino acids, which provides interaction with peptides or proteins.

"Avimers" are well known in the art and refer to antibody mimetic technology. The term "DARPins" (Designed Ankyrin Repeat Proteins) is well known in the art and refers to DRP (Designed Repeat Protein) antibody mimetic technology developed to utilize the binding ability of non-antibody polypeptides.

The term "diabodies" refers to small antibody fragments obtained by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that interchain but not intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, then there is a fragment that has two antigen-binding sites. Bispecific diabodies are heterodimers of two "cross" scFv fragments, in which the VH and VL domains of the two antibodies are present in different polypeptide chains. Diabodies are more fully described, for example, in EP 0404097; WO 93/11161; and Holliger et al., Proc. Natl. Acad. sci. USA, 90: 6444-6448 (1993).

The term "evasin" is well known in the art and refers to a class of chemokine-binding proteins.

The term "phenomer" is well known in the art and refers to proteins that belong to the class of antibody mimetics. They are attractive binding molecules due to their high thermal stability and reduced immunogenicity.

The term "knottin" (which may also be referred to as an inhibitory cystine knot) refers to an antibody mimetic containing a protein structural motif containing three disulfide bridges.

The term "Kunitz domain peptide" refers to a type of antibody mimetic and is based on the active domains of proteins that inhibit the function of proteases.

The term "nanobody" is well known in the art and refers to an antibody-derived therapeutic protein that incorporates the unique structural and functional properties of naturally occurring heavy chain-only antibodies. These heavy chain-only antibodies contain one variable domain (VHH) and two constant domains (CH2 and CH3).

The term "unibody" is well known in the art and refers to an antibody fragment lacking the hinge region of IgG4 antibodies. Removal of the hinge region results in a molecule that is substantially half the size of traditional IgG4 antibodies and has a univalent binding region, as opposed to the bivalent binding region of IgG4 antibodies.

The term "versatelo" is well known in the art and refers to other antibody mimetic technology. These are small 3-5 kD proteins with >15% cysteines that form a high disulfide density scaffold that replaces the hydrophobic core that typical proteins have.

The term "flexible polypeptide linker" or "linker" as used in the context of scFv refers to a peptide linker that consists of amino acids, such as glycine and/or serine residues, used alone or in combination, to link heavy and light chain variable regions together. . In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and contains the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In another embodiment, the flexible polypeptide linker is a Gly/Ser linker and contains the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n= 2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 or (Gly4 Ser)3. In another embodiment, the linkers include multiple repeats (Gly2Ser), (GlySer) or (Gly3Ser). Also included within the scope of the invention are the linkers described in WO2012/138475, incorporated herein by reference).

The term "heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their natural conformations, and which usually defines the class to which the antibody belongs.

The term "light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their natural conformations. The kappa (K) and lambda (λ) light chains are the two major antibody light chain isotypes.

As used herein, the term "hypervariable region", "HVR" or "HV" refers to regions of the variable domain of an antibody that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six hypervariable regions; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). A number of hypervariable region transdifferentiations are used and embraced by the present invention. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., 1991). In contrast, Chothia refers to the localization of structural loops (Chothia et al., 1987). Chothia's CDR-H1 loop end when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places inserts in H35A and H35B; if 35A or 35B is missing, the loop ends at 32, if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody molecular modeling program. "Contact" hypervariable regions are based on the analysis of available complex crystal structures. Residues from each of these hypervariable regions are noted below. Residues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact

------- -------------- -------------- -------------- - ------------

L1 L24-L34 L24-L34 L24-L34 L30-L36

L2 L50-L56 L50-L56 L50-L56 L46-L55

L3 L89-L97 L89-L97 L89-L97 L89-L96

H1 H31-H35B H26-H35B H26-H32.34 H30-H35B

(Numbering according to Kabat)

H1 H31-H35 H26-H35 H26-H32 H30-H35

(Numbering by Chothia)

H2 H50-H65 H50-H58 H52-H56 H47-H58

H3 H95-H102 H95-H102 H95-H102 H93-H101

The terms "hypervariable region" and "complementarity determining region" and their respective abbreviations (HVR, HV, CDR) are used interchangeably herein. In addition, the following pairs of terms are also used interchangeably in this document:

"VH CDR1" and "HCDR1";

"VH CDR2" and "HCDR2";

"VH CDR3" and "HCDR3";

"VL CDR1" and "LCDR1";

"VL CDR2" and "LCDR2"; and

"VL CDR3" and "LCDR3".

Hypervariable regions may contain "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in VL and 26-35B (HI ). 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al. (Kabat et al., 1991) for each of these definitions.

"Frame" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein.

The term "variable residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refers to the numbering system used for heavy chain variable domains or for light chain variable domains of antibody assembly in (Kabat et al., 1991) . Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion in the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 in FR heavy chains. The Kabat numbering of residues can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residue in a variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (eg, Kabat et al., 1991). "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (eg, the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in the variable domain of antibodies mean residue numbering using the Kabat numbering system.

The term “specifically binds” refers to a ligand (e.g., a humanized anti-HLA-A2 antibody) that recognizes and binds to a cognate binding partner protein (e.g., HLA-A2) present in a sample, but that ligand does not significantly recognize or bind the molecules. in the sample. Non-specific binding will refer to binding with an affinity of less than 10 ^-7 M, eg binding with an affinity of 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, etc.

An antibody that "specifically binds" an antigen or epitope of interest is an antibody that binds an antigen or epitope with sufficient affinity that is markedly different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule.

The term "recombinant antibody" refers to an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast-based expression system. The term should also be construed as meaning an antibody that has been produced by the synthesis of a DNA molecule encoding an antibody, and which DNA molecule expresses the antibody protein, or amino acid sequence defining the antibody, where the DNA sequence or amino acid sequence was obtained using recombinant DNA technology or technology amino acid sequences that are available and well known in the art.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of partially homogeneous antibodies, i.e. individual antibodies that make up a population that are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The "monoclonal" modifier should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies can be made using the hybridoma technique first described by Kohler et al., Nature, 256: 495 (1975), or can be made using recombinant DNA techniques in bacterial, eukaryotic animal or plant cells (see, e.g., US Pat. No. 4816567). A "monoclonal antibody" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve either the production of antibodies or the activation of specific immunologically competent cells, or both. One skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. One of ordinary skill in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as the term is used herein. In addition, one of skill in the art will appreciate that an antigen need not be encoded solely by the full nucleotide sequence of a gene. Obviously, the present invention includes, without limitation, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. In addition, one skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. Obviously, the antigen may be designed, synthesized or obtained from a biological sample or may be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell or fluid with other biological components.

Used in this document, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). The affinity can be at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold at least 6 times more, at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more, at least 20 times more times more, at least 30 times more, at least 40 times more, at least 50 times more, at least 60 times more, at least 70 times more, at least at least 80 times greater, at least 90 times greater, at least 100 times, or at least 1000 times greater, or more than the affinity of the antibody for unrelated amino acid sequences. The affinity of the antibody for the target protein may be, for example, from about 100 nM to about 0.1 nM, from about 100 nM to about 1 picomole (pM), or from about 100 nM to about 1 femtomol (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive", "preferably binds" and "specifically binds" are used interchangeably herein with respect to antibodies and fragments thereof. The anti-HLA-A2 antibodies of the invention, including humanized anti-HLA-A2 antibodies, as well as fragments thereof as used herein, specifically bind to HLA-A2.

The term "bonding" refers to a direct association between two molecules, for example, due to covalent, electrostatic, hydrophobic and ionic and/or hydrogen bonds, including interactions such as salt and water bridges. Non-specific binding will refer to binding with an affinity of less than 10 ^-7 M, eg binding with an affinity of 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, etc.

As used herein, the term "reactivity" refers to the ability of an antibody to react (ie, bind) to a molecule (eg, specifically bind to a molecule). The first antibody is "less reactive" to the molecule (eg, HLA molecule) than the second antibody when the first antibody exhibits reduced binding to the molecule compared to the second antibody. Approaches are known for rapidly comparing the reactivity of the first and second antibodies with one or more specific HLA molecules. One exemplary approach is presented in the Examples section of this document, where FlowPRA® Single Antigen (One Lambda) beads were used to test antibodies for their ability to react (or bind) to specific HLA molecules. Such flow cytometric approaches allow high-throughput analysis of antibody reactivity.

As used herein, the term "hinge region" refers to a flexible polypeptide junction region (also referred to herein as a "hinge" or "spacer") providing structural flexibility and distance from flanking polypeptide regions, and may be composed of naturally occurring or synthetic polypeptides. The hinge region can influence the activity of an immune cell expressing CAR (see e.g. Watanabe et al. (2016)). A "hinge region" derived from an immunoglobulin (eg, IgG1) is commonly defined as the Glu216 to Pro230 region of human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be natural or non-natural, including, without limitation, the modified hinge region described in US Pat. USA No. No. 5,677,425. The hinge region may include a complete hinge region derived from an antibody of a different class or subclass than that of the CH1 domain. The term "hinge region" may also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and distance to flanking regions.

As used herein, the term "immune cells" generally includes white blood cells (leukocytes) that are derived from hematopoietic stem cells (HSC) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

"T cells" includes all types of CD3-expressing immune cells, including T helper cells (CD4+ cells), CD8+ T cells (eg, cytotoxic CD8+ T cells, regulatory CD8+ T cells), regulatory T cells (Treg ), gamma delta T cells, and double negative T cells.

"Cytotoxic cell" includes cytotoxic CD8+ T cells, natural killer (NK), and neutrophils that are capable of mediating cytotoxic reactions.

As used herein, the term "regulatory immune cell" refers to an immune cell that acts in a "regulatory" manner to suppress immune system activation and thus maintain immune system homeostasis and tolerance to self antigens. "Regulatory immune cells" can also have an effect on non-immune cells, resulting in clinical improvement, such as promoting tissue repair or regeneration. Regulatory immune cells may include regulatory T cells, CD4+ regulatory T cells, CD8+ regulatory T cells, regulatory γδ T cells, regulatory DN T cells, regulatory B cells, regulatory NK cells, regulatory macrophages, and regulatory dendritic cells. cells.

"Regulatory T-lymphocyte", "regulatory T-cell", "regulatory T-cell", "Treg-cell" or "Treg", as used in the present description, are synonymous and have a standard definition used in this field. Treg cells are a specialized subpopulation of T cells that act in a "regulatory" manner to suppress immune system activation and thereby maintain immune system homeostasis and tolerance to self antigens. Tregs are sometimes referred to as suppressor T cells. Treg cells often, but not always, express the forkhead family transcription factor, Foxp3 (forkhead box p3). They can also express CD4 or CD8 surface proteins. They usually also express CD25. As used herein and in the claims, and unless otherwise indicated, Tregs include "natural" Tregs that develop in the thymus, induced/adaptive/peripheral Tregs that result from a differentiation process that occurs outside the thymus (e.g., in tissues or secondary lymphoid organ, or in vitro under certain culture conditions), and Tregs that have been generated using recombinant DNA technology, such as engineered to express FOXP3. Naturally occurring Treg cells (CD4+ CD25+ Foxp3+) arise like all other T cells in the thymus. In contrast, induced/adaptive/peripheral Tregs (which include CD4+ CD25+ Foxp3+ Tregs, Tr1 cells, Th3 cells, and others) arise outside the thymus. One way to induce Treg is by exposing IL-10 or TGF-β to effector T cells. T cells can also be converted to Treg cells by transfection or transduction of the Foxp3 gene into a mixed population of T cells. A T cell in which Foxp3 expression is induced adopts the Treg phenotype, and such recombinant Tregs are also referred to herein as "Treg".

As used herein, the term "immune effector cell" refers to a cell of the immune system that is in a form capable of eliciting a specific immune response.

As used herein, the term "immune response" includes immune responses mediated by T cells and/or B cells. Typical immune responses include T cell responses such as cytokine production and cellular cytotoxicity. In addition, the term "immune response" includes immune responses that are indirectly affected by T cell activation, such as the production of antibodies (humoral responses) and the activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (eg professional antigen-presenting cells such as dendritic cells, macrophages, B-lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells such as macrophages, eosinophils, mast cells, basophils and granulocytes.

The term "rejection" refers to a condition in which a transplanted organ or tissue is not accepted by the recipient's body. Rejection occurs as a result of the effect of the recipient's immune system on the transplanted organ or tissue. Rejection can occur days to weeks after transplant (acute) or months to years after transplant (chronic).

The term "graft-versus-host disease" or "GVHD" as used herein refers to a medical complication after receiving transplanted tissue from a genetically different individual. Immune cells in the donor tissue (graft) recognize the recipient (host) as foreign. The transplanted immune cells then attack the host cells. GVHD is commonly associated with stem cell transplantation; however, the term includes GVHD due to other forms of tissue graft. GVHD can also occur after a blood transfusion.

As used herein, the term "immunological tolerance" or "immune tolerance" refers to methods performed on a portion of treated subjects compared to untreated subjects, where: a) a reduced level of specific immunological response (which is considered mediated at least in part by antigen -specific effector T-lymphocytes, B-lymphocytes, antibodies or their equivalents); b) delaying the onset or progression of a specific immunological response; or c) reduced risk of occurrence or progression of a specific immunological response. "Specific" immunological or immune tolerance occurs when immunological or immune tolerance is elicited preferentially against certain antigens over others.

As used herein, the term "functional tolerance" refers to the clinical situation where there is stable graft function without histological evidence of rejection, including acute or chronic rejection, in the absence of any immunosuppressive drug therapy for at least 1 year. , in an immunocompetent host capable of responding to other challenges, including infections.

As used herein, the term "immune accommodation" refers to the condition of a transplant recipient in which the organ or tissue graft functions normally despite the presence of antibodies specific for the organ or tissue graft in the recipient.

As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. "Stem cells" include, without limitation, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); cells derived from bone marrow, etc.).

The term "treatment", "treat", etc. relate to obtaining the desired pharmacological and/or physiological effect. The effect may be therapeutic in terms of partial or complete treatment of the disease and/or adverse effect inherent in the disease. The term "treatment", as used herein, encompasses any treatment of a disease in a mammal, eg, a human, and includes alleviation of the disease, ie causing regression of the disease and/or amelioration of one or more symptoms of the disease.

As used herein, the terms "preventing", "preventing", and "preventing" mean providing a prophylactic or protective treatment for a disease or disease state. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof. "Prevention" as used herein encompasses any prophylactic effect on a disease in a mammal, such as a human, and includes: (a) preventing the occurrence of a disease in an entity that may be predisposed to the disease but has not yet been diagnosed as having it; and (b) suppressing the disease, that is, stopping its development.

The terms "patient", "object", "individual", "host" and the like are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, amenable to the methods described herein. The terms "patient", "subject", "individual", "host" and the like are intended to include living organisms in which an immune response can be elicited (eg, mammals). Examples of "patient", "subject", "individual", "host" include mice (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses). , cattle, sheep, pigs, goats, etc. and their transgenic varieties In some non-limiting embodiments, the patient, subject, host, or individual is a human.

The term "effective amount" or "therapeutically effective amount" is used interchangeably herein and refers to an amount of a therapeutic agent, or combined amounts of more than one therapeutic agent, that will elicit a biological or medical response in a tissue, system, or entity that is being sought by a researcher, veterinarian, physician or other clinical physician. The term "therapeutically effective amount" includes that amount of a therapeutic agent which, when administered, is sufficient to prevent the development or to some extent ameliorate one or more signs or symptoms of the disorder or disease being treated. A therapeutically effective amount will vary depending on the therapeutic agent, the disease and its severity, as well as age, weight, etc. object to be treated.

As used herein, the term "activation" refers to the state of a T cell (eg, a regulatory T cell) that has been sufficiently stimulated to induce a detectable cellular response. Activation can also be associated with detectable(s) effector(s) function(s), such as the production of cytokines or suppressive activity. The term "activated" regulatory T cells refers, inter alia, to regulatory T cells that are capable of suppressing an immune response.

The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct containing an extracellular domain containing an antigen-binding domain; transmembrane domain; and a cytoplasmic domain containing an intracellular signaling domain. In one embodiment, the CAR optionally includes a hinge. The terms "chimeric receptor" or "chimeric antigen receptor" or "CAR" may particularly refer to a single polypeptide or a set of polypeptides, usually two in the simplest embodiments, which, when present in an immune cell, confer ligand specificity on the cell. targets and generate an intracellular signal. In some embodiments, a set of polypeptides is adjacent to each other. In some embodiments, the chimeric receptor is a chimeric fusion protein containing a set of polypeptides. In some embodiments, the set of polypeptides includes a dimerization switch, which, in the presence of a dimerization molecule, can bind polypeptides to each other, for example, can bind a ligand-binding domain to an intracellular signaling domain. In one embodiment, the chimeric receptor includes an optional leader sequence at the amino-terminus (N-ter) of the chimeric receptor fusion protein. In one embodiment, the chimeric receptor further includes a leader sequence at the N-terminus of the extracellular ligand-binding domain, where the leader sequence is optionally cleaved from the ligand-binding domain during cellular processing and localization of the chimeric receptor to the cell membrane.

The term "signaling domain" refers to a functional portion of a protein that acts by relaying information within a cell to regulate cellular activity through certain signaling pathways, generating second messengers or acting as effectors responsive to such messengers.

The term "autologous" refers to any material derived from the same subject to which it is later reintroduced.

The term "allogeneic" refers to any material obtained from another object of the same species as the object to which this material is introduced. Two or more entities are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from objects of the same species may be genetically dissimilar enough to interact antigenically. The term "allograft" refers to a graft obtained from another object of the same species.

The term "xenogeneic" refers to any material obtained from an object of a different species. The term "xenograft" refers to a transplant obtained from an object of another species.

As used herein, the term "instructions" includes a publication, record, diagram, or any other medium of expression that can be used to communicate the applicability of the compositions and methods of the invention. The instructions for a kit of the invention may, for example, be attached to a container that includes a nucleic acid, peptide, cell, and/or composition of the invention, or supplied with a container that includes a nucleic acid, peptide, cell, and/or composition. Alternatively, the instructions may be delivered separately from the container for the recipient to share the instructions and the nucleic acid, peptide, cell, and/or composition.

"Modification" of an amino acid residue/position, as used herein, refers to a change in the primary amino acid sequence from the original amino acid sequence, where the change is the result of a sequence change involving the specified amino acid residue/positions. For example, typical modifications include replacing a residue (or at a specified position) with a different amino acid (e.g., a conservative or non-conservative substitution), inserting one or more (typically less than 5 or 3) amino acids adjacent to a specified residue/position, and deleting said residue/position. "Amino acid substitution" or a variation thereof refers to the replacement of an existing amino acid residue in a predetermined (original) amino acid sequence with a different amino acid residue. Typically and preferably, the modification results in a change in at least one physico-biochemical activity of the variant polypeptide compared to a polypeptide containing the original (or "wild-type") amino acid sequence. For example, in the case of an antibody, the physicochemical activity that changes may be the binding affinity, binding capacity, and/or effect of binding to the target molecule.

The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or an antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention may be replaced with other amino acid residues from the same side chain family, and the altered CAR may be tested using the functional assays described herein.

The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (eg, TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as upregulation or downregulation of cytokines and cell surface proteins and/or reorganization of cytoskeletal structures, and the like.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence(s) that regulates the primary activation of the TCR complex in a stimulatory manner for at least some aspect of the T cell signaling pathway. In one aspect, a signal is a primary signal that is initiated, for example, by binding of a TCR/CD3 complex to an MHC molecule loaded with a peptide, and that results in the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. . The primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM).

The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., B cell, dendritic cell, macrophages, Langerhans cells, etc.) that presents a foreign antigen that forms a complex with basic complexes histocompatibility (MHC) on its surface for recognition by certain lymphocytes such as T cells. T cells can recognize these complexes using their T cell receptors (TCRs). Antigen-presenting cells can process antigens for presentation in combination with the MHC. As used herein, the term "antigen-presenting cell" or "APC" includes states in which APCs present antigen and states in which APCs do not present antigen. In some cases, APCs process antigens and present them to T cells. In other cases, T cells can recognize APCs in the absence of antigen presentation when the TCR binds directly to the MHC protein. For example, in the context of transplantation, APCs can directly stimulate T cells through the expression of foreign MHC proteins.

"Intracellular signaling domain", as used herein, refers to the intracellular portion of the CAR. The intracellular signaling domain generates a signal that promotes the immune effector function of a CAR containing cell, such as a CAR Treg cell. Examples of immune effector function, for example in a CAR Treg cell, may include the suppression or down regulation of effector function of other immune cells. Other immune cells include white blood cells of any type, for example, (but not limited to), T cells, B cells, NK cells. In addition, the immune effector function of Treg may include the effect on non-immune cells, which leads to an improvement in the clinical condition, for example, promotes repair or tissue regeneration.

The term "zeta" or alternatively "zeta chain" or "CD3 zeta" is defined as the protein provided as GenBank Acc. no. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, great ape, etc., and "zeta-stimulating domain" or "CD3-zeta-stimulating domain" is defined as amino acid residues of the cytoplasmic zeta chain domain, which are sufficient for the functional transmission of the initial signal required for the activation of T cells. In one embodiment, the cytoplasmic zeta domain includes residues 52 to 164 of GenBank Acc. no. BAG36664.1 or equivalent residues from a non-human species, eg mouse, rodent, monkey, great ape, etc., which are their functional orthologues.

A "costimulatory ligand" as used herein includes an antigen presenting cell molecule (e.g., APC, dendritic cell, B cell, and the like) that specifically binds a cognate costimulatory molecule on a T cell, thereby providing a signal that, in addition to the primary signal provided, for example, by binding of the TCR/CD3 complex to the MHC molecule loaded with the peptide, mediates T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The co-stimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonist or antibody that binds Toll ligand receptor and ligand , which specifically binds to B7-H3. A costimulatory ligand also includes, but is not limited to, a ligand, including an antibody that specifically binds to a costimulatory molecule present on a T cell, such as, but not limited to, MHC class I, BTLA, Toll Ligand receptor, OX40, CD27, CD28 , lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4- 1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, GITR, HVEM, SLAMF7, NKp80, CD160, IL2ra, IL6Ra, IL-7Ra, IL-13RA1 /RA2, IL-33R(IL1RL1), IL-10RA/RB, IL-4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1/2/3, common gamma chain, ligand that binds specifically to CD83, and any combination thereof.

The term "costimulatory molecule" refers to a related binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that promote an effective immune response. The costimulatory molecule can be present in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecule, BTLA, Toll ligand receptor, OX40, CD27, CD28, Lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A ), TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, GITR, HVEM, SLAMF7, NKp80, CD160, IL2ra, IL6Ra, IL-7Ra, IL-13RA1/RA2, IL-33R(IL1RL1), IL-10RA/RB, IL-4R, IL-5R (CSF2RB ), ARHR, BAFF receptor, IL-21R, TGFbR1/2/3, common gamma chain, a ligand that specifically binds to CD83, and any combination thereof.

A "costimulatory intracellular signaling domain" or "costimulatory domain" may be an intracellular portion of a costimulatory molecule. Examples of such molecules include MHC class I molecule, BTLA, Toll ligand receptor, OX40, CD27, CD28, Lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 ( CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, GITR, HVEM, SLAMF7, NKp80, CD160, IL2ra, IL6Ra, IL-7Ra, IL-13RA1/RA2, IL-33R(IL1RL1), IL-10RA/RB, IL-4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1/2/3, a common gamma chain, a ligand that specifically binds to CD83, and the like.

As currently used, the term "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR/CD3 ligation, results in T cell proliferation and/or up- or down-regulation of key molecules.

An intracellular signaling domain may include the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

"Disease" is a state of health in an animal in which the animal cannot maintain homeostasis, and if the state of the disease does not improve, then the health of the animal continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, the disorder does not necessarily cause a further decline in the health of the animal.

The term "coding" refers to the inherited property of specific nucleotide sequences in a polynucleotide, such as, for example, a gene, cDNA or mRNA, which serve as templates for the synthesis of other polymers and macromolecules in biological processes, having either a specific nucleotide sequence (for example, rRNA, tRNA and mRNA), or a certain sequence of amino acids and biological properties derived from them. Thus, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both a coding strand whose nucleotide sequence is identical to an mRNA sequence and is commonly listed in sequence listings, and a non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise indicated, "nucleotide sequence or nucleic acid sequence encoding an amino acid sequence" includes all nucleotides or nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding a protein may, in some versions, contain intron(s).

The term "graft" as used herein refers to cells, tissues, or organs that are introduced into an object. The source of the transplanted material may be cultured cells, cells from a different entity, or cells from the same entity (eg, after the cells have been cultured in vitro). Examples of organ transplantation are kidney, liver, heart, lung and pancreas. An example of tissue transplantation is islet cells. An example of cell transplantation is allogeneic hematopoietic stem cell transplantation.

The term "exogenous" refers to any material introduced or produced outside the body, cell, tissue or system.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "expression vector" refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector includes a sufficient number of cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (eg, "naked" or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that include the recombinant polynucleotide.

The term "lentivirus" refers to a genus in the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of the host cell, so they are one of the most efficient ways for a gene delivery vector. HIV, SIV and FIV are examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of the lentivirus genome, including in particular the self-inactivating lentiviral vector presented in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

The term "homologous" or "identity" refers to the sequence identity of a subunit between two polymeric molecules, for example between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the position of a subunit in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half (eg, five positions in a polymer of ten subunits long) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are the same or homologous, the two sequences are 90% homologous.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as, for example, Fv, scFv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and their antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or a rabbit having the desired specificity, affinity and potential. In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications can further improve and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment thereof will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs match those of a non-human immunoglobulin and all or significant a portion of the FR regions are human immunoglobulin sequences. The humanized antibody or antibody fragment may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more information, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

"Human" immunoglobulin, antibody or antibody fragment refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from coexisting materials in their natural state is "isolated". An isolated nucleic acid or peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell. An "isolated" antibody is an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that will interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutions.

In the context of the present invention, the following abbreviations of the most frequently occurring nucleic acid bases are used. "A" refers to adenine, "C" refers to cytosine, "G" refers to guanine, "T" refers to thymine, and "U" refers to uracil.

The term "operably linked" refers to a functional relationship between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is operably linked to a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects transcription or expression of the coding sequence. Functionally linked DNA sequences can be adjacent to each other and, for example, if necessary, connect two protein-coding regions that are in the same reading frame.

The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (SC), intradermal, intranodal, intramedullary, intraperitoneal, intravenous (IV), intramuscular (IM) or epigastric injections, intratumoral or infusion methods. .

The terms "nucleic acid" or "polynucleotide" are used interchangeably and refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. Thus, this term includes, without limitation, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA fusion or polymer containing purine and pyrimidine bases or other natural, chemical or biochemically modified, non-natural or derivatized nucleotide bases. Except where specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly specified sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that a protein or peptide sequence may include. Polypeptides include any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art, such as peptides, oligopeptides, and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, this sequence may be the sequence of the main promoter, and in other cases, this sequence may also include the sequence of the enhancer and other regulatory elements that are necessary for the expression of the gene product. The promoter/regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

The term "constitutive promoter" refers to a nucleotide sequence which, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be generated in a cell under most or all physiological conditions of the cell.

The term "inducible promoter" refers to a nucleotide sequence which, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer is present that corresponds to a promoter present in the cell.

As used herein, the term "transient" refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, where the expression time period is less than the time period for gene expression if integrated into the genome or contained in a stable plasmid replicon in the host cell. .

The term "substantially purified" cell refers to a cell that is substantially free of other cell types. As such, a purified cell also refers to a cell that has been separated from other types of cells with which it is normally associated in its natural state. In some cases, the population of essentially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that have been separated from the cells they are naturally associated with in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

The term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. A cell includes the object's primary cell and its offspring.

Various aspects of the invention may be presented throughout this disclosure in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to be specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and etc., as well as individual numbers in that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98%, or 99% identity and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the latitude of the range.

III. ANTI-HLA-A2 ANTIBODIES

In one aspect, the present invention relates to anti-HLA-A2 antibodies. Exemplary antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, human antibodies, humanized antibodies, and antigen-binding fragments thereof.

In one embodiment, the invention relates to humanized anti-HLA-A2 antibodies. The humanized anti-HLA-A2 antibodies provided herein specifically bind to HLA-A2. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein specifically bind to HLA-A*02:01. As will be appreciated by one of skill in the art, the ability of an antibody to bind to HLA-A2 can be detected using methods known in the art. For example, antibody binding to HLA-A2 can be detected using the HLA-A2 tetramer as illustrated herein. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein compete for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein bind to the same HLA-A2 epitope as the antibody, comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein compete for binding to HLA-A2 with antibody BB7.2.

In one embodiment, the humanized anti-HLA-A2 antibodies provided herein bind to the same HLA-A2 epitope as the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from three or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from four or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from five or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from six or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from seven or more of HLA-A*03, HLA-A*25 , HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from each of HLA-A*03, HLA-A*25, HLA -A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29 , HLA-A*30 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*25, HLA-A*29 , HLA-A*30 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from each of HLA-A*25, HLA-A*29, HLA -A*30 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least two of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least three of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least four of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least five of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least six of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least seven of HLA-A*03:01, HLA-A*25:01, HLA-A*29 :02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30 :01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least one of HLA-A*25:01, HLA-A*29:02 and HLA-A*30 :01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein are less reactive to at least two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30 :01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01 compared to the BB7 antibody. .2. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*25:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*29:02 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*30:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*03:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*31:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*33:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*36:01 compared to the BB7.2 antibody. In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*68:01 compared to the BB7.2 antibody. The BB7.2 antibody can be isolated from the BB7.2 hybridoma (ATCC deposit number HB-82).

Methods for determining the reactivity of humanized anti-HLA-A2 antibodies to HLA-A subtypes will be known to those skilled in the art. For example, the reactivity of humanized anti-HLA-A2 antibodies to HLA-A subtypes can be determined by assay using individual antigen bead assay. Such individual antigen bead assays are commercially available (eg, FlowPRA Single Antigen Antibody; ONE LAMBDA).

In one embodiment, the humanized anti-HLA-A2 antibodies provided herein have less reactivity to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, and any combination thereof compared to BB7.2 antibody, e.g. compared to the BB7.2 scFv antibody as measured under test A conditions. of one or more of HLA-A*25, HLA-A*29, HLA-A*30, and any combination thereof compared to a BB7.2 antibody, e.g. compared to a BB7.2 scFv when measured under Test A conditions .

Test A:

0.25×10 ⁶ T cells expressing a CAR containing a humanized anti-HLA-A2 antibody or a BB7.2 antibody, e.g. BB7.2 scFv (mA2 CAR)), were incubated with a panel of FlowPRA individual antigen beads (FL1HD01, FL1HD02, FL1HD03). FL1HD04, FL1HD06 and FL1HD08, One Lambda) and fixed viability stain (FVD, ThermoFisher, 65-0865-14, eBioscience) for 30 minutes at room temperature. Samples were washed, fixed with 0.5% formaldehyde and analyzed by flow cytometry. Two hundred negative control beads were used per sample. Pure granules were used as a negative control. For analysis, dead cells were first removed with fixed dye to assess viability. Beads with individual antigen were then selected for a signal above the threshold after the exclusion of dead cells and doublets. The number of beads per HLA was then determined by their respective PE intensity peak. Data were normalized by multiplying the number of beads of interest in each HLA peak by 200 divided by the number of negative beads in the sample. For each HLA peak, the percentage of relative binding of CAR Treg compared to control (cells not expressing CAR) was determined by subtracting the number of beads in CAR-Treg from the number of beads in the control, then dividing the average number of beads in the non-CAR-expressing control by 100 .

In one embodiment, the humanized anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA -A*30, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 statistically inferior BB7.2 antibody reactivity, e.g. when measured under Test A conditions .

In one embodiment, the anti-HLA-A2 antibody of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30, statistically inferior to the reactivity of the BB7.2 antibody, for example, when measured under test A conditions.

In one embodiment, the term "statistically inferior" means that the reactivity (i.e., for example, relative binding under Test A conditions) measured for an anti-HLA-A2 antibody of the invention is lower than that measured for a BB7.2 antibody, with p- a value of at most about 0.05, preferably at most about 0.01, more preferably at most about 0.005, and even more preferably at most about 0.001, particularly when analyzed with using two-way ANOVA, Dunnett's post hoc test.

In one embodiment, the anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA- A*30, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 inferior BB7.2 antibody reactivity. In some embodiments, such an anti-HLA-A2 antibody has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA- A*30, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 inferior to BB7.2 antibody binding when measured under test A conditions. In certain aspects, relative the binding measured for such an anti-HLA-A2 antibody is at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less relative binding, measured for the BB7.2 antibody.

In one embodiment, the anti-HLA-A2 antibody of the invention has inferior reactivity to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30 BB7.2 antibodies. In some embodiments, such an anti-HLA-A2 antibody has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30 inferior to binding BB7.2 antibody when measured under test A conditions. In certain aspects, the relative binding measured for such an anti-HLA-A2 antibody is at most about 90%, 80%, 70%, 60%, 50%, 40 %, 30%, 20%, 10% or less relative binding as measured for antibody BB7.2.

In addition, the humanized anti-HLA-A2 antibodies provided herein having antigen-binding activity are capable of forming chimeric antigen receptor (CAR) antigen-binding domains, wherein such CARs are capable of being expressed in human cells such that the CARs specifically bind to HLA-A2. In one embodiment, CARs specifically bind to HLA-A*02:01. As will be appreciated by one of skill in the art, the ability of an antibody to bind to HLA-A2 can be detected using methods known in the art. For example, CAR binding to HLA-A2 can be detected using the HLA-A2 tetramer as illustrated herein. In one embodiment, the human cell is an immune cell. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg. In addition, the humanized anti-HLA-A2 antibodies provided herein having antigen-binding activity are capable of forming chimeric antigen receptor (CAR) antigen-binding domains, wherein such CARs are capable of being expressed in regulatory T cells such that the CARs specifically bind to HLA- A2. In one embodiment, CARs specifically bind to HLA-A*02:01. In one embodiment, the Treg is a human Treg.

In one embodiment, the humanized anti-HLA-A2 antibody is capable of constituting an antigen-binding domain of a CAR, wherein such CAR is capable of being expressed in an immune cell such that the immune cell is activated by HLA-A2. In one embodiment, the immune cell is activated by HLA-A*02:01. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the T cell is a human T cell. In one embodiment, the Treg is a human Treg.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SYHIQ (SEQ ID NO: 1) and GYTFTSY (SEQ ID NO: 2).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 1 (VH CDR1) selected from SEQ ID NOs: 1-2. In one embodiment, a humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a VH CDR1 represented by SEQ ID NO: 1. In one embodiment, a humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a VH CDR1 represented by SEQ ID NO: 2.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: YPGDGS (SEQ ID NO: 4) and WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G (SEQ ID NO: 10), where in SEQ ID NO: 10, the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G (SEQ ID NO: 10) wherein the amino acid at position 10 (X ¹⁰ ) is Q or K , the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: YPGDGS (SEQ ID NO: 4) and WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G (SEQ ID NO: 10), where in SEQ ID NO: 10, the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing complementarity determining region 2 (VH CDR2) containing the amino acid sequence WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G (SEQ ID NO: 10), where the amino acid in position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is S, the amino acid at position 13 is Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is N or S, the amino acid at position 13 is Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is S, the amino acid at position 13 is E or Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is S, the amino acid at position 13 is Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is N or S, the amino acid at position 13 is Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is S, the amino acid at position 13 is E or Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is S, the amino acid at position 13 is Q, and the amino acid at position 16 is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is N or S, the amino acid at position 13 is E or Q, and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is N or S, the amino acid at position 13 is Q, and the amino acid at position 16 is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is S, the amino acid at position 13 is E or Q, and the amino acid at position 16 is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is N or S, the amino acid at position 13 is E or Q and the amino acid at position 16 is Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is N or S, the amino acid at position 13 is Q and the amino acid at position 16 is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is Q or K, the amino acid at position 12 is S, the amino acid at position 13 is E or Q and the amino acid at position 16 is K or Q.

In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, wherein the amino acid at position 10 is K, the amino acid at position 12 is N or S, the amino acid at position 13 is E or Q and the amino acid at position 16 is K or Q.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: WIYPGDGSTQYNEKFKG (SEQ ID NO: 3), YPGDGS (SEQ ID NO: 4) and WIYPGDGSTKYSQKFQG (SEQ ID NO: : 5). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: WIYPGDGSTQYNEKFKG (SEQ ID NO: 3) and YPGDGS (SEQ ID NO: 4). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: YPGDGS (SEQ ID NO: 4) and WIYPGDGSTKYSQKFQG (SEQ ID NO: 5).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 4. In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) selected from SEQ ID NOs: 3-5. In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) selected from SEQ ID NOs: 3-4. In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) selected from SEQ ID NOs: 4-5. In one embodiment, a humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) represented by SEQ ID NO: 5. In one embodiment, a humanized anti-HLA-A2 antibody comprises a heavy chain variable region, containing the VH CDR2 represented by SEQ ID NO: 3. In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing the VH CDR2 represented by SEQ ID NO: 4.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing the amino acid sequence EGTYYAMDY (SEQ ID NO: 6).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 3 (VH CDR3) represented by SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 1; or

VH CDR2 shown in SEQ ID NO: 10, wherein the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 1; or

VH CDR2 shown in SEQ ID NO: 3; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 1; or

VH CDR2 shown in SEQ ID NO: 5; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 1; one VH CDR2 shown in SEQ ID NO: 10, where the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q; and one VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 1; one VH CDR2 shown in SEQ ID NO: 3; and one VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 1; one VH CDR2 shown in SEQ ID NO: 5; and one VH CDR3 shown in SEQ ID NO: 6.

According to the present invention, any of the heavy chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 1, 3, 5, 6 and 10.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 2; or

VH CDR2 shown in SEQ ID NO: 4; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 2; one VH CDR2 shown in SEQ ID NO: 4; and one VH CDR3 shown in SEQ ID NO: 6.

According to the present invention, any of the heavy chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 2, 4 and 6.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing the complementarity determining region 1 (VH CDR1) shown in SEQ ID NO: 7.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence KVSNRFS (SEQ ID NO: 8).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) shown in SEQ ID NO: 8.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence FQGSHVPRT (SEQ ID NO: 9).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 3 (VH CDR3) shown in SEQ ID NO: 9.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing at least one of the following CDRs:

VL CDR1 shown in SEQ ID NO: 7; or

VL CDR2 shown in SEQ ID NO: 8; or

VL CDR3 shown in SEQ ID NO: 9.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region comprising one VL CDR1 as set forth in SEQ ID NO: 7; one VL CDR2 shown in SEQ ID NO: 8; and one VL CDR3 shown in SEQ ID NO: 9.

According to the present invention, any of the light chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 7, 8 and 9.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) comprising an amino acid sequence selected from the group consisting of: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 11) and QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO : 12).

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 11). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 12). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing an amino acid sequence selected from the group consisting of:

WVRQAPGQX ⁹ LEWMGX ¹⁵ (SEQ ID NO: 13),

WVRQAPGQX ⁹ LEWMGX ¹⁵ WI (SEQ ID NO: 17),

HIQWVRQAPGQX ¹² LEWMGX ¹⁸ WI (SEQ ID NO: 21), and

HIQWVRQAPGQX ¹² LEWMGX ¹⁸ (SEQ ID NO: 25), where:

X ⁹ is R or G and X15 is I or not present in SEQ ID NO: 13;

X ⁹ is R or G and X15 is I or not present in SEQ ID NO: 17;

X ¹² is R or G and X18 is I or not present in SEQ ID NO: 21; and

X ¹² is R or G and X ¹⁸ is I or not present in SEQ ID NO: 25.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence WVRQAPGQX ⁹ LEWMGX ¹⁵ (SEQ ID NO: 13), where the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 13, where the amino acid at position 9 is R or G and the amino acid at position 15 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 14, 15, or 16. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 14, 15 or 16.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence HIQWVRQAPGQX ¹² LEWMGX ¹⁸ (SEQ ID NO: 25), where the amino acid at position 12 (X ¹² ) is is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 25, where the amino acid at position 12 is R or G and the amino acid at position 18 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 26, 27, or 28. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 26, 27 or 28.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence WVRQAPGQX ⁹ LEWMGX ¹⁵ WI (SEQ ID NO: 17), where the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 17, where the amino acid at position 9 is R or G and the amino acid at position 15 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 18, 19, or 20. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 18, 19 or 20.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence HIQWVRQAPGQX ¹² LEWMGX ¹⁸ WI (SEQ ID NO: 21), where the amino acid at position 12 (X ¹² ) is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 25, where the amino acid at position 12 is R or G and the amino acid at position 18 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 22, 23, or 24. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 22, 23 or 24.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR2) containing an amino acid sequence selected from the group consisting of:

X ¹ VTX ⁴ TX ⁶ DTSX ¹⁰ STAYMX ¹⁶ LSX ¹⁹ LRSX ²³ DX ²⁵ AVYYCAR (SEQ ID NO: 29),

TX ² YX ⁴ X ⁵ KFX ⁸ GX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴

AVYYCAR (SEQ ID NO: 35),

TQYNEKFKGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴

AVYYCAR (SEQ ID NO: 36) and

TKYSQKFQGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR (SEQ ID NO: 37), where:

X ¹ is R or absent, X ⁴ is I or M, X ⁶ is R or A, X ¹⁰ is A, T or I, X ¹⁶ is E or L, X ¹⁹ is S or R, X ²³ is E or D and X ²⁵ is T or M in SEQ ID NO: 29;

X ² is Q or K, X ⁴ is N or S, X ⁵ is E or Q, X8 is K or Q, X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO : 35;

X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO: 36; and

X ¹⁰ is R or absent, X ¹³ is I or M, X ¹⁵ is R or A, X ¹⁹ is A, T or I, X ²⁵ is E or L, X ²⁸ is S or R, X ³² is E or D and X ³⁴ is T or M in SEQ ID NO: 37.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR3) containing the amino acid sequence X ¹ VTX ⁴ TX ⁶ DTSX ¹⁰ STAYMX ¹⁶ LSX ¹⁹ LRSX ²³ DX ²⁵ AVYYCAR (SEQ ID NO: 29), where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 (X ¹⁰ ) is A, T or I, amino acid at position 16 (X ¹⁶ ) is E or L, amino acid at position 19 (X ¹⁹ ) is S or R, amino acid at position 23 (X ²³ ) is E or D, and the amino acid at position 25 (X ²⁵ ) is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, where the amino acid at position 1 is R, the amino acid at position 4 is I or M, the amino acid at position 6 is R, the amino acid at position 10 is A or I, the amino acid at position 16 is E, the amino acid at position 19 is S or R, the amino acid at position 23 is E or D, and the amino acid at position 25 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, where the amino acid at position 1 is R, the amino acid at position 4 is I or M, the amino acid at position 6 is R, the amino acid at position 10 is A or I, the amino acid at position 16 is E, the amino acid at position 19 is S or R, the amino acid at position 23 is E or D, and the amino acid at position 25 is T.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, where the amino acid at position 1 is R, the amino acid at position 4 is I, the amino acid at position 6 is is R, the amino acid at position 10 is A, the amino acid at position 16 is E, the amino acid at position 19 is S, the amino acid at position 23 is E, and the amino acid at position 25 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NOs: 30, 31, 32, 33, or 34. In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3 ) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 30, 31, 32, 33 or 34.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR3) containing the amino acid sequence TX ² YX4X ⁵ KFX ⁸ GX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR (SEQ ID NO: 35) wherein the amino acid at position 2 (X ² ) is Q or K, the amino acid at position 4 (X ⁴ ) is N or S, the amino acid at position 5 (X ⁵ ) is E or Q, amino acid at position 8 (X ⁸ ) is K or Q, amino acid at position 10 (X ¹⁰ ) is R or absent, amino acid at position 13 (X ¹³ ) is I or M, amino acid at position 15 (X ¹⁵ ) is R or A, the amino acid at position 19 (X ¹⁹ ) is A, T or I, the amino acid at position 25 (X ²⁵ ) is E or L, the amino acid at position 28 (X ²⁸ ) is S or R, the amino acid at position 32 (X ³² ) is E or D, and the amino acid at position 34 (X ³⁴ ) is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I, the amino acid at position 15 is R, the amino acid at position 19 is A , the amino acid at position 25 is E, the amino acid at position 28 is S, the amino acid at position 32 is E, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N, the amino acid at position 5 is E, amino acid at position 8 is K, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I, amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N or S, the amino acid at position 5 is E, amino acid at position 8 is K, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I, amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N, the amino acid at position 5 is is E or Q, the amino acid at position 8 is K, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N, the amino acid at position 5 is is E, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E, the amino acid at position 8 is K, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N, the amino acid at position 5 is E or Q, amino acid at position 8 is K, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N, the amino acid at position 5 is E, amino acid at position 8 is K or Q, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, amino acid at position 8 is K, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N or S, the amino acid at position 5 is E, amino acid at position 8 is K or Q, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N, the amino acid at position 5 is is E or Q, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, the amino acid at position 8 is K, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N or S, the amino acid at position 5 is E, the amino acid at position 8 is K or Q, the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q or K, the amino acid at position 4 is N, the amino acid at position 5 is E, amino acid at position 8 is K or Q, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I , the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 35, where the amino acid at position 2 is Q, the amino acid at position 4 is N or S, the amino acid at position 5 is E or Q, amino acid at position 8 is K or Q, amino acid at position 10 is R, amino acid at position 13 is I or M, amino acid at position 15 is R, amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR3) containing the amino acid sequence TQYNEKFKGX ¹⁰ VTX13TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR (SEQ ID NO: 36) where the amino acid at position 10 (X ¹⁰ ) is R or absent, the amino acid at position 13 (X ¹³ ) is I or M, the amino acid at position 15 (X ¹⁵ ) is R or A, the amino acid at position 19 (X ¹⁹ ) is A, T or I, the amino acid at position 25 (X ²⁵ ) is E or L, the amino acid at position 28 (X ²⁸ ) is S or R, the amino acid at position 32 (X ³² ) is E or D, and the amino acid at position 34 (X ³⁴ ) is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 36, where the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 36, where the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 36, where the amino acid at position 10 is R, the amino acid at position 13 is I, the amino acid at position 15 is is R, the amino acid at position 19 is A, the amino acid at position 25 is E, the amino acid at position 28 is S, the amino acid at position 32 is E, and the amino acid at position 34 is T or M.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR3) containing the amino acid sequence TKYSQKFQGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR (SEQ ID NO: 37), where the amino acid at position 10 (X ¹⁰ ) is R or absent, the amino acid at position 13 (X ¹³ ) is I or M, the amino acid at position 15 (X ¹⁵ ) is R or A, the amino acid at position 19 (X ¹⁰ ) is A, T or I, amino acid at position 25 (X ²⁵ ) is E or L, amino acid at position 28 (X ²⁸ ) is S or R, amino acid at position 32 (X ³² ) is E or D, and the amino acid at position 34 (X ³⁴ ) is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 37, where the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 37, where the amino acid at position 10 is R, the amino acid at position 13 is I or M, the amino acid at position 15 is R, the amino acid at position 19 is A or I, the amino acid at position 25 is E, the amino acid at position 28 is S or R, the amino acid at position 32 is E or D, and the amino acid at position 34 is T.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 37, where the amino acid at position 10 is R, the amino acid at position 13 is I, the amino acid at position 15 is is R, the amino acid at position 19 is A, the amino acid at position 25 is E, the amino acid at position 28 is S, the amino acid at position 32 is E, and the amino acid at position 34 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NOs: 38, 39, 40, 41, 42, or 43. In one embodiment, the heavy chain variable region comprises framework region 3 ( VH FR3) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 38, 39, 40, 41, 42 or 43.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 4 (VH FR1) containing the amino acid sequence WGQGTTVTVSS (SEQ ID NO: 44). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR4) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 shown in SEQ ID NO: 1;

VH CDR2 shown in SEQ ID NO: 10, wherein the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 12;

VH FR2 shown in SEQ ID NO: 13, where the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent;

VH FR3 shown in SEQ ID NO: 29, where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 (X ¹⁰ ) is A, T or I, the amino acid at position 16 (X ¹⁶ ) is E or L, the amino acid at position 19 (X ¹⁹ ) is S or R, the amino acid at position 23 (X ²³ ) is E or D and the amino acid at position 25 (X ²⁵ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 represented by SEQ ID NO: 2;

VH CDR2 shown in SEQ ID NO: 10, wherein the amino acid at position 10 (X ¹⁰ ) is Q or K, the amino acid at position 12 (X ¹² ) is N or S, the amino acid at position 13 (X ¹³ ) is E or Q, and the amino acid at position 16 (X ¹⁶ ) is K or Q;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 11;

VH FR2 shown in SEQ ID NO: 25, wherein the amino acid at position 12 (X ¹² ) is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent;

VH FR3 shown in SEQ ID NO: 29, where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 (X ¹⁰ ) is A, T or I, the amino acid at position 16 (X ¹⁶ ) is E or L, the amino acid at position 19 (X ¹⁹ ) is S or R, the amino acid at position 23 (X ²³ ) is E or D and the amino acid at position 25 (X ²⁵ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 represented by SEQ ID NO: 2;

VH CDR2 shown in SEQ ID NO: 4;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 11;

VH FR2 shown in SEQ ID NO: 21, wherein the amino acid at position 12 (X ¹² ) is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent;

VH FR3 shown in SEQ ID NO: 35, wherein the amino acid at position 2 (X ² ) is Q or K, the amino acid at position 4 (X ⁴ ) is N or S, the amino acid at position 5 (X ⁵ ) is E or Q, amino acid at position 8 (X ⁸ ) is K or Q, amino acid at position 10 (X ¹⁰ ) is R or absent, amino acid at position 13 (X ¹³ ) is I or M, amino acid at position 15 (X ¹⁵ ) is R or A, the amino acid at position 19 (X ¹⁹ ) is A, T or I, the amino acid at position 25 (X ²⁵ ) is E or L, the amino acid at position 28 (X ²⁸ ) is S or R, the amino acid at position 32 (X ³² ) is E or D, and the amino acid at position 34 (X ³⁴ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 shown in SEQ ID NO: 1;

VH CDR2 shown in SEQ ID NO: 4;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 12;

VH FR2 shown in SEQ ID NO: 17, wherein the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent;

VH FR3 shown in SEQ ID NO: 35, wherein the amino acid at position 2 (X ² ) is Q or K, the amino acid at position 4 (X ⁴ ) is N or S, the amino acid at position 5 (X ⁵ ) is E or Q, amino acid at position 8 (X ⁸ ) is K or Q, amino acid at position 10 (X ¹⁰ ) is R or absent, amino acid at position 13 (X ¹³ ) is I or M, amino acid at position 15 (X ¹⁵ ) is R or A, the amino acid at position 19 (X ¹⁹ ) is A, T or I, the amino acid at position 25 (X ²⁵ ) is E or L, the amino acid at position 28 (X ²⁸ ) is S or R, the amino acid at position 32 (X ³² ) is E or D, and the amino acid at position 34 (X ³⁴ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

In one embodiment of the invention, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQX ⁴⁴ LEWMGX ⁵⁰ WIYPG DGSTX ⁶⁰ YX ⁶² X ⁶³ KFX ⁶⁶ GX ⁶⁸ VTX ⁷¹ TX ^{73 DTSX 90 DXLR90 83 LSX} AVYYCARE GTYYAMDYWGQGTTVTVSS (SEQ ID NO: 45) wherein the amino acid at position 44 (X ⁴⁴ ) is R or G, the amino acid at position 50 (X ⁵⁰ ) is I or absent, the amino acid at position 60 (X ⁶⁰ ) is Q or K, the amino acid at position 62 (X ⁶² ) is N or S, the amino acid at position 63 (X ⁶³ ) is E or Q, the amino acid at position 66 (X ⁶⁶ ) is K or Q, the amino acid at position 68 ( X ⁶⁸ ) is R or absent, amino acid at position 71 (X ⁷¹ ) is I or M, amino acid at position 73 (X ⁷³ ) is R or A, amino acid at position 77 (X ⁷⁷ ) is oh A, T, or I, the amino acid at position 83 (X ⁸³ ) is E or L, the amino acid at position 86 (X ⁸⁶ ) is S or R, the amino acid at position 90 (X ⁹⁰ ) is E or D, and the amino acid at position 92 (X ⁹² ) is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G; the amino acid at position 50 is missing; the amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; the amino acid at position 68 is R; the amino acid at position 71 is I or M; the amino acid at position 73 is R; the amino acid at position 77 is A or I; the amino acid at position 83 is E; the amino acid at position 86 is S or R; the amino acid at position 90 is E or D; and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G; the amino acid at position 50 is missing; the amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; the amino acid at position 68 is R; the amino acid at position 71 is I or M; the amino acid at position 73 is R; the amino acid at position 77 is A or I; the amino acid at position 83 is E; the amino acid at position 86 is S or R; the amino acid at position 90 is E or D; and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R, no amino acid at position 50, amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I, amino acid at position 73 is R, amino acid at position 77 is A, amino acid at position 83 is E, amino acid at position 86 is S, amino acid at position 90 is E and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q; the amino acid at position 62 is N; the amino acid at position 63 is E; the amino acid at position 66 is K; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q; the amino acid at position 62 is N; the amino acid at position 63 is E; the amino acid at position 66 is K; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R, no amino acid at position 50, amino acid at position 60 is Q; the amino acid at position 62 is N; the amino acid at position 63 is E; the amino acid at position 66 is K; amino acid at position 68 is R, amino acid at position 71 is I, amino acid at position 73 is R, amino acid at position 77 is A, amino acid at position 83 is E, amino acid at position 86 is S, amino acid at position 90 is E and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, there is no amino acid at position 50, the amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, the amino acid at position 50 is absent, the amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I, amino acid at position 73 is R, amino acid at position 77 is A, amino acid at position 83 is E, amino acid at position 86 is S, amino acid at position 90 is E and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q or K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is N or S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is E or Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, the amino acid at position 50 is absent, the amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, there is no amino acid at position 50, the amino acid at position 60 is Q or K; the amino acid at position 62 is S; the amino acid at position 63 is E or Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q or K; the amino acid at position 62 is S; the amino acid at position 63 is Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is N or S; the amino acid at position 63 is Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, there is no amino acid at position 50, the amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein the amino acid at position 44 is R or G, there is no amino acid at position 50, the amino acid at position 60 is Q or K; the amino acid at position 62 is N or S; the amino acid at position 63 is Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is Q or K; the amino acid at position 62 is S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45, wherein amino acid at position 44 is R or G, no amino acid at position 50, amino acid at position 60 is K; the amino acid at position 62 is N or S; the amino acid at position 63 is E or Q; the amino acid at position 66 is K or Q; amino acid at position 68 is R, amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 61, 62, 63, 64, 65, or 66. In one embodiment, the humanized anti-HLA-A2 antibody comprises the variable a heavy chain region containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 61, 62, 63, 64, 65 or 66.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing framework region 1 (VL FR1) containing the amino acid sequence DX ² VMTQX ⁷ PLSX ¹¹ X ¹² VTX ¹⁵ GQPASISX ²³ (SEQ ID NO: 46), where the amino acid at position 2 (X ² ) is V or I, the amino acid at position 7 (X ⁷ ) is S or T, the amino acid at position 11 (X ¹¹ ) is L or S; the amino acid at position 12 (X ¹² ) is P or S; the amino acid at position 15 (X ¹⁵ ) is L or P, and the amino acid at position 23 (X ²³ ) is C or F.

In one embodiment, the light chain variable region comprises framework region 1 (VL FR1) comprising the amino acid sequence shown in SEQ ID NO: 46, wherein the amino acid at position 2 is V or I; the amino acid at position 7 is S or T; the amino acid at position 11 is L or S; the amino acid at position 12 is P or S; the amino acid at position 15 is L or P; and the amino acid at position 23 is C.

In one embodiment, the light chain variable region comprises framework region 1 (VL FR1) comprising the amino acid sequence shown in SEQ ID NO: 46, where the amino acid at position 2 is I; the amino acid at position 7 is T; the amino acid at position 11 is L or S; the amino acid at position 12 is P or S; the amino acid at position 15 is L or P; and the amino acid at position 23 is C.

In one embodiment, the light chain variable region comprises framework region 1 (VL FR1) comprising the amino acid sequence shown in SEQ ID NO: 46, wherein the amino acid at position 2 is V or I; the amino acid at position 7 is S or T; the amino acid at position 11 is L; the amino acid at position 12 is P or S; the amino acid at position 15 is L or P; and the amino acid at position 23 is C.

In one embodiment, the light chain variable region comprises framework region 1 (VL FR1) comprising the amino acid sequence shown in SEQ ID NO: 46, wherein the amino acid at position 2 is V or I; the amino acid at position 7 is S or T; the amino acid at position 11 is L or S; the amino acid at position 12 is P; the amino acid at position 15 is L; and the amino acid at position 23 is C.

In one embodiment, the light chain variable region comprises framework region 1 (VL FR1) comprising the amino acid sequence shown in SEQ ID NOs: 47, 48, 49, or 50. In one embodiment, the light chain variable region comprises framework region 1 (VL FR1), containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 47, 48, 49 or 50.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing framework region 2 (VL FR2) containing the amino acid sequence WX ² X ³ QX ⁵ PGQX ⁹ PX ¹¹ X ¹² LIY (SEQ ID NO: 51), where amino acid at position 2 (X ² ) is F or Y, amino acid at position 3 (X ³ ) is Q or L, amino acid at position 5 (X ⁵ ) is R or K, amino acid at position 9 (X ⁹ ) is S or P, the amino acid at position 11 (X ¹¹ ) is R or Q, and the amino acid at position 12 (X ¹² ) is R or L.

In one embodiment, the light chain variable region comprises framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NO: 51, wherein the amino acid at position 2 is Y, the amino acid at position 3 is Q or L, the amino acid at position 5 is is R or K, the amino acid at position 9 is S or P, the amino acid at position 11 is R or Q, and the amino acid at position 12 is L.

In one embodiment, the light chain variable region comprises framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NO: 51, wherein the amino acid at position 2 is Y, the amino acid at position 3 is Q or L, the amino acid at position 5 is is R or K, the amino acid at position 9 is S, the amino acid at position 11 is R or Q, and the amino acid at position 12 is L.

In one embodiment, the light chain variable region comprises a framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NO: 51, wherein the amino acid at position 2 is Y, the amino acid at position 3 is Q, the amino acid at position 5 is R , the amino acid at position 9 is S or P, the amino acid at position 11 is R, and the amino acid at position 12 is L.

In one embodiment, the light chain variable region comprises framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NO: 51, wherein the amino acid at position 2 is F or Y, the amino acid at position 3 is Q or L, the amino acid at position 5 is R or K, the amino acid at position 9 is S, the amino acid at position 11 is R or Q, and the amino acid at position 12 is R or L.

In one embodiment, the light chain variable region comprises framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NO: 51, where the amino acid at position 2 is F or Y, the amino acid at position 3 is Q, the amino acid at position 5 is R, the amino acid at position 9 is S or P, the amino acid at position 11 is R, and the amino acid at position 12 is R or L.

In one embodiment, the light chain variable region comprises framework region 2 (VL FR2) comprising the amino acid sequence shown in SEQ ID NOs: 52, 53, 54, or 55. In one embodiment, the light chain variable region comprises framework region 1 (VL FR2), containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 52, 53, 54 or 55.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing framework region 3 (VL FR3) containing the amino acid sequence GVPDRFSGSGX ¹¹ GTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 56), where the amino acid at position 11 (X ¹¹ ) is S or A.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing framework region 3 (VL FR3) comprising the amino acid sequence shown in SEQ ID NO: 57 or 58. In one embodiment, the humanized anti-HLA-A2 antibody comprises heavy chain variable region containing framework region 3 (VL FR3) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 57 or 58.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region containing framework region 4 (FR4 VL) containing the amino acid sequence FGGGTKVEIK (SEQ ID NO: 59). In one embodiment, the humanized anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 4 (VL FR4) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59.

В одном воплощении изобретения гуманизированным анти-HLA-A2-антитело содержит вариабельный участок легкой цепи, содержащий аминокислотной последовательности DX ² VMTQX ⁷ PLSX ¹¹ X ¹² VTX ¹⁵ GQPASISX ²³ RSSQSIVHSNGNTYLEWX ⁴¹ X ⁴² QX ⁴⁴ PGQX ⁴⁸ PX ⁵⁰ X ⁵¹ LIYKVSNRFSGVPDRFSGSGX ⁷² GTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGG GTKVEIK ( SEQ ID NO: 60) wherein the amino acid at position 2 (X ² ) is B or I, the amino acid at position 7 (X ⁷ ) is S or T, the amino acid at position 11 (X ¹¹ ) is L or S , the amino acid at position 12 (X ¹² ) is P or S, the amino acid at position 15 (X ¹⁵ ) is L or P, the amino acid at position 23 (X ²³ ) is C or F, the amino acid at position 41 (X ⁴¹ ) is F or Y, the amino acid at position 42 (X ⁴² ) is Q or L, the amino acid at position 44 (X ⁴⁴ ) is R or K, the amino acid at position 48 (X ⁴⁸ ) is S or P, the amino acid in p position 50 (X ⁵⁰ ) is R or Q, the amino acid at position 51 (X ⁵¹ ) is R or L, and the amino acid at position 72 (X ⁷² ) is S or A.

In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, wherein the amino acid at position 2 is V or I, the amino acid at position 7 is S or T, the amino acid at position 11 is L or S, amino acid at position 12 is P or S, amino acid at position 15 is L or P, amino acid at position 23 is C, amino acid at position 41 is Y, amino acid at position 42 is Q or L, amino acid at position 44 is R or K, the amino acid at position 48 is S or P, the amino acid at position 50 is R or Q, the amino acid at position 51 is L, and the amino acid at position 72 is S or A.

In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, wherein the amino acid at position 2 is I, the amino acid at position 7 is T, the amino acid at position 11 is L or S, the amino acid at position 12 is P or S, the amino acid at position 15 is L or P, the amino acid at position 23 is C, the amino acid at position 41 is Y, the amino acid at position 42 is Q or L, the amino acid at position 44 is R or K, the amino acid at position 48 is S or P, the amino acid at position 50 is R or Q, the amino acid at position 51 is L, and the amino acid at position 72 is S or A.

In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, where the amino acid at position 2 is V or I, the amino acid at position 7 is S or T, the amino acid at position 11 is L, the amino acid at position 12 is P or S, amino acid at position 15 is L or P, amino acid at position 23 is C, amino acid at position 41 is Y, amino acid at position 42 is Q or L, amino acid at position 44 is R or K, the amino acid at position 48 is S, the amino acid at position 50 is R or Q, the amino acid at position 51 is L, and the amino acid at position 72 is S.

In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, wherein the amino acid at position 2 is V or I, the amino acid at position 7 is S or T, the amino acid at position 11 is L or S, amino acid at position 12 is P or S, amino acid at position 15 is L, amino acid at position 23 is C, amino acid at position 41 is Y, amino acid at position 42 is Q, amino acid at position 44 is R, the amino acid at position 48 is S or P, the amino acid at position 50 is R, the amino acid at position 51 is L, and the amino acid at position 72 is S or A.

In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, wherein the amino acid at position 2 is V or I, the amino acid at position 7 is S or T, the amino acid at position 11 is L or S, the amino acid at position 12 is P or S, the amino acid at position 15 is L or P, the amino acid at position 23 is C, the amino acid at position 41 is F or Y, the amino acid at position 42 is Q or L, the amino acid at position 44 is R or K, the amino acid at position 48 is S or P, the amino acid at position 50 is R or Q, the amino acid at position 51 is R or L, and the amino acid at position 72 is S or A.

In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 67, 68, 69, 70, or 71. In one embodiment, the humanized anti-HLA-A2 antibody comprises a light chain variable region. a chain containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:67, 68, 69, 70 or 71.

In one embodiment, the humanized anti-HLA-A2 antibody is a scFv, scFab, or sdAb. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv or scFab. In one embodiment, the humanized anti-HLA-A2 antibody is an sdAb. In one embodiment, the humanized anti-HLA-A2 antibody is a scFab. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv containing the amino acid sequence shown in SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91.

In one embodiment, the humanized anti-HLA-A2 antibody contains an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO above. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv or scFab containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO above.

In one aspect, the invention provides an anti-HLA antibody comprising a heavy chain variable region comprising the amino acid sequence WIYPGDGSTKYSQKFQG (SEQ ID NO: 5).

In one embodiment, said antibody is an antibody mimetic selected from the group consisting of affitel, alpha body, armadillo repeat protein scaffold, knottin, Kunitz domain peptide, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, finomer, versatel and duocalin.

The anti-HLA-A2 antibodies provided herein specifically bind to HLA-A2. In one embodiment, the anti-HLA-A2 antibodies provided herein specifically bind to HLA-A*02:01. As will be appreciated by one of skill in the art, the ability of an antibody to bind to HLA-A2 can be detected using methods known in the art. For example, antibody binding to HLA-A2 can be detected using the HLA-A2 tetramer as illustrated herein. In one embodiment, the anti-HLA-A2 antibodies provided herein compete for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, the anti-HLA-A2 antibodies provided herein bind to the same HLA-A2 epitope as an antibody comprising : heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, the anti-HLA-A2 antibodies provided herein compete for binding to HLA-A2 with antibody BB7.2. In one embodiment, the anti-HLA-A2 antibodies provided herein bind to the same HLA-A2 epitope as the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) represented by SEQ ID NO: 5. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31 , HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from three or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from four or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from five or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from six or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from seven or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from each of HLA-A*03, HLA-A*25, HLA- A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29, HLA-A*30 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*25, HLA-A*29, HLA-A*30 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from each of HLA-A*25, HLA-A*29, HLA- A*30 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to at least one of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least two of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least three of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least four of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least five of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least six of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least seven of HLA-A*03:01, HLA-A*25:01, HLA-A*29: 02, HLA-A*30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30: 01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to at least one of HLA-A*25:01, HLA-A*29:02, and HLA-A*30: 01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30: 01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*25:01, HLA-A*29:02 and HLA-A*30:01 compared to the BB7 antibody. 2. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*25:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*29:02 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*30:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*03:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*31:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*33:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*36:01 compared to the BB7.2 antibody. In one embodiment, the anti-HLA-A2 antibodies provided herein have less reactivity to HLA-A*68:01 compared to the BB7.2 antibody. The BB7.2 antibody can be isolated from the BB7.2 hybridoma (ATCC deposit number HB-82). Methods for determining the reactivity of anti-HLA-A2 antibodies to HLA-A subtypes will be known to those skilled in the art. For example, the reactivity of anti-HLA-A2 antibodies to HLA-A subtypes can be determined using a single antigen bead assay. Such single antigen bead assays are commercially available (eg, FlowPRA Single Antigen Antibody; ONE LAMBDA).

In one embodiment, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 versus BB7.2 antibody when measured under test A conditions.

For example, in some embodiments, the anti-HLA-A2 antibodies provided herein are less reactive to at least one HLA-A subtype selected from the group consisting of HLA-A* 25, HLA-A* 29, HLA -A* 30 and any combination thereof compared to BB7.2 antibody when measured under Test A conditions.

Test A:

^0.25⋅106 T cells expressing a CAR containing an anti-HLA-A2 antibody or a BB7.2 antibody, e.g. BB7.2 scFv (mA2 CAR)), were incubated with a panel of FlowPRA individual antigen beads (FL1HD01, FL1HD02, FL1HD03 ). FL1HD04, FL1HD06 and FL1HD08, One Lambda) and fixed viability stain (FVD, ThermoFisher, 65-0865-14, eBioscience) for 30 minutes at room temperature. Samples were washed, fixed with 0.5% formaldehyde and analyzed by flow cytometry. Two hundred negative control beads were used per sample. Pure granules were used as a negative control. For analysis, dead cells were first removed with fixed dye to assess viability. Beads with individual antigens were then selected for a signal above the threshold after the exclusion of dead cells and doublets. The number of beads per HLA was then determined from their respective PE intensity peaks. Data were normalized by multiplying the number of beads of interest in each HLA peak by 200 divided by the number of negative beads in the sample. For each HLA peak, the percentage of relative binding of CAR Treg compared to control (cells not expressing CAR) was determined by subtracting the number of beads in CAR-Treg from the number of beads in the control, then dividing the average number of beads in the non-CAR-expressing control by 100 In one embodiment, the anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA -A*30, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, statistically inferior to BB7.2 antibody reactivity, e.g. when measured under test conditions BUT.

In one embodiment, the anti-HLA-A2 antibody of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30, statistically inferior to reactivity of the BB7.2 antibody, e.g. as measured under test A conditions.

In one embodiment, the term "statistically inferior" means that the reactivity (i.e., for example, relative binding under Test A conditions) measured for an anti-HLA-A2 antibody of the invention is lower than that measured for a BB7.2 antibody, with p- a value of at most about 0.05, preferably at most about 0.01, more preferably at most about 0.005, and even more preferably at most about 0.001, particularly when analyzed with using two-way ANOVA, Dunnett's post hoc test.

In one embodiment, the anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA- A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, inferior to BB7.2 antibody. In some embodiments, such an anti-HLA-A2 antibody has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA- A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 inferior to BB7.2 antibody when measured under test A conditions. In certain aspects, the relative binding for such anti-HLA is measured The -A2 antibody is at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the relative binding measured for the BB7.2 antibody.

In one embodiment, the anti-HLA-A2 antibody of the invention has inferior reactivity to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30 BB7.2 antibodies. In some embodiments, such an anti-HLA-A2 antibody has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30 inferior to binding BB7.2 antibody when measured under test A conditions. In certain aspects, the relative binding measured for such an anti-HLA-A2 antibody is at most about 90%, 80%, 70%, 60%, 50%, 40 %, 30%, 20%, 10% or less of the relative binding measured for the BB7.2 antibody.

In addition, anti-HLA-A2 antibodies provided herein with antigen-binding activity are capable of constituting CAR antigen-binding domains, wherein such CARs are capable of being expressed in human cells such that the CARs specifically bind to HLA-A2. In one embodiment, CARs specifically bind to HLA-A*02:01. As will be appreciated by one of skill in the art, the ability of an antibody to bind to HLA-A2 can be detected using methods known in the art. For example, CAR binding to HLA-A2 can be detected using the HLA-A2 tetramer as illustrated herein. In one embodiment, the human cell is an immune cell. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg.

In addition, the anti-HLA-A2 antibodies provided herein having antigen-binding activity are capable of forming chimeric antigen receptor (CAR) antigen-binding domains, wherein such CARs are capable of being expressed in regulatory T cells (Treg), so that the CARs specifically bind to HLA-A2. In one embodiment, CARs specifically bind to HLA-A*02:01. In one embodiment, the Treg is a human Treg.

In one embodiment, the anti-HLA-A2 antibody is capable of constituting an antigen-binding domain of a CAR, wherein such CAR is capable of being expressed in an immune cell such that the immune cell is activated by HLA-A2. In one embodiment, the immune cell is activated by HLA-A*02:01. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the T cell is a human T cell. In one embodiment, the Treg is a human Treg.

In one embodiment, such an anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SYHIQ (SEQ ID NO: 1) and GYTFTSY (SEQ ID NO: 2).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 1 (VH CDR1) selected from SEQ ID NOs: 1-2. In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a VH CDR1 represented by SEQ ID NO: 1. In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a VH CDR1 represented by SEQ ID NO: 2.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing the amino acid sequence EGTYYAMDY (SEQ ID NO: 6).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 3 (VH CDR3) represented by SEQ ID NO: 6.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR2 as set forth in SEQ ID NO: 5 and at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 1; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 1; one VH CDR2 shown in SEQ ID NO: 5; and one VH CDR3 shown in SEQ ID NO: 6.

According to the present invention, any of the heavy chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 1, 5 and 6.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR2 as set forth in SEQ ID NO: 5 and at least one of the following CDRs:

VH CDR1 shown in SEQ ID NO: 2; or

VH CDR3 shown in SEQ ID NO: 6.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising one VH CDR1 as set forth in SEQ ID NO: 2; one VH CDR2 shown in SEQ ID NO: 5; and one VH CDR3 shown in SEQ ID NO: 6.

According to the present invention, any of the heavy chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 2, 5 and 6.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 7).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 1 (VH CDR1) shown in SEQ ID NO: 7.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence KVSNRFS (SEQ ID NO: 8).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 2 (VH CDR2) shown in SEQ ID NO: 8.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region containing the amino acid sequence FQGSHVPRT (SEQ ID NO: 9).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising a complementarity determining region 3 (VH CDR3) shown in SEQ ID NO: 9.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region containing at least one of the following CDRs:

VL CDR1 shown in SEQ ID NO: 7; or

VL CDR2 shown in SEQ ID NO: 8; or

VL CDR3 shown in SEQ ID NO: 9.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region containing one VL CDR1 as set forth in SEQ ID NO: 7; one VL CDR2 shown in SEQ ID NO: 8; and one VL CDR3 shown in SEQ ID NO: 9.

According to the present invention, any of the light chain CDRs 1, 2, or 3 can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identity with the specific CDR sets listed in the respective SEQ ID NOs: 7, 8 and 9.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising framework region 1 (VH FR1) comprising an amino acid sequence selected from the group consisting of: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 11) and QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 12).

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 11). In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 12). In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 1 (VH FR1) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) comprising an amino acid sequence selected from the group consisting of: WVRQAPGQX ⁹ LEWMGX ¹⁵ (SEQ ID NO: 13) and HIQWVRQAPGQX ¹² LEWMGX ¹⁸ (SEQ ID NO: 25) where: X ⁹ is R or G and X ¹⁵ is I or not present in SEQ ID NO: 13; and X ¹² is R or G and X ¹⁸ is I or not present in SEQ ID NO: 25.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence WVRQAPGQX ⁹ LEWMGX ¹⁵ (SEQ ID NO: 13), where the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 13, where the amino acid at position 9 is R or G and the amino acid at position 15 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 14, 15, or 16. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 14, 15 or 16.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 2 (VH FR2) containing the amino acid sequence HIQWVRQAPGQX ¹² LEWMGX ¹⁸ (SEQ ID NO: 25), where the amino acid at position 12 (X ¹² ) is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 25, where the amino acid at position 12 is R or G and the amino acid at position 18 is absent.

In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence shown in SEQ ID NO: 26, 27, or 28. In one embodiment, the heavy chain variable region comprises framework region 2 (VH FR2) comprising the amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity with SEQ ID NO: 26, 27 or 28.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 3 (VH FR3) containing the amino acid sequence X ¹ VTX ⁴ TX ⁶ DTSX ¹⁰ STAYMX ¹⁶ LSX ¹⁹ LRSX ²³ DX ²⁵ AVYYCAR (SEQ ID NO: 29 ), where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 ( X ¹⁰ ) is A, T or I, the amino acid at position 16 (X ¹⁶ ) is E or L, the amino acid at position 19 (X ¹⁹ ) is S or R, the amino acid at position 23 (X ²³ ) is E or D, and the amino acid at position 25 (X ²⁵ ) is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, where the amino acid at position 1 is R, the amino acid at position 4 is I or M, the amino acid at position 6 is R, the amino acid at position 10 is A or I, the amino acid at position 16 is E, the amino acid at position 19 is S or R, the amino acid at position 23 is E or D, and the amino acid at position 25 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, where the amino acid at position 1 is R, the amino acid at position 4 is I or M, the amino acid at position 6 is R, the amino acid at position 10 is A or I, the amino acid at position 16 is E, the amino acid at position 19 is S or R, the amino acid at position 23 is E or D, and the amino acid at position 25 is T.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NO: 29, wherein the amino acid at position 1 is R, the amino acid at position 4 is I, the amino acid at position 6 is is R, the amino acid at position 10 is A, the amino acid at position 16 is E, the amino acid at position 19 is S, the amino acid at position 23 is E, and the amino acid at position 25 is T or M.

In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3) comprising the amino acid sequence shown in SEQ ID NOs: 30, 31, 32, 33, or 34. In one embodiment, the heavy chain variable region comprises framework region 3 (VH FR3 ) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 30, 31, 32, 33 or 34.

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 4 (VH FR4) containing the amino acid sequence WGQGTTVTVSS (SEQ ID NO: 44). In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region containing framework region 4 (VH FR4) containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 shown in SEQ ID NO: 1;

VH CDR2 shown in SEQ ID NO: 5;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 12;

VH FR2 shown in SEQ ID NO: 13, where the amino acid at position 9 (X ⁹ ) is R or G and the amino acid at position 15 (X ¹⁵ ) is I or absent;

VH FR3 shown in SEQ ID NO: 29, where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 (X ¹⁰ ) is A, T or I, the amino acid at position 16 (X ¹⁶ ) is E or L, the amino acid at position 19 (X ¹⁹ ) is S or R, the amino acid at position 23 (X ²³ ) is E or D and the amino acid at position 25 (X ²⁵ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

In one embodiment, the heavy chain variable region includes:

VH CDR1 represented by SEQ ID NO: 2;

VH CDR2 shown in SEQ ID NO: 5;

VH CDR3 shown in SEQ ID NO: 6;

VH FR1 shown in SEQ ID NO: 11;

VH FR2 shown in SEQ ID NO: 25, wherein the amino acid at position 12 (X ¹² ) is R or G and the amino acid at position 18 (X ¹⁸ ) is I or absent;

VH FR3 shown in SEQ ID NO: 29, where the amino acid at position 1 (X ¹ ) is R or absent, the amino acid at position 4 (X ⁴ ) is I or M, the amino acid at position 6 (X ⁶ ) is R or A, the amino acid at position 10 (X ¹⁰ ) is A, T or I, the amino acid at position 16 (X ¹⁶ ) is E or L, the amino acid at position 19 (X ¹⁹ ) is S or R, the amino acid at position 23 (X ²³ ) is E or D and the amino acid at position 25 (X ²⁵ ) is T or M; and

VH FR4 shown in SEQ ID NO: 44.

В одном воплощении изобретения антитело анти-HLA-A2 включает вариабельную область тяжелой цепи, содержащую аминокислотную последовательность QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQX ⁴⁴ LEWMGX ⁵⁰ WIYPGDGSTKYSQKFQGX ⁶⁸ VTX ⁷¹ TX ⁷³ DTSX ⁷⁷ STAYMX ⁸³ LSX ⁸⁶ LRSX ⁹⁰ DX ⁹² AVYYCAREGTYYAMDYWGQGTTVTVSS (SEQ ID NO: 92), в которой the amino acid at position 44 is R or G, the amino acid at position 50 is I or absent, the amino acid at position 68 is R or absent, the amino acid at position 71 is I or M, the amino acid at position 73 is R or A, the amino acid at position 77 is A, T, or I, the amino acid at position 83 is E or L, the amino acid at position 86 is S or R, the amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 92, wherein the amino acid at position 44 is R or G, the amino acid at position 50 is absent, the amino acid at position 68 is R, the amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, amino acid at position 90 is E or D, and the amino acid at position 92 is T or M.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 92, wherein the amino acid at position 44 is R or G, the amino acid at position 50 is absent, the amino acid at position 68 is R, the amino acid at position 71 is I or M, amino acid at position 73 is R, amino acid at position 77 is A or I, amino acid at position 83 is E, amino acid at position 86 is S or R, amino acid at position 90 is E or D, and the amino acid at position 92 is T.

In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 92, wherein the amino acid at position 44 is R, the amino acid at position 50 is absent, the amino acid at position 68 is R, the amino acid at position 71 is I, the amino acid at position 73 is R, the amino acid at position 77 is A, the amino acid at position 83 is E, the amino acid at position 86 is S, the amino acid at position 90 is E, and the amino acid at position 92 is T or M .

In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66. In one embodiment, the anti-HLA-A2 antibody comprises a heavy chain variable region comprising an amino acid sequence having about 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% identity with SEQ ID NO: 66.

In one embodiment, the anti-HLA-A2 antibody comprises a light chain variable region as defined herein. For example, in one embodiment, an anti-HLA-A2 antibody may comprise a light chain variable region as defined herein for a humanized anti-HLA-A2 antibody.

In one embodiment, the anti-HLA-A2 antibody is a scFv, scFab, or sdAb. In one embodiment, the anti-HLA-A2 antibody is a scFv or scFab. In one embodiment, the anti-HLA-A2 antibody is a sdAb. In one embodiment, the anti-HLA-A2 antibody is a scFab. In one embodiment, the anti-HLA-A2 antibody is scFv. In one embodiment, the humanized anti-HLA-A2 antibody is a scFv containing the amino acid sequence shown in SEQ ID NO: 91. In one embodiment, the anti-HLA-A2 antibody is a scFv containing an amino acid sequence having about 80%, 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity with SEQ ID NO: 91.

In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody. In one embodiment, the anti-HLA-A2 antibody is a human antibody. In one embodiment, the anti-HLA-A2 antibody is a non-humanized antibody. In one embodiment, the anti-HLA-A2 antibody is a non-human antibody.

In one embodiment, the anti-HLA-A2 antibody comprises an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO above. In one embodiment, the anti-HLA-A2 antibody is a scFv or scFab containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO above.

Also provided is a composition comprising, essentially consisting of, or consisting of at least one anti-HLA-A2 antibody of the invention.

As used herein, the term "consisting essentially of" with reference to a composition means that at least one anti-HLA-A2 antibody of the invention, as described herein above, is the only therapeutic agent or agent with biological activity in specified composition.

In another embodiment, a pharmaceutical composition is provided comprising at least one anti-HLA-A2 antibody of the invention and a pharmaceutically acceptable carrier.

In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody.

Examples of pharmaceutically acceptable carriers include, but are not limited to, media, diluents, coatings, isotonic and absorption delaying agents, additives, stabilizers, preservatives, surfactants, agents that inhibit enzymatic degradation, alcohols, pH adjusting agents, antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); preservatives and propellants.

Examples of pharmaceutically acceptable media include, but are not limited to, water, neutral saline, phosphate buffered saline, saline or other physiologically buffered saline or other solvent such as glycol, glycerin, and oil such as olive oil or organic ether for injection. The pharmaceutically acceptable medium may also contain liposomes or micelles.

Examples of coating materials include, but are not limited to, lecithin.

Examples of isotonic agents include, without limitation, sugars, sodium chloride, and the like.

Examples of agents that delay absorption include, but are not limited to, aluminum monostearate and gelatin.

Examples of supplements include, but are not limited to, mannitol, dextran, carbohydrates (such as, for example, glucose, mannose, sucrose, or dextran); glycine, lactose, or polyvinylpyrrolidone; or other additives such as antioxidants or inert gas, stabilizers, or recombinant proteins (eg, d. human serum albumin) suitable for in vivo administration.

Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-amine or NZ-amine AS.

Pharmaceutically acceptable carriers that may be used in these compositions further include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose , polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and lanolin.

Also provided is a medicament comprising, consisting of, or consisting essentially of at least one anti-HLA-A2 antibody of the invention as described above. In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody.

IV. Chimeric antigen receptors (CAR)

In one aspect, the present invention relates to chimeric antigen receptors (CARs). CARs are chimeric protein molecules that combine antibody specificity for a target antigen with a receptor-activated intracellular domain of an immune cell.

The CARs of the invention contain an extracellular domain that specifically binds to HLA-A2. The extracellular domain includes an anti-HLA-A2 antibody of the invention. In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody. The CARs of the invention further comprise a transmembrane domain and a cytoplasmic domain including an intracellular signaling domain. In one embodiment, the CARs of the invention are capable of being expressed in a human cell such that the CARs specifically bind to HLA-A2. In other embodiments, the CARs of the invention are capable of being expressed in an immune cell such that the CARs specifically bind to HLA-A2. In one embodiment, CARs specifically bind to HLA-A*02:01. As will be appreciated by one of skill in the art, the ability of an antibody to bind to HLA-A2 can be detected using methods known in the art. For example, CAR binding to HLA-A2 can be detected using the HLA-A2 tetramer as illustrated herein. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from three or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from four or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from five or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from six or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from seven or more of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from each of HLA-A*03, HLA-A*25, HLA-A*29, HLA- A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from one or more of HLA-A*25, HLA-A*29, HLA-A*30, compared to CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one HLA-A subtype selected from two or more of HLA-A*25, HLA-A*29, HLA-A*30, compared to CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to at least one HLA-A subtype selected from each of HLA-A*25, HLA-A*29, HLA-A*30 compared to with CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least one of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least two of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least three of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least four of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least five of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least six of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least seven of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30:01, HLA-A* 31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01 compared to CAR containing BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to at least one of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01 compared to CARs, containing the BB7.2 antibody. In one embodiment, the CARs provided herein are less reactive to at least two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01 compared to CARs. containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01 compared to the CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*25:01 compared to the CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*29:02 compared to the CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*30:01 compared to CARs containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*03:01 compared to CARs containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*31:01 compared to the CAR containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*33:01 compared to CARs containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*36:01 compared to CARs containing the BB7.2 antibody. In one embodiment, the CARs provided herein have less reactivity to HLA-A*68:01 compared to CARs containing the BB7.2 antibody. In one embodiment, the human cell is an immune cell. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg.

Methods for determining the reactivity of the CARs of the invention to HLA-A subtypes will be known to those skilled in the art. For example, the reactivity of the CARs of the invention to HLA-A subtypes can be determined by assay using individual antigen bead assay. Such individual antigen bead assays are commercially available (eg, FlowPRA Single Antigen Antibody; ONE LAMBDA).

In one embodiment, the CAR of the invention is less reactive to an HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, and any combination thereof, compared to CARs containing the BB7.2 antibody when measured under Test A conditions as described above.

In one embodiment, a CAR of the invention is less reactive to an HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30, and any combination thereof, compared to CARs containing the BB7 antibody. .2 when measured under Test A conditions as described above.

In one embodiment, the CAR of the invention of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30 , HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68, statistically inferior to BB7.2 antibody reactivity, e.g. when measured under Test A conditions.

In one embodiment, the CAR of the invention of the invention has reactivity to at least one subtype of HLA-A*25, HLA-A*29, HLA-A*30 statistically inferior to that of the BB7.2 antibody, e.g., when measured under conditions test A.

In one embodiment, the term "statistically inferior" means that the reactivity (i.e., for example, relative binding under test A conditions) measured for the CAR of the invention is lower than that measured for the CAR comprising the BB7.2 antibody, with a p-value, at most about 0.05, preferably at most about 0.01, more preferably at least about 0.005, and more preferably at most about 0.001, in particular when analyzed by two-way analysis of variance, Dunnett's posterior test.

In one embodiment, the CAR of the invention is reactive to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA -A*31, HLA-A*33, HLA-A*36, HLA-A*68, inferior BB7.2 antibody reactivity. In some embodiments, such a CAR has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A*30, HLA -A*31, HLA-A*33, HLA-A*36, HLA-A*68, inferior to the binding of a CAR comprising the BB7.2 antibody when measured under test A conditions. In certain aspects, the relative binding measured for such a CAR, is at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the relative binding measured for a CAR comprising the BB7.2 antibody.

In one embodiment, the CAR of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30, inferior to the reactivity of the BB7.2 antibody. In some embodiments, such a CAR has relative binding to at least one HLA-A subtype selected from the group consisting of HLA-A*25, HLA-A*29, HLA-A*30, inferior to that of a CAR comprising the BB7 antibody .2 when measured under test A conditions. In certain aspects, the relative binding measured for such a CAR is at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% , 10% or less of the relative binding measured for a CAR comprising the BB7.2 antibody.

In one embodiment, the CAR is capable of being expressed in an immune cell such that the immune cell is activated by HLA-A2. In one embodiment, the immune cell is activated by HLA-A*02:01. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the T cell is a human T cell. In one embodiment, the Treg is a human Treg.

In one embodiment, the CAR competes for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, CAR binds to the same HLA-A2 epitope as an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, CAR competes for binding to HLA-A2 with antibody BB7.2. In one embodiment, the CAR binds to the same HLA-A2 epitope as the BB7.2 antibody. The BB7.2 antibody can be isolated from the BB7.2 hybridoma (ATCC deposit number HB-82).

In one embodiment, the CAR of the invention includes: an extracellular domain containing an anti-HLA-A2 antibody; transmembrane domain; and a cytoplasmic domain containing an intracellular signaling domain. In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody.

In one embodiment, the chimeric receptor further includes a label and/or a leader sequence.

In one embodiment, the chimeric receptor further comprises a label, such as, for example, a label for quality control, enrichment, in vivo tracking, and the like. Said label may be localized at the N-terminus, C-terminus and/or internally. Examples of labels that can be used in the chimeric receptor of the invention are well known to those skilled in the art. For example, without limitation, a label used in the invention may be a label selected from the group consisting of or consisting of a haemagglutinin tag, polyarginine tag, polyhistidine tag, Myc tag, Strep tag, S tag, HAT tag, 3x Flag tags, calmodullin binding peptide tags, SBP tags, chitin binding domain tags, GST tags, maltose binding protein tags, fluorescent protein (e.g. eGFP) tags, T7 tags, V5 tags, and Xpress tags.

The extracellular domain is a target-specific binding element, which is also sometimes referred to as the targeting arm of a CAR. The extracellular domain is chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. The CAR of the present invention is designed to target an HLA-A2 presenting cell by creating an appropriate extracellular domain that specifically binds to an HLA-A2 epitope. The target-specific binding element or antigen-binding domain of the CAR of the present invention may be referred to herein as an anti-HLA-A2 binding domain. In some embodiments, the anti-HLA-A2 binding domain may be a humanized anti-HLA-A2 binding domain.

The transmembrane domain is attached to the extracellular domain and the cytoplasmic domain of CAR. The transmembrane domain is capable of signaling to the intracellular signaling domain(s) of the cytoplasmic domain whenever the extracellular domain of CAR binds to a target.

The cytoplasmic domain, which includes the CAR intracellular signaling domain, is responsible for activating at least one of the physiological effector functions of the immune cell (eg, regulatory T cell) into which the CAR has been placed. The term "effector function" refers to a specialized function of an immune cell. For example, the effector function of a regulatory T cell may include the suppression or down regulation of the induction and/or proliferation of other immune cells. In addition, the effector function of Treg may include the effect on non-immune cells, which leads to an improvement in the clinical condition, for example, promotes repair or tissue regeneration. Thus, the term "intracellular signaling domain" refers to the portion of the protein that transduces the effector function signal and directs the immune cell to carry out its specialized function. Although it is usually possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact strand if it signals an effector function. The term "intracellular signaling domain" thus implies the inclusion of any truncated portion of an intracellular signaling domain sufficient to signal an effector function.

In one embodiment, there may be a spacer domain (or linker or hinge) included between the antigen-binding domain and the CAR transmembrane domain, or between the intracellular signaling domain and the CAR transmembrane domain. As defined above, the spacer domain, linker, and hinge are oligo- or polypeptides that function to link the transmembrane domain to either an antigen-binding domain or an intracellular signaling domain in the polypeptide chain. The spacer domain may include, for example, up to 300 amino acids, 10 to 100 amino acids, 25 to 75 amino acids, or 25 to 50 amino acids, or amino acids of any sub-ranges or individual numerical values within these ranges.

Extracellular domain

The extracellular domain contains the anti-HLA-A2 antibody of the present invention. In one embodiment, the extracellular domain comprises a humanized anti-HLA-A2 antibody of the present invention.

In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFv, scFab or sdAb. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFv or scFab. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a sdAb. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFv. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFab. In one embodiment, the extracellular domain comprises any humanized anti-HLA-A2 antibody of the present invention, wherein the humanized anti-HLA-A2 antibody is scFv, scFab, or sdAb. In one embodiment, the extracellular domain comprises any humanized anti-HLA-A2 antibody of the present invention, wherein the humanized anti-HLA-A2 antibody is scFv or sdAb. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a sdAb. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFv. In one embodiment, the extracellular domain contains an anti-HLA-A2 antibody of the present invention, which is a scFab.

In some embodiments, the extracellular domain may comprise a hinge, where the transmembrane domain is attached to the extracellular region of the CAR, such as the antigen-binding domain of the CAR, via the hinge. In one embodiment, the hinge may be from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8α hinge. In some cases, the extracellular domain of the CAR of the invention may contain a CD8α hinge. In one embodiment, the hinge region comprises a CD8α stalk region. In one embodiment, the CD8 hinge may be encoded by the nucleic acid sequence of SEQ ID NO: 15 in US Pat. No. 9,102,760. In one embodiment, the CD8 hinge may comprise the amino acid sequence of SEQ ID NO: 21 in US Pat. No. 9,102,760. SEQ ID NO: 21 in US Pat. No. 9,102,760. In one embodiment, the hinge or spacer may include the amino acid sequence of SEQ ID NO: 115 or 219 in Table 3. In one embodiment, the hinge or spacer may be encoded by the nucleic acid sequence of SEQ ID NO: 159 or 220 in table 4.

transmembrane domain

The transmembrane domain can be obtained from either a natural source or a synthetic source. In one embodiment, the transmembrane domain can be obtained from a natural source, for example, from any membrane-bound or transmembrane protein.

In one embodiment, the CAR transmembrane domain can be derived from a transmembrane domain that is naturally associated with one of the CAR domains. In other embodiments, the transmembrane domain may be selected or modified by amino acid substitution to avoid such domains from binding to transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex.

In one embodiment, the transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the cytoplasmic region of the protein? from which the transmembrane domain is derived (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids of the cytoplasmic region).

In one embodiment, the transmembrane domain may comprise a transmembrane domain of a protein selected from the group consisting of CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, T-cell receptor alpha chain, T-cell receptor beta chain, gamma -chain of T-cell receptor, delta-chain of T-cell receptor, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, KIRDS2, OX40, CD2 , CD27, LFA-1 (CD1a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, PD1, ITGAX, CDl1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LT BR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D and/or NKG2C and CD154 and any combination thereof. In one embodiment, the transmembrane domain may comprise the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 160 in Table 4. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 116 in Table 3. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 116 in table 3.

In one embodiment, the transmembrane domain may comprise a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 16 in US Pat. No. 9,102,760. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 in US Pat. SEQ ID NO: 22 in US Pat. No. 9,102,760.

In one embodiment, the CD8 transmembrane domain is encoded by the nucleic acid sequence SEQ ID NO: 224 in Table 4. In one embodiment, the CD8 transmembrane domain contains the amino acid sequence SEQ ID NO: 223 in Table 3.

In other embodiments, the transmembrane domain may be synthetic, in which case it may contain predominantly hydrophobic residues, including leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan and valine can be found at each end of the synthetic transmembrane domain.

In one embodiment, a short oligo- or polypeptide linker can form a link between the transmembrane domain and the cytoplasmic domain of CAR. In one embodiment, the linker may be in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In one embodiment, the linker may contain a glycine-serine doublet.

Cytoplasmic domain

In one embodiment, intracellular signaling domains for use in the CARs of the invention may include cytoplasmic T cell receptor (TCR) and co-receptor sequences that act together to initiate signal transduction upon antigen receptor incorporation, as well as any derivative or variant of these sequences, and any synthetic sequence that has the same functionality.

Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may include signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences that are particularly useful in the invention include sequences derived from CD3-zeta, FcR-gamma, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b and CD66d. In one embodiment, the primary signaling domain contains a modified ITAM domain, eg, a mutated ITAM domain that has changed (eg, increased or decreased) activity from its own ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, for example, an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, FcR-gamma (e.g., FCγRI, RCγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, or FcγRIIIB), FcR alpha (eg, FcαRI), FcR epsilon (eg, FcεRI or FcεRII), CD5, CD22, CD79a, CD79b, DAP10, DAP12, and CD66d, and any combination thereof. In one embodiment, the intracellular signaling domain comprises or consists of a primary CD3-zeta signaling domain.

It is known that signals generated only through the TCR may not be sufficient to fully activate T cells and that a secondary or co-stimulatory signal may also be required. Thus, in some embodiments, T cell activation may be mediated by two classes of cytoplasmic signal sequence: those that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner to provide secondary or a costimulatory signal (secondary cytoplasmic signal sequences). In one embodiment, the intracellular signaling domain may further comprise a costimulatory domain.

In one embodiment, the CAR intracellular signaling domain may comprise the CD3 zeta signaling domain by itself, or it may be combined with any other desired intracellular signaling domain(s) useful in the context of the CAR of the invention. For example, a CAR intracellular signaling domain may contain a region of the CD3 zeta chain and a co-stimulatory signaling domain. A costimulatory signaling domain refers to the portion of a CAR containing the intracellular domain of a costimulatory molecule. A co-stimulatory molecule is a cell surface molecule, other than the antigen receptor or its ligands, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include OX40, CD27, CD28, lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, ligand that specifically binds to CD83, IL2ra (CD25) , IL6Ra (CD126), IL-7Ra (CD127), IL-13RA1, IL-13RA2, IL-33R (IL1RL1), IL-10RA, IL-10RB, IL-4R, IL-5R (CSF2RB), ARHR, receptor BAFF, IL-21R, TGFbR1, TGFbR2, TGFbR3, common gamma chain, MHC class I molecule, BTLA and Toll ligand receptor, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160 (BY55), CD19, CD19a, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, IT4, CD49D, IT4, CD49 V, IT4, CD49 V, IT4, CD49A, IT4, CD49 -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CitgbD18, ITGB7, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150 , IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKG2D, etc.

The intracellular signal sequences in the cytoplasmic portion of the CARs of the invention may be linked to each other in a random or predetermined order. Optionally, a short oligo- or polypeptide linker, eg 2 to 10 amino acids long (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can form a link between intracellular signal sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, can be used as a suitable linker.

In one embodiment, the intracellular signaling domain is designed to include two or more, eg 2, 3, 4, 5 or more co-stimulatory signaling domains. In one embodiment, two or more, eg 2, 3, 4, 5 or more co-stimulatory signaling domains are separated by a linker molecule, eg the linker molecule described herein. In one embodiment, the intracellular signaling domain contains two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In one embodiment, the cytoplasmic domain may include the CD3-zeta signaling domain and the CD28 signaling domain. In another embodiment, the cytoplasmic domain may include the CD3-zeta signaling domain and the 4-IBB signaling domain. In yet another embodiment, the cytoplasmic domain may include the CD3-zeta signaling domain and the CD28 and 4-1BB signaling domains.

In one embodiment, the cytoplasmic domain may include a CD28 signaling domain and a CD3-zeta signaling domain, wherein the CD28 signaling domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 161 in Table 4 and the CD3-zeta signaling domain is encoded by the nucleic acid sequence shown by in SEQ ID NO: 162 in Table 4.

In one embodiment, the cytoplasmic domain may include a CD28 signal domain and a CD3 zeta signal domain, where the CD28 signal domain contains the amino acid sequence of SEQ ID NO: 117 in Table 3 and the CD3 zeta signal domain contains the amino acid sequence of SEQ ID NO: 118 in Table 3.

In one embodiment, the cytoplasmic domain may include a CD28 signal domain and a CD3 zeta signal domain, wherein the CD28 signal domain contains the amino acid sequence shown in SEQ ID NO: 117 in Table 3 and the CD3 zeta signal domain contains the amino acid sequence shown in SEQ ID NO: 118 in table 3.

In one embodiment, the cytoplasmic domain may include a 4-1BB signal domain and a CD3-zeta signal domain, where the 4-1BB signal domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 17 of US Pat. US No. 9102760 and the CD3-zeta signal domain is encoded by the nucleic acid sequence shown in SEQ ID NO: 162 in Table 4.

In one embodiment, the cytoplasmic domain may include the 4-1BB signal domain and the CD3-zeta signal domain, where the 4-1BB signal domain contains the amino acid sequence of SEQ ID NO: 23 in US Pat. No. 9,102,760 and the CD3-zeta signal domain contains the amino acid sequence of SEQ ID NO : 118 in table 3.

In one embodiment, the cytoplasmic domain may include the 4-1BB signal domain and the CD3-zeta signal domain, where the 4-1BB signal domain contains the amino acid sequence shown in SEQ ID NO: 23 in US Pat. No. 9,102,760 and the CD3-zeta signal domain contains the amino acid sequence shown in SEQ ID NO: 118 in Table 3.

In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NOs: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 213. In one embodiment, the CAR contains an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 or 213.

V. NUCLEIC ACIDS AND VECTORS

In some embodiments, the invention relates to a nucleic acid encoding an anti-HLA-A2 antibody of the present invention. In some embodiments, the invention provides a nucleic acid encoding an anti-HLA-A2 antibody of the present invention. In some embodiments, the invention provides a nucleic acid encoding a protein comprising the amino acid sequence shown in SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, the invention provides a nucleic acid encoding a protein comprising the amino acid sequence shown in SEQ ID NOs: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112. In some embodiments, the invention relates to a nucleic acid encoding a protein containing an amino acid sequence having about 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % identity to SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, The invention relates to a nucleic acid encoding a protein containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity with SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112. In some embodiments, the invention provides a nucleic acid comprising the nucleic acid sequence shown in SEQ ID NOs: 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 or 158. In some embodiments, the invention relates to a nucleic acid containing a nucleic acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 139, 140 , 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, or 158. In some embodiments, the invention relates to a protein encoded by a nucleic acid containing the nucleic acid sequence shown in SEQ ID NOs: 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 or 158. In some embodiments, the invention relates to a protein encoded by a nucleic acid containing a nucleic acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, or 158. In some embodiments, the invention provides a nucleic acid encoding a protein comprising the amino acid sequence shown in SEQ ID NOs: 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, or 71. In some embodiments, the invention relates to a nucleic acid encoding a protein containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61, 62, 63, 64 65, 66, 67, 68, 69, 70 or 71.

In some embodiments, the invention relates to a nucleic acid encoding a chimeric antigen receptor (CAR) of the present invention. Nucleic acid sequences encoding CARs of the invention may encode CARs that include the extracellular domain described elsewhere herein.

The extracellular domain may contain an anti-HLA-A2 antibody of the present invention. The extracellular domain may comprise a humanized anti-HLA-A2 antibody of the present invention. In one embodiment, the extracellular domain may further include a leader sequence. In one embodiment, the leader sequence includes the amino acid sequence shown in SEQ ID NO: 113. In one embodiment, the extracellular domain contains a hinge region, where the anti-HLA-A2 binding domain is connected to the transmembrane domain via a hinge region. In one embodiment, the hinge region comprises a CD8α stalk region.

The nucleic acid sequences of the invention may encode CARs that contain the transmembrane domain described elsewhere herein. For example, in one embodiment, the transmembrane domain includes the transmembrane domain of a protein selected from the group consisting of CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, T-cell receptor alpha chain, T-cell receptor beta chain, T cell receptor gamma chain, T cell receptor delta chain, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, PD1, ITGAX, CDl1c , ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55) , PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LT BR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D and/or NKG2C and CD154 and any combination thereof. In one embodiment, the transmembrane domain contains the CD28 transmembrane domain.

The nucleic acid sequences of the invention may encode CARs that contain a cytoplasmic domain as described herein anywhere. In addition, the cytoplasmic domain may include an intracellular signaling domain, as described herein, anywhere. For example, in one embodiment, the intracellular signaling domain includes a functional signaling domain of a protein selected from the group consisting of CD3-gamma, CD3-delta, CD3-epsilon, CD3-zeta, FcR-gamma (e.g., FCγRI, RCγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA or FcγRIIIB), FcR alpha (eg, FcαRI), FcR epsilon (eg, FcεRI or FcεRII), CD5, CD22, CD79a, CD79b, DAP10, DAP12, and CD66d, and any combination thereof. In one embodiment, the intracellular signaling domain includes a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain further includes a costimulatory domain. The costimulatory domain of the CARs encoded by the nucleic acid sequences of the invention may be a costimulatory domain as described herein anywhere. For example, in one embodiment, the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A) , TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM -1, a ligand that specifically binds to CD83, IL2ra (CD25), IL6Ra (CD126), IL-7Ra (CD127), IL-13RA1, IL-13RA2, IL-33R (IL1RL1), IL-10RA, IL-10RB , IL-4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1, TGFbR2, TGFbR3, common gamma chain, MHC class I molecule, BTLA and Toll ligand receptor, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160 (BY55), CD19, CD19a, CD4, CD8-alpha, CD8-beta, IL2R-beta , IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, IT4, CD49D, IT4, CD49 V, IT4, CD49 V, IT4, CD49A, IT4, CD49-6, CD49f, ITGAD, CD11d, ITGAE , CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CitgbD18, ITGB7, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKG2D and any combination thereof. In one embodiment, the costimulatory domain contains a functional signaling domain of a protein selected from the group consisting of CD28, 4-1BB, and combinations thereof. In one embodiment, the co-stimulatory domain contains a functional CD28 signaling domain. In one embodiment, the costimulatory domain contains a functional 4-1BB signaling domain. In one embodiment, sequences containing an intracellular signaling domain are expressed in the same framework and as a single polypeptide chain.

The nucleic acid sequences of the present invention may be isolated nucleic acid sequences.

In one embodiment, the nucleic acid is provided as a messenger RNA transcript. In one embodiment, the nucleic acid is provided as a DNA construct.

In one embodiment, a recombinant DNA construct is provided comprising sequences encoding a CAR, wherein the CAR comprises: (i) an extracellular domain containing an anti-HLA-A2 antibody; (ii) a transmembrane domain; and (iii) a cytoplasmic domain containing an intracellular signaling domain, wherein the encoded CAR is capable of being expressed in a human cell such that the CAR is capable of specifically binding to HLA-A2. In one embodiment, the CAR is capable of specifically binding to HLA-A*02:01. In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody. In one embodiment, the human cell is an immune cell. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg.

In one embodiment, a recombinant DNA construct is provided comprising sequences encoding a CAR, wherein the CAR comprises: (i) an extracellular domain containing an anti-HLA-A2 antibody; (ii) a transmembrane domain; and (iii) a cytoplasmic domain containing an intracellular signaling domain, wherein the encoded CAR is capable of being expressed in a human cell such that the CAR is capable of specifically binding to HLA-A2. In one embodiment, the CAR is able to specifically bind to HLA-A*02:01. In one embodiment, CAR binding to HLA-A2 can be detected by using the HLA-A2 tetramer as illustrated herein. In one embodiment, the anti-HLA-A2 antibody is a humanized anti-HLA-A2 antibody. In one embodiment, the Treg is a human Treg.

In one embodiment, the encoded CAR is capable of being expressed in an immune cell such that the immune cell is activated by HLA-A2. In one embodiment, the immune cell is activated by HLA-A*02:01. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is a Treg. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the T cell is a human T cell. In one embodiment, the Treg is a human Treg.

In one embodiment, the encoded CAR competes for binding to HLA-A2 with an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, CAR binds to the same HLA-A2 epitope as an antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 183; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 185; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 187; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 188; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 189; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 190. In one embodiment, the encoded CAR competes for binding to the HLA-A2 antibody BB7.2. In one embodiment, the encoded CAR binds to the same HLA-A2 epitope as the BB7.2 antibody.

In some embodiments, the invention provides a nucleic acid encoding a protein comprising the amino acid sequence shown in SEQ ID NOs: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 213. In some embodiments, the invention relates to a nucleic acid encoding a protein containing an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 119, 120 , 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 213. In some embodiments, the invention relates to a nucleic acid containing the sequence nucleic acid set forth in SEQ ID NOs: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, or 214 In some embodiments, the invention relates to a nucleic acid containing a sequence nucleic acid content having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 178, 179, 180, 181, 182, or 214. In some embodiments, the invention provides a protein encoded by a nucleic acid comprising the nucleic acid sequence shown in SEQ ID NOs: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, or 214. In some embodiments, the invention relates to a protein encoded by a nucleic acid containing a nucleic acid sequence having about 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% identity with SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 or 214.

Nucleic acid sequences encoding the molecules of interest can be obtained using recombinant methods known in the art, such as, for example, screening libraries from cells expressing the gene by obtaining the gene from a vector known to contain it, or by isolation directly from cells and tissues containing it, using standard methods. Alternatively, the nucleic acid of interest can be obtained synthetically rather than cloned.

The present invention also relates to a vector containing a nucleic acid molecule encoding an anti-HLA-A2 antibody or a nucleic acid molecule encoding a CAR. In one embodiment, the invention relates to a vector containing a nucleic acid molecule encoding a humanized anti-HLA-A2 antibody. In one embodiment, the invention relates to vectors into which such a nucleic acid molecule is inserted.

In one embodiment, the present invention relates to a vector comprising a nucleic acid sequence described elsewhere in this document.

In some embodiments, the present invention provides a vector comprising a nucleic acid that encodes any of the anti-HLA-A2 antibodies described herein. In other embodiments, the present invention relates to a vector comprising a nucleic acid that encodes any of the humanized anti-HLA-A2 antibodies described herein. In some embodiments, the present invention provides a vector comprising a nucleic acid comprising the nucleic acid sequence shown in SEQ ID NOs: 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, or 158. In some embodiments, the invention relates to a vector comprising a nucleic acid containing a nucleic acid sequence having about 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, or 158. In certain aspects, a host cell is provided that includes such a vector .

In one embodiment, the present invention relates to a vector containing a nucleic acid encoding a CAR according to the invention. In some embodiments, the present invention relates to a vector containing the nucleic acid sequence shown in SEQ ID NOs: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, or 214. In some embodiments, the invention relates to a vector comprising a nucleic acid containing a nucleic acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 or 214.

In one embodiment, the vectors of the invention may be transduced into a cell. In one embodiment, the vectors of the invention may be transduced or constructed via non-viral vector methods into a human cell. In one embodiment, the vectors of the invention may be transduced into an immune cell. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the immune cell is a regulatory T cell (Treg). In one embodiment, the immune cell is a T cell. In one embodiment, the T cell may be a Treg. In one embodiment, the vector is capable of expressing CAR in mammalian immune cells. In one embodiment, the mammalian immune cell is a human immune cell. In one embodiment, the vector is capable of expressing CAR in mammalian immune regulatory cells. In one embodiment, the mammalian immune regulatory cell is a human regulatory immune cell. In one embodiment, the vector is capable of expressing CAR in mammalian T cells. In one embodiment, the mammalian T cells are mammalian regulatory T cells. In one embodiment, the mammalian T cell is a human T cell. In one embodiment, the mammalian T cell is a human regulatory T cell.

In one embodiment, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral and lentiviral vector constructs.

The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into a cell. Vectors derived from retroviruses such as lentivirus are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that such vectors can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

Briefly, expression of natural or synthetic CAR-encoding nucleic acids is typically achieved by operably linking the CAR polypeptide-encoding nucleic acid, or portions thereof, to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration into eukaryotes. Exemplary cloning vectors include transcription and translation terminators, initiation sequences, and promoters useful in regulating expression of the nucleic acid sequence of interest.

In addition to the methods described above, the following methods may be used.

The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, Pat. USA No. 5399346, 5580859, 5589466, incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector. In another embodiment, genome editing methods such as CRISPR-Cas9 or TALEN methods can be used to introduce nucleic acids encoding the CARs of the present invention into the genome of immune recipient cells (Delhove, JMKM et al., 2017; Eyquem, J. et al. al., 2017).

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe vectors, and sequencing vectors.

In addition, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, poxviruses, and lentiviruses (see, for example, US Pat. No. 5350674 and 5585362). In general, a suitable vector contains an origin of replication operable in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and US Pat. No. 6326193).

A number of viral systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional transcriptionally active elements, such as promoters and enhancers, can regulate the frequency of transcription initiation. As a rule, in the case of the main promoter, they are located in the region of 30-110 bp. upstream of the start site, although a number of promoters have recently been shown to also contain functional elements downstream of the start site, and enhancer elements are typically located at -2000 bp. up the sequence from the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the distance between the promoter elements can be extended up to 50 bp before activity begins to decline. Depending on the promoter, it appears that the individual elements may function together or independently to activate transcription.

One example of a suitable promoter is the cytomegalovirus (CMV) immediate-early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor-1α (EF-1α). Another example of a suitable promoter is the phosphoglycerate kinase (PGK) promoter. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 early promoter (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, the metalthionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

In order to evaluate the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells that are attempting to transfect or infect via viral vectors. In other aspects, the selectable marker may be applied to a single DNA fragment and used in a co-transfection procedure. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to allow expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or not expressed in the host organism or tissue and which encodes a polypeptide whose expression exhibits some readily detectable property, such as enzymatic activity. The expression of the reporter gene is analyzed at the appropriate time after the introduction of the DNA into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a green fluorescent protein gene (eg, Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be obtained using known methods or obtained commercially. In general, a construct with a minimal 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, eg a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for obtaining cells containing vectors and/or exogenous nucleic acids are well known in the art (see, for example, Sambrook et al., 2001). For example, a polynucleotide can be introduced into a host cell by transfection with calcium phosphate. Alternatively, the host cell can be transfected with a polynucleotide, such as RNA, by electroporation (see, for example, WO 2007/065957).

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). Other methods of modern targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable submicron delivery system.

In the case where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous interior of the liposome, placed in the lipid bilayer of the liposome, attached to the liposome through a binding molecule that is associated with both the liposome and the oligonucleotide entrapped in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, is mixed with a lipid, combined with a lipid, suspended in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Compositions associated with a lipid, lipid/DNA, or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be placed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may occur naturally or synthetic lipids. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) is available from K&K Laboratories (Plain View, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). The original solutions of lipids in chloroform or chloroform/methanol can be stored at a temperature of about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term encompassing a plurality of monolayer and multilayer lipid carriers formed as a result of the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilamellar liposomes have several lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components have self-organized before forming closed structures and trap water and solutes between lipid bilayers (Ghosh et al., 1991). However, compositions are also contemplated which have other structures in solution than the normal vesicular structure. For example, lipids may have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

Regardless of the method used to introduce exogenous nucleic acids into the host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blotting) or assays described herein, to identify agents falling within the scope of the invention.

VI. CAR-MODIFIED IMMUNE CELLS

Nucleic acids encoding CARs of the invention can be introduced into immune cells to produce CAR-modified immune cells expressing the CARs of the present invention, where the modified immune cells find use as described herein. CAR-modified immune cells may also be referred to herein as "CAR-engineered immune cells". In an embodiment, a modified immune cell is provided containing CAR as described herein anywhere. In another embodiment, an immune cell is provided that contains an expression vector as described elsewhere herein. The immune cell may be any immune cell suitable for use in cell therapy. In one embodiment, the immune cell may be a human immune cell.

In one embodiment, the immune cell is selected from the group consisting of a lymphocyte, a myeloid cell, and any combination thereof. In one embodiment, the lymphocyte is selected from the group consisting of T cells, B cells, natural killer (NK) cells, and any combination thereof. In one embodiment, the immune cell is a T cell. In one embodiment, the T cell is selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a γδ T cell, a double negative (DN) T cell, and any combination thereof. In one embodiment, the CD4+ T cell is selected from the group consisting of a T helper cell, a regulatory T cell, and any combination thereof. In one embodiment, the immune cell is a regulatory T cell. In one embodiment, the Treg is a thymus-derived Treg or an adaptive or induced Treg. In one embodiment, the Treg is a CD4+FOXP3+ regulatory T cell or a CD4+FOXP3- regulatory T cell (TR1 cell). In one embodiment, the Treg is a regulatory CD4+FOXP3+ T cell. In one embodiment, the Treg is a D4+FOXP3 regulatory T cell (TR1 cell). In one embodiment, the CD8+ T cell is a cytotoxic CD8+ T cell or a CD8+ regulatory T cell. In one embodiment, the CD8+ T cell is a CD8+ regulatory T cell. In one embodiment, the CD8+ regulatory T cell is selected from the group consisting of CD8+ CD28 regulatory T cell, CD8+CD103+ regulatory T cell, CD8+ FoxP3+ regulatory T cell, CD8+CD122+ regulatory T cell, and any combination thereof. In one embodiment, the CD8+ regulatory T cell is an INFγ+IL10+IL34+CD8+CD45RClow regulatory T cell. In one embodiment, the γδ T cell is a regulatory γδ T cell. In one embodiment, the DN T cell is a regulatory DN T cell. In one embodiment, the immune cell is a B cell. In one embodiment, the B cell is a regulatory B cell. In one embodiment, the regulatory B cell is a CD19+CD24hiCD38hi B cell. In one embodiment, the NK cell is a regulatory NK cell. In one embodiment, the myeloid cell is selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, or any combination thereof. In one embodiment, the macrophage is a regulatory macrophage. In one embodiment, the dendritic cell is a regulatory dendritic cell.

In one embodiment, the immune cell is a regulatory immune cell. The immune cell may be any regulatory immune cell suitable for use in cell therapy (see, for example, Wood, K.J. et al., 2012; Papp, G. et al., 2017). In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the regulatory immune cell is a CD8+ regulatory T cell (see, for example, Guillonneau, C. et al., 2010). In one embodiment, the CD8+ regulatory T cell is selected from the group consisting of a CD8+ CD28 regulatory T cell, a CD8+ CD103+ regulatory T cell, a CD8+ FoxP3+ regulatory T cell, a CD8+ CD122+ regulatory T cell, and any combination thereof. In one embodiment, the CD8+ regulatory T cell is an INFγ+IL10+IL34+ CD8+CD45RClow regulatory T cell. In one embodiment, the regulatory immune cell is a regulatory γδ T cell (see, for example, Wesch, D., et al., 2014). In one embodiment, the immune cell is a DN T cell (see, for example, Juvet, S.C., et al., 2012). In one embodiment, the regulatory immune cell is a regulatory B cell (see, for example, Chong, A.S., et al., 2017). In one embodiment, the regulatory B cell is a CD19+CD24hiCD38hi B cell. In one embodiment, the regulatory immune cell is a regulatory NK cell (see, for example, Fu, B. et al., 2013; Crome, S. Q., et al., 2013; Crome, S. Q., et al., 2017). In one embodiment, the regulatory immune cell is a regulatory macrophage (see, for example, Hutchinson, J.A. et al., 2017). In one embodiment, the regulatory immune cell is a regulatory dendritic cell. In one embodiment, the regulatory immune cell is a regulatory T cell. In one embodiment, the Treg is a thymus-derived Treg or an adaptive or induced Treg. In one embodiment, the Treg is a CD4+ FOXP3+ regulatory T cell or a CD4+FOXP3 regulatory T cell (TR1 cell). In one embodiment, the Treg is a regulatory CD4+FOXP3+ T cell. In one embodiment, the Treg is a D4+FOXP3 regulatory T cell (TR1 cell).

In one embodiment, the regulatory immune cell has the following phenotype: CD4+ CD25+, such as, for example, CD4+ CD25+ CD127-, such as, for example, CD4+ CD25+ CD127-CD45RA+. In additional embodiments, the regulatory immune cell has the following phenotype: FoxP3+CD4+CD25+, such as, for example, FoxP3+CD4+CD25+CD127-, such as, for example, FoxP3+CD4+CD25+CD127-CD45RA+.

In one embodiment, the immune regulatory cell has at least one of the following phenotypes: CD4+CD25+, FoxP3+, CD127lo/-, CTLA-4+, CD39+, Helios+, CD62L+/hi, VLA4+, LFA1+, CD49bint, ITGb7int, PSGL- 1+, ICOS+, GITR+, PD-1int, Perflo/-, CCR7+. In one embodiment, the immune regulatory cell does not express granzyme A and/or granzyme B.

In one embodiment, determination of the level of expression of molecules is carried out using flow cytometry, immunofluorescence, or image analysis, for example, high content analysis. Preferably, determination of the level of expression of molecules is carried out by flow cytometry. In one embodiment, cells are fixed and permeable prior to flow cytometry analysis to allow detection of intracellular proteins.

In one embodiment, determining the level of expression of a molecule in a cell population includes determining the percentage of cells in the cell population expressing the molecule (ie, "+" cells for the molecule). Preferably, said percentage of cells expressing the molecule is measured by FACS.

The terms "expressing (or +)" and "non-expressing (or -)" are well known in the art and refer to the level of expression of a cell marker of interest, in which the expression level of the cell marker corresponding to "+" is high or medium, also referred to as " +/-”, and the expression level of the cell marker corresponding to “-” is equal to zero.

The terms "low" or "lo" or "lo/-" are well known in the art and refer to the level of expression of a cellular marker of interest in which the level of expression of the cellular marker is low compared to the level of expression of that cellular marker in a population of analyzed cells in in general. More specifically, the term "lo" refers to a distinct population of cells that express a cellular marker at a lower level than one or more other distinct cell populations.

The term "high" or "hi" or "bright" is well known in the art and refers to the level of expression of a cellular marker of interest in the sense that the level of expression of the cellular marker is high compared to the level of expression of that cellular marker in a population of cells, which were analyzed as a whole.

Typically, cells in the top 2, 3, 4, or 5% of the staining intensity are labeled "hi" and those in the top half of the population are classified as "+". Cells showing below 50% fluorescence intensity are labeled "lo" and cells below 5% - as "-" cells.

The level of expression of a cell marker of interest was determined by comparing the mean fluorescence intensity (MFI) of cells from a population of cells stained with a fluorescently labeled antibody specific for that marker with the fluorescence intensity (FI) of cells from the same cell population stained with a fluorescently labeled antibody with irrelevant specificity, but with the same isotype, the same fluorescent probe, and from the same species (referred to as an isotype control). Cells from a population stained with fluorescently labeled antibodies specific for this marker and showing an equivalent MFI or lower MFI than cells stained with isotype controls do not express this marker and are therefore denoted (-) or negative. Cells from a population stained with a fluorescently labeled antibody specific for this marker and exhibiting an MFI value greater than that of cells stained with isotype controls express this marker and are therefore denoted (+) or positive.

In one embodiment, the immune cell of the invention expresses on its cell surface the CAR of the invention and another receptor (herein referred to as a "second receptor") that binds to a ligand other than HLA-A2. According to the invention, this second receptor includes an extracellular ligand-binding domain, optionally a hinge, optionally a transmembrane domain and an intracellular signaling domain, as previously described.

In one embodiment, the second receptor is endogenous (such as, for example, endogenous TCR). In another embodiment, the second receptor is exogenous and its expression is induced in the immune cell of the invention by transformation or transduction of the nucleic acid encoding it. Said exogenous receptor may be an exogenous TCR or a chimeric antigen receptor. Therefore, in one embodiment, the immune cell of the invention expresses two chimeric antigen receptors, where the first recognizes HLA-A2 and the second recognizes a separate ligand.

In another embodiment, the immune cell of the invention expresses on its cell surface the CAR of the invention and another receptor (referred to herein as a "second receptor") that binds to a different epitope in HLA-A2. According to the invention, this second receptor includes an extracellular ligand-binding domain, optionally a hinge, optionally a transmembrane domain and an intracellular signaling domain, as previously described.

In another embodiment, the immune cell of the invention expresses two CARs, the first recognizing a first HLA-A2 epitope and the second recognizing a non-HLA-A2 epitope.

In one embodiment, the CAR of the invention includes a first intracellular signaling domain and the second receptor includes a separate second intracellular signaling domain. In a first embodiment, a CAR of the invention includes a primary T cell signaling domain (such as, for example, CD3zeta) and a second receptor includes a costimulatory signaling domain (such as, for example, 4-1BB, CD28, or a combination of the costimulatory signaling domain 4-1BB and CD28 ). In a second embodiment, the CAR of the invention includes a costimulatory signaling domain (such as, for example, 4-1BB, CD28, or a combination of the costimulatory signaling domain 4-1BB and CD28), and a second receptor comprising a primary T cell signaling domain (such as, for example, CD3zeta ).

Therefore, according to these embodiments, complete activation of the immune cell of the invention requires both the binding of the CAR of the invention to HLA-A2 and the binding of the second receptor to the ligand it targets.

In one embodiment, the ligand recognized by the second receptor is expressed or present in the affected tissue or organ or at the site of an autoimmune response.

Examples of ligands that can be recognized by the second receptor include, but are not limited to, dietary antigens from a person's normal diet, self antigens, inhaled allergens, ingested allergens, or contact allergens.

The term "food antigen from normal human diet" refers to an immunogenic peptide that comes from foods common to humans, such as food antigens from the following non-limiting list: bovine antigens such as lipocalin, Ca-binding S100, alpha-lactalbumin, lactoglobulins such as beta-lactoglobulin, bovine serum albumin, caseins. Dietary antigens can also be Atlantic salmon antigens such as parvalbumin; chicken antigens such as, for example, ovomucoid, ovalbumin, Ag22, conalbumin, lysozyme or chicken serum albumin; peanut antigens; shrimp antigens such as tropomyosin; wheat antigens such as agglutinin or gliadin; celery antigens such as celery profilin; carrot antigens such as carrot profilin; apple antigens such as thaumatin, apple lipid transfer protein or apple profilin; pear antigens such as pear profilin or isoflavone reductase; avocado antigens such as endochitinase; apricot antigens such as apricot lipid transfer protein; peach antigens such as peach lipid transfer protein or peach profilin; soy antigens such as HPS, soy profilin or (SAM22) PR-10 pro; fragments, their variants and mixtures.

In one embodiment, said self-antigen is a multiple sclerosis-associated antigen, a joint-associated antigen, an eye-associated antigen, a human HSP antigen, a skin-associated antigen, or an antigen involved in transplant rejection or GVHD.

The term "multiple sclerosis-associated antigen" refers to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), oligodendrocyte myelin oligoprotein (OMGP), myelin-associated oligodendrocyte basic protein (MOBP), oligodendrocyte-specific protein (OSP/Claudin 1), heat shock proteins, oligodendrocyte-specific proteins (OSP), NOGO A, Po glycoprotein, peripheral myelin protein 22 (PMP22), 3'-phosphodiesterase 2, 3-cyclic nucleotides (CNPase), their fragments, variants and mixtures.

The term “joint-associated antigen” refers to citrulline-substituted cyclic and linear filaggrin peptides, type II collagen peptides, human cartilage glycoprotein 39 (HCgp39), HSP, heterogeneous nuclear ribonucleoprotein (hnRNP) peptides A2, hnRNP B1, hnRNP D, Ro60/52 , HSP60, HSP65, HSP70 and HSP90, BiP, keratin, vimentin, fibrinogen, collagen type I, III, IV and V peptide, annexin V, glucose-6-phosphate isomerase (GPI), acetyl-calpastatin, pyruvate dehydrogenase (PDH) , aldolase, topoisomerase I, snRNP, PARP, Scl-70, Scl-100, phospholipid antigens, including anionic cardiolipin and phosphatidylserine, neutrally charged phosphatidylethanolamine and phosphatidylcholine, matrix metalloproteinase, fibrillin, aggrecan, fragments, variants and mixtures thereof.

The term "ocular antigen" refers to collagen type II, retinal arrestin, S-arrestin, interforeceptor retinoid-binding proteins (IRBP1), beta-crystallin B1, retinal proteins, choroid proteins, and fragments, variants, and mixtures thereof.

The term "human HSP antigen" refers to human HSP60, HSP70, HSP90, fragments, variants and mixtures thereof.

Examples of skin-associated antigens include, but are not limited to, keratinocyte antigens, an antigen present in the dermis or epidermis, a melanocyte antigen (such as, for example, melanin or tyrosinase), desmoglein (for example, desmoglein 1 or 3, which may also be referred to as Dsg1 /3), BP180, BP230, plectins, integrins (eg integrin α4β6), collagens (eg collagen type VII), laminins (eg laminin 332 or laminin γ1), plakins (eg envoplakin, periplakin or desmoplakin), keratins (eg, KRT5, KRT8, KRT15, KRT17, and KRT31), keratin filament-associated proteins, filaggrin, corneodesmosin, and elastin.

In one embodiment, the ligand is an antigen involved in transplant rejection or GVHD. Examples of such antigens include, but are not limited to, graft or host specific MHC, β2-microglobulin, antigens from the ABO system, antigens from the Rhesus system (particularly antigens from the C, c, E et e and D systems), and isohemagglutinins. . Other examples of antigens that may be involved in transplant rejection or GVHD include, but are not limited to, HLA-DR (particularly during the first six months after transplantation), HLA-B (particularly during the first two years after transplantation). ), minor histocompatibility antigens (miHA, e.g. HLA-E, HLA-F and HLA-G), HLA corresponding to MHC class I (B and C), HLA corresponding to MHC class II (DP, DM, DOA, DOB, DQ and DR) and HLA corresponding to MHC class III (for example, components of the complement system).

Other examples of autoantigens include, but are not limited to, aquaporin water channels (such as, for example, aquaporin-4 (AQP4) water channel), Hu, Ma2, collapsin response mediator protein 5 (CRMP5), and amphiphysin, voltage-gated potassium channel (VGKC ), N-methyl-d-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR), thyroid peroxidase, thyroglobulin, anti-N-methyl-D-aspartate receptor ( NR1 subunit), Rh blood group antigens, antigen I, desmoglein 1 or 3 (Dsg1/3), BP180, BP230, nicotinic postsynaptic acetylcholine receptors, platelet integrin, GpIIb:IIIa, collagen (e.g., alpha-3(IV) chain collagen), rheumatoid factor, calpastatin, citrullinated proteins, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) peptides, alpha-beta-crystallin, DNA, histone, ribosomes, RNP, tissue transglutaminase (TG2), intrinsic factor, antigen 65 kDa, phosphatidylserine, ribosomal phosphoproteins, anti neutrophil cytoplasmic antibody, Scl-70, U1-RNP, ANA, SSA, anti-SSB, antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), Jo-1, antimitochondrial antibodies, gp210, p62, sp100, antiphospholipid antibodies, U1 -70 kd snRNP, ganglioside GQ1b, GM1, asialo-GM1, GD1b, anti-smooth muscle antibodies (ASMA), antibodies to liver and kidney microsomes-1 (ALKM-1), antibodies against liver cytosol-1 (ALC-1), IgA anti-endomysal antibodies, neutrophil granule proteins, streptococcal cell wall antigen, gastric parietal cell intrinsic factor, insulin (IAA), glutamic acid decarboxylase (GAA or GAD) and protein tyrosine phosphatase (such as, for example, IA2 or ICA512) PLA2R1 and THSD7A1.

In one embodiment, said ligand is selected from the group consisting of fragments of ovalbumin, MOG, type II collagen, variants and mixtures thereof.

In one embodiment, said ligand is ovalbumin, fragments, variants, and mixtures thereof.

In another embodiment, said ligand is MOG, fragments, variants and mixtures thereof.

In another embodiment, said ligand is type II collagen, fragments, variants and mixtures thereof.

In another embodiment, said ligand is IL23R, fragments, variants and mixtures thereof.

In one embodiment, the CAR of the invention further comprises an extracellular ligand-binding domain that recognizes a ligand other than HLA-A2. In one embodiment, said ligand-binding domain is an antibody or an antigen-binding fragment thereof.

In one embodiment, the CAR ligand-binding domain of the invention is a multifunctional antibody that recognizes many different epitopes on HLA-A2. In one embodiment, the CAR ligand-binding domain of the invention is a bifunctional antibody that recognizes two different epitopes on HLA-A2.

In one embodiment of the invention, the CAR of the invention comprises an extracellular ligand-binding domain comprising an HLA-A2 binding domain and another ligand-binding domain that recognizes a ligand other than HLA-A2. In one embodiment, said ligand-binding domain is a bifunctional antibody that recognizes both HLA-A2 and a single ligand.

Examples of ligands other than HLA-A2 that can be recognized by the CAR of the invention are listed above.

In one embodiment, the CAR-modified immune cells of the invention can be obtained by introducing into the cells a lentiviral vector containing the desired CAR. In one embodiment, a method is provided for obtaining an immune cell modified to express CAR, the method comprising transducing the immune cell with a vector described elsewhere herein, thereby forming said modified immune cell.

In one embodiment, the genetically modified immune cells of the invention are modified by introducing RNA. In one embodiment, the in vitro transcribed CAR RNA can be introduced into a cell in the form of transient transfection. RNA can be obtained by in vitro transcription using a polymerase chain reaction (PCR) generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, a synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is the CAR of the present invention.

In one embodiment, the DNA intended for PCR includes an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the organism's genome. In one embodiment, the DNA is a full length gene or part of a gene of interest. The gene may include some or all of the 5' and/or 3' untranslated regions (UTRs). A gene may include exons and introns. In one embodiment, the DNA used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene comprising the 5' and 3' UTR. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a natural organism. An example of an artificial DNA sequence is one that includes portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The pieces of DNA that are ligated together may be from the same organism or from more than one organism.

PCR can be used to generate an in vitro mRNA transcription template that is used for transfection. PCR methods are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of DNA to be used as a template for PCR. "Substantially complementary", as used herein, refers to nucleotide sequences where most or all of the bases in the primer sequence are complementary, or one or more bases are not complementary or do not match. Significantly complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify the portion of a gene that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify the portion of a gene that encodes for a particular domain of interest. In one embodiment, the primers are designed to amplify the human cDNA coding region, including all or parts of the 5' and 3' UTR. Primers suitable for PCR are prepared by synthetic methods which are well known in the art.

"Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence to be amplified. "Up the surface" is used herein to refer to the location of the 5' DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. The term "downstream" is used herein to refer to the location of the 3' DNA sequence to be amplified relative to the coding strand.

Any DNA polymerase suitable for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

Can also be used chemical structures capable of ensuring the stability and/or efficiency of translation. The RNA preferably has a 5' and a 3' UTR. In one embodiment, the 5'-UTR is zero to 3000 nucleotides in length. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied in various ways, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA. The 5' and 3' UTRs may be the naturally occurring endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by including UTR sequences in the forward and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest may be useful in altering the stability and/or efficiency of RNA translation. For example, it is known that AU-rich elements in the 3' sequences of UTRs can reduce mRNA stability. Therefore, 3'-UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of the UTRs, which are well known in the art.

In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the Kozak consensus sequence can be altered by adding the 5'UTR sequence. Kozak sequences can increase the translation efficiency of some RNA transcripts, but it does not appear to be required that all RNAs provide efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, different nucleotide analogs can be used in the 3' or 5' UTR to prevent degradation of the mRNA by the exonuclease.

To enable the synthesis of RNA from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template before the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes included in the PCR product ahead of the open reading frame to be transcribed. In one preferred embodiment, the promoter is the T7 polymerase promoter described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA has both a cap at the 5' end and a 3' poly(A) tail, which determined ribosome binding, translation initiation, and stability of the mRNA in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in a normal-sized mRNA that is not efficient for eukaryotic transfection, even if it polyadenylates after transcription.

On a linear DNA template, T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J Biochem., 270: 1485-65 (2003).

A common method for integrating polyA/T regions into a DNA template is molecular cloning. However, the polyA/T sequence integrated into plasmid DNA can cause plasmid instability, so plasmid DNA templates derived from bacterial cells are often heavily contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and lengthy, but often unreliable. This is why a method that allows the construction of DNA templates with a polyA/T 3' region without cloning is highly desirable.

A segment of a polyA/T transcription DNA template can be obtained during PCR using a reverse primer containing a polyT tail, such as a 100T tail (size can be 50-5000 T) or after PCR by any other method, including, but not limited to, ligation DNA or in vitro recombination. Poly(A) tails also provide RNA stability and reduce their degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is from 100 to 5000 adenosines.

RNA poly(A) tails can be further extended after in vitro transcription using a poly(A) polymerase such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides results in an approximately twofold increase in the efficiency of RNA translation. In addition, the attachment of different chemical groups to the 3' end can increase the stability of the mRNA. Such attachment may contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of RNA.

5' caps on RNA also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is proposed using methods known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7 : 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or engineered sequence that initiates cap-independent binding of the ribosome to mRNA and facilitates translation initiation. Any solutions suitable for cell electroporation may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into target cells using any of a number of different methods, for example, commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)) (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or ballistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

In one embodiment, the CAR sequences are delivered to cells using a retroviral or lentiviral vector. CAR-expressing retroviral and lentiviral vectors can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transduced cells as carriers or cell-free local or systemic delivery of encapsulated, linked or naked vectors. The method used can be used for any purpose where stable expression is required or sufficient.

In another embodiment, the CAR sequences are delivered to cells using transcribed mRNA in vitro. Transcribed CAR mRNA can be delivered in vitro to various types of eukaryotic cells, as well as tissues and whole organisms, using transfected cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked mRNA. The method used can be used for any purpose where transient expression is required or sufficient.

In another embodiment, the desired CAR can be expressed in cells via transposons.

In one embodiment, the immune cell of the invention is a T cell. Prior to propagation and genetic modification of T cells according to the invention, a source of T cells is obtained from the subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art may be used. In some embodiments of the present invention, T cells can be obtained from a unit of blood taken from the subject, using any number of techniques known to the person skilled in the art, such as separation on Ficoll™ or the Sepax separation system. In one preferred embodiment, cells from the individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells were washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution is deficient in calcium and may be deficient in magnesium, or may be deficient in many if not all of the divalent cations. After washing, the cells can be resuspended in various biocompatible buffers such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in culture medium.

Sources, activation and expansion of T cells

In one embodiment, the immune cell may be a T cell. Prior to propagation and genetic modification of T cells according to the invention, a source of T cells or progenitor cells from which T cells can be obtained is obtained from the subject. T cells can be obtained from any source containing these cells, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, germinal centers, tissues from the site of infection, ascites, pleural effusion, spleen, tumors and transplanted organs/tissues. In certain embodiments of the present invention, T cells can be obtained from a unit of blood taken from a subject using any number of methods known to one of skill in the art, such as Ficoll™ separation. In one embodiment, cells from the subject's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells may be washed with a wash solution (eg, phosphate buffered saline (PBS)). It will be readily understood by those skilled in the art that the wash step can be performed by methods known to those skilled in the art, such as using a semi-automated "flow through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the instructions manufacturer. After washing, the cells can be resuspended in various biocompatible buffers such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in culture medium.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysis of erythrocytes and depletion of monocytes, for example by PERCOLL™ gradient centrifugation or countercurrent centrifugation. A specific subset of T cells, such as CD3+, CD25+, CD127neg, HLA-DR+, CD28+, CD4+, CD8+, CD45RA+ and/or CD45RO+ T cells, can be further isolated by positive or negative selection methods. One skilled in the art will recognize that multiple rounds of selection may also be used in the context of this invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells may also be subject to additional rounds of selection.

Enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells. One way is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry, which uses a set of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich or positively select for regulatory T cells, typically expressing CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.

To isolate the desired population of cells using positive or negative selection, you can vary the concentration of cells and surfaces (for example, particles, such as granules). In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed with each other (ie, increase the concentration of cells) in order to maximize the contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, more than 100 million cells/ml are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In other embodiments, concentrations of 125 or 150 million cells/ml may be used. The use of high concentrations may result in increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, or from samples where many tumor cells are present (ie, leukemic blood, tumor tissue, etc.). Such populations of cells may be of therapeutic value and would be desirable.

In a related embodiment, it may be desirable to use lower concentrations of cells. By greatly diluting the mixture of T cells and the surface (eg, particles such as beads), interaction between particles and cells is minimized. Thus, cells are selected that express a large amount of the desired antigens to be associated with the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently taken up than CD8+ T cells at dilute concentrations. In one embodiment, the cell concentration used is 5×10 ⁶ /ml. In other embodiments, the concentration used may be from about 1×10 ⁵ /ml to 1×10 ⁶ /ml, and any integer value in between.

In other embodiments, the cells can be incubated on a rotating device for various periods of time at different speeds, either at 2-10°C or at room temperature.

T cells for stimulation can also be frozen after washing. Without wishing to be bound by theory, it is believed that the freezing and subsequent thawing step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% plasmalite-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7 5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, the cells were then frozen to -80°C at 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing directly at -20 °C or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed as described herein and left for one hour at room temperature before being activated using the methods of the present invention.

Also contemplated within the context of the invention is the collection of blood or apheresis product samples from the subject at a time prior to when expanded cells may be required, as described herein. As such, a source of cells to be expanded can be collected at any point in time when needed, and the cells of interest, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions. which would be useful in T cell therapy such as that described herein. In some embodiments, a blood sample or apheresis is taken from a subject who is at risk of developing a disease but has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, samples are taken from the subject shortly after diagnosis of a particular disease, as described herein, but prior to any treatment. In a further embodiment, cells are isolated from a blood sample or apheresis from a subject prior to any number of appropriate treatments including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids and radiation. These drugs inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al. , Immun 73: 316-321, 1991; Bierer et al., Curr Opin Immun 5:763-773, 1993). In a further embodiment, cells are isolated for a patient and frozen for later use in combination with (e.g., before, concurrently, or after) bone marrow or stem cell transplantation, T-cell ablative therapy using any chemotherapeutic agents such as fludarabine, external beam beam radiation therapy ( XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are recovered prior to and may be frozen for subsequent use for treatment following B-cell ablative therapy, eg, agents that react with CD20, eg, rituximab.

In a further embodiment of the present invention, T cells are obtained from the patient immediately after treatment. In this regard, it has been noted that after certain immunosuppressive procedures, in particular treatment with drugs that damage the immune system, shortly after treatment during the period when patients usually recover from treatment, the quality of T cells obtained may be optimal or improved in their ability to propagate ex vivo. Likewise, after ex vivo manipulations using the methods described herein, these cells may be in a preferred state for enhanced engraftment and expansion in vivo. Thus, in the context of the present invention, it is intended to collect immune cells during this extraction phase. In addition, in certain embodiments, mobilization schemes (e.g., mobilization with GM-CSF) and conditioning can be used to create a state in the subject in which repopulation, recycling, regeneration and/or expansion of certain cell types is preferred, especially during a certain time interval. after therapy.

Before or after genetic modification of T cells to express the desired CAR, T cells can be activated and propagated, as a rule, using methods as described, for example, in US Pat. USA No. 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and U.S. Patent Application Publication No. 20060121005.

Typically, T cells of the invention proliferate upon contact with a surface on which is attached an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator ( for example, bryostatin) in combination with a calcium ionophore. To costimulate a helper molecule on the surface of T cells, a ligand is used that binds the helper molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate the proliferation of CD4+ T cells, anti-CD3 antibodies and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France) can be used, as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In some embodiments, the primary stimulatory signal and the co-stimulatory signal for T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or associated with the surface. When they are bound to a surface, the agents may be bound to the same surface (ie, in a "cis" formation) or to other surfaces (ie, in a "trans" formation). Alternatively, one agent may be associated with the surface and the other agent may be in solution. In one embodiment, the agent providing the costimulatory signal is associated with the cell surface, and the agent providing the primary activation signal is in solution or associated with the surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing Fc receptors, or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, U.S. Patent Application Publication No. 20040101519 and 20060034810 on artificial antigen presenting cells (aAPC) which are contemplated for use in T cell activation and expansion in the present invention.

In one embodiment, the two agents are immobilized on the beads, either on the same bead, i.e. "cis", or on separate beads, i.e. "trans". For example, the agent providing the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent providing the costimulation signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio of each bead-bound antibody is used to expand CD4+ T cells and grow T cells. In some aspects of the present invention, a ratio of anti-CD3:CD28 antibodies associated with the beads is used such that an increase in T cell expansion is observed compared to that observed using a 1:1 ratio. In one particular embodiment, there is an increase from about 1 to about 3 times compared to reproduction observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values in between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibodies, ie the CD3:CD28 ratio is less than one. In some embodiments of the invention, the ratio of anti-CD28 antibody to bead-bound anti-CD3 antibody is greater than 2:1. In one particular embodiment, a 1:100 ratio of CD3:CD28 bead-bound antibodies is used. In another embodiment, a 1:75 ratio of CD3:CD28 bead-bound antibodies is used. In a further embodiment, a 1:50 ratio of CD3:CD28 bead-bound antibodies is used. In another embodiment, a 1:30 ratio of CD3:CD28 bead-bound antibodies is used. In one preferred embodiment, a 1:10 ratio of CD3:CD28 bead-bound antibodies is used. In another embodiment, a 1:3 ratio of CD3:CD28 bead-bound antibodies is used. In yet another embodiment, a 3:1 ratio of CD3:CD28 bead-bound antibodies is used.

Particle to cell ratios from 1:500 to 500:1 and any integer values in between can be used to stimulate T cells or other target cells. As those skilled in the art can readily appreciate, the ratio of particles to cells may depend on the size of the particles relative to the target cell. For example, small granules may bind only a few cells, while large granules may bind many cells. In some embodiments, the ratio of cells to particles is in the range of 1:100 to 100:1, and any integer values in between, and in other embodiments, the ratio is from 1:9 to 9:1, and any integer values in between can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 bound particles to T cells that result in T cell stimulation can vary, as noted above. For example, such values might include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10: 1, 15:1 and 1:1 particles per T cell. In one embodiment, a particle to cell ratio of 1:1 or less is used. In one particular embodiment, the particle:cell ratio may be 1:5. In other embodiments, the ratio of particles to cells may vary depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or a day thereafter for up to 10 days in final ratios. from 1:1 to 1:10 (based on the number of cells on the day of addition). In one specific embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment of the invention, the particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment of the invention, the particles are added daily or every other day to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that many other ratios may be suitable for use in the present invention. In particular, the ratios will vary with particle size as well as cell size and type.

In other embodiments of the present invention, cells such as T cells (eg, Treg cells) are combined with agent-coated beads, the beads and cells are then separated, and the cells are then cultured. In an alternative embodiment, before culturing, the agent-coated beads and cells are not separated, but cultured together. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, which results in increased ligation of the cell surface markers, thereby causing stimulation of the cells.

For example, cell surface proteins can be ligated to allow paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 x 28 beads) to contact T cells. In one embodiment, cells (eg, 10 ⁴ -10 ⁹ T cells) and beads (eg, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (without divalent cations such as like calcium and magnesium). Again, those skilled in the art can readily appreciate that any concentration of cells can be used.

For example, the target cell may be very rare in the sample and contain only 0.01% of the sample, or the entire sample (ie 100%) may contain the target cell of interest. Accordingly, any number of cells is within the scope of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed (ie, increase the concentration of cells) in order to maximize cell-particle contact. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, more than 100 million cells/ml are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In other embodiments, concentrations of 125 or 150 million cells/ml may be used. The use of high concentrations may result in increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express the target antigens of interest. Such cell populations may be of therapeutic value and would be desirable in certain embodiments.

In one embodiment of the present invention, the mixture may be cultured for several hours (about 3 hours) to about 14 days, or any integer hour in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, the beads and T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Multiple cycles of stimulation may also be desirable, so that the T cell culture time may be 60 days or more. Suitable conditions for T cell culture include suitable media (eg, minimal growth media or RPMI 1640 or X-vivo 15 (Lonza) media) that may contain factors necessary for proliferation and viability, including serum (eg, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other cell growth supplements known to the person skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, Immunocult (StemCell), AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with or without added amino acids, sodium pyruvate and vitamins serum or supplemented with an appropriate amount of serum (or plasma) or a certain set of hormones and / or an amount of cytokines sufficient for the growth and reproduction of T cells. Antibiotics, such as penicillin and streptomycin, are only included in the experimental cultures, not in the cell cultures to be administered to the subject. Target cells are maintained under conditions necessary to support growth, eg at a suitable temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ).

T cells that have been subjected to different stimulation times may have different characteristics. For example, typical blood mononuclear cell or peripheral blood peripheral blood products have a helper T cell population (Th, CD4+) that is larger than a population of cytotoxic or suppressor T cells (Tc, CD8+). Ex vivo expression of T cells by stimulation of the CD3 and CD28 receptors results in a T cell population that up to about 8-9 days consists predominantly of Th cells, while after about 8-9 days the T cell population includes all large population of Ts cells.

In addition, in addition to the CD4 and CD8 markers, other phenotypic markers vary greatly but are largely reproducible during the process of cell reproduction. Thus, this reproducibility makes it possible to tailor the activated T cell product for specific purposes.

In one embodiment, regulatory T cells can be obtained by isolating CD4+ T cells from HLA-A2- donors via RosetteSep (Stemcell) and enriched for CD25+ cells (Miltenyi) before sorting live CD4+CD127loCD25hi Tregs with FACSAria II (BD Biosciences ). Sorted Tregs can be stimulated with L cells and αCD3 mAb (OCT3; 200 ng/ml) in 1000 U/ml IL-2.

In one embodiment of the invention, T cells can be cultured in the presence of rapamycin to obtain regulatory T cells, as described, for example, in WO2007110785, incorporated herein by reference. Another method for producing regulatory T cells is described in US2016024470, incorporated herein by reference, wherein the T cells are cultured with a T cell receptor (TCR)/CD3 activator such as, for example, TCR/CD3 antibodies, a TCR costimulatory activator such as eg antibodies CD28, CD137 (4-1 BB), GITR, B7-1/2, CD5, ICOS, OX40, CD40 or CD137 and rapamycin.

In one embodiment of the invention, T cells genetically modified by CAR expression can also be genetically modified by expression of at least one intracellular factor such as ROR-C, Foxo1, T-bet or Gata 3, c-Maf, AhR . In one embodiment, the genetically modified immune cell of the invention expresses Foxo1.

In one embodiment, the genetically modified cells of the present invention may be allogeneic immune cells such as, for example, allogeneic T or Treg cells. For example, an allogeneic immune cell may be an immune cell that lacks expression of a functional human leukocyte antigen (HLA), such as HLA class I and/or HLA class II, or lacks expression of a functional T cell receptor (TCR).

In one embodiment, a T cell lacking a functional TCR may be engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that contain a functional TCR, or designed in such a way that it produced very little functional TCR on its surface. Alternatively, the T cell may express a substantially attenuated TCR, for example, by expressing mutated or truncated forms of one or more TCR subunits. The term "substantially attenuated TCR" means that the TCR will not elicit an adverse immune response in the host.

In another embodiment, the genetically modified cells described herein can be engineered such that they do not express functional HLA on their surface. For example, the immune cell described herein can be engineered to reduce expression of HLA on the cell surface, eg, HLA class 1 and/or HLA class II, or non-classical HLA molecules.

In another embodiment, the T cell may lack a functional TCR and functional HLA, such as HLA class I and/or HLA class II.

Modified immune cells (such as, for example, modified T or Treg cells) that lack functional TCR and/HLA expression can be generated by any suitable method, including knocking out or knocking down one or more TCR and/or HLA subunits. For example, an immune cell may include TCR and/or HLA knockdown using siRNA, shRNA, Clustered Regularly Spaced Short Palindromic Repeats (CRISPR) such as transcription activator-like effector nuclease (TALEN), zinc finger endonuclease (ZFN), meganuclease (mn, also known as homing endonuclease) or megaTAL (integrating TAL effector with mn cleavage domain) (Osborn et al, "Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases" Mol Ther. 2016 Mar; 24(3):570-81).

In one embodiment, TCR and/or HLA expression can be inhibited using siRNA or shRNA that targets a TCR and/or HLA-encoding nucleic acid in a T cell. Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system such as, for example, a lentivirus expression system. Exemplary siRNAs and shRNAs that repress the expression of HLA class I and/or HLA class II genes are described, for example, in US 2007/0036773. Exemplary shRNAs that repress the expression of TCR components are described, for example, in US2012/0321667.

As used herein, the term "CRISPR" or "CRISPR for TCR and/or HLA" or "CRISPR for TCR and/or HLA inhibition" refers to a set of clustered short palindromic repeats spaced at regular intervals, or a system containing such a set of repeats. "Cas", as used herein, refers to a CRISPR-associated protein. A "CRISPR/Cas" system refers to a system derived from CRISPR and Cas that can be used to suppress or mutate the TCR and/or HLA gene.

Naturally occurring CRISPR/Cas systems are found in about 40% of sequenced eubacteria genomes and in 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8:172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845. The CRISPR/Cas system has been modified for use in gene editing (silencing, amplification or alteration of certain genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This is achieved by introducing into a eukaryotic cell a plasmid containing a specially designed CRISPR and one or more suitable Cas. The CRISPR sequence, sometimes referred to as the CRISPR locus, includes alternating repeats and spacers. Naturally occurring CRISPR spacers typically contain sequences foreign to bacteria, such as a plasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, spacers are derived from the TCR or HLA gene sequence. RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. They contain a spacer flanked by a repeating sequence. RNAs direct other Cas proteins to cause exogenous genetic elements to be turned off at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Spacers thus serve as templates for RNA molecules, similar to miRNAs. Pennisi (2013) Science 341: 833-836. Thus, the CRISPR/Cas system can be used to edit the TCR and/or HLA gene (add or remove a base pair) or to introduce a premature stop codon, which thus reduces TCR and/or HLA expression. Alternatively, the CRISPR/Cas system can be used, similar to RNA interference, reversibly knocking out the HLA gene. For example, in a mammalian cell, RNA can direct the Cas protein to the TCR and/or HLA promoter, sterically blocking RNA polymerases.

Artificial CRISPR/Cas systems can be created that inhibit TCR and/or HLA using technology known in the art, such as those described in US 20140068797 and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems known in the art that inhibit TCR and/or HLA can also be made, such as those described in Tsai (2014) Nature Biotechnol., 32: 6 569-576, US 8,871,445; 8865406; 8795965; 8771945; and 8 697 359.

"TALEN" or "TALEN for TCR and/or HLA" or "TALEN for TCR and/or HLA inhibition" refers to a transcription activator-like effector nuclease, an artificial nuclease that can be used to edit TCR and/or HLA. TALEN is produced artificially by fusion of a TAL effector DNA binding domain with a DNA cleavage domain. Transcriptional activators (TALE) like activators can be designed to bind any DNA sequence of interest, including part of the TCR and/or HLA gene. By combining the engineered TALE with a DNA cleavage domain, one can generate a restriction enzyme that is specific for any DNA sequence of interest, including the TCR and/or HLA sequence. They can then be introduced into the cell where they can be used to edit the genome. Boch (2011) Nature Biotech. 29:135-6; and Boch et al. (2009) Science 326: 1509-12; Moscow et al. (2009) Science 326: 3501.

TALE are proteins secreted by Xanthomonas bacteria. The DNA-binding domain includes a repetitive highly conserved 33-34 amino acid sequence, except for the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with the recognition of specific nucleotides. Thus, they can be designed to bind to the desired DNA sequence. To obtain TALEN, the TALE protein is fused to a nuclease (N), which is a wild-type or mutated Fokl endonuclease. Several Fokl mutations have been generated for use in TALEN; for example, they improve the specificity or activity of the cleavage. Cermak et al. (2011) Nucl. Acids Res. 39:e82; Miller et al. (2011) Nature Biotech. 29:143-8; Hockemeyer et al. (2011) Nature Biotech. 29:731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25:786-793; and Guo et al. (2010)/. Mol. Biol. 200:96. The Fokl domain functions as a dimer, requiring two constructs with unique DNA-binding domains for sites in the target genome with correct orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the Fokl cleavage domain, as well as the number of bases between two separate TALEN binding sites, appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29:143-8. TCR and/or HLA TALEN can be used intracellularly to create a double strand break (DSB). A mutation may be introduced at the site of the break if the repair mechanisms do not correctly repair the break through a non-homologous end connection. For example, incorrect repair can lead to a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with TALEN; depending on the foreign DNA sequences and chromosomal sequence, this process can be used to correct a defect in the TCR and/or HLA gene, or to introduce such a defect in the wt TCR and/or HLA gene, thereby reducing TCR and/or HLA expression. TALENs specific for sequences in TCR and/or HLA can be created using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6: el9509.

"ZFN" or "zinc finger nuclease" or "ZFN for TCR and/or HLA" or "ZFN for TCR and/or HLA inhibition" refers to a zinc finger nuclease, an artificial nuclease that can be used to edit the TCR gene and/or HLA. Like TALEN, ZFN includes the Fokl nuclease domain (or a derivative thereof) fused to a DNA-binding domain. In the case of ZFN, the DNA binding domain includes one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. sci. USA 93: 1156-1160. A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may contain, for example, Cys2His2 and may recognize a sequence of about 3 bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of 6, 9, 12, 15, or 18 bp in length. Various selection and modular assembly methods are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, which include phage display, yeast single-hybrid systems, bacterial single-hybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must dimerize in order to cleave DNA. Thus, a ZFN pair is required to target non-palindromic DNA sites. Two separate ZFNs must bind opposite DNA strands with their nucleases correctly spaced relative to each other. Bitinaite et al. (1998) Proc. Natl. Acad. sci. USA 95: 10570-5. Like TALEN, ZFN can create a double-strand break in DNA, which can create a frameshift mutation when not properly repaired, resulting in decreased expression and amount of TCR and/or HLA in the cell. ZFNs can also be used in homologous recombination to mutate in the TCR and/or HLA gene. ZFNs specific for sequences in TCR and/or HLA can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16:1200-7; Quo et al. (2010) /. Mol. Biol. 400:96; US2011/0158957; and US2012/0060230.

"Meganuclease" or "meganuclease for TCR and/or HLA" or "meganuclease for TCR and/or HLA inhibition" refers to a monomeric endonuclease with large (>14 bp) recognition sites that can be used to edit the TCR and/or HLA gene . Meganucleases (mn) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site. Homing endonucleases are DNA-cleaving enzymes that can generate double-strand breaks at single loci in their host genomes and thus drive site-specific gene conversion events. (Stoddard, Structure. 2011 Jan 12;19(1):7-15). Despite their small size, homing endonucleases recognize long DNA sequences (typically 20 to 30 base pairs). Homing endonucleases are extremely common and found in microbes as well as phages and viruses. The box enzymes LAGLIDADG and His-Cys (which are the most sequence-specific of these enzymes) rely on antiparallel β-sheets that anchor in the major grooves of the DNA of their target sites (Flick et al., 1998; Jurica et al., 1998 ). There, they establish a collection of sequence-specific and non-sequence-specific contacts that are unevenly distributed over several consecutive base pairs (Chevalier et al., 2003; Scalley-Kim et al., 2007).

The LAGLIDADG (LHE) family of homing endonucleases is the main source of engineered enzymes used in gene targeting applications. The LHE family is mainly encoded in archaea and in the chloroplast and mitochondrial genomes of algae and fungi (Chevalier et al., 2005; Dalgaard et al., 1997; Sethuraman et al., 2009). Meganucleases that have one conserved LAGLIDADG motif (SEQ ID NO: 85) per protein strand form homodimeric proteins that cleave palindromic and near-palindromic DNA target sequences, while those containing two such motifs per protein strand form larger pseudo-symmetrical monomers that can target completely asymmetric DNA sequences.

Meganucleases can be engineered to target TCR and/or HLA and thus create a double-strand break in DNA that can create a frameshift mutation upon misrepair, resulting in decreased expression and amount of TCR and/or HLA in the cell.

"MegaTAL" or "megaTAL for TCR and/or HLA" or "megaTAL for TCR and/or HLA inhibition" refers to an artificial nuclease that can be used to edit a TCR and/or HLA gene. MegaTAL are hybrid monomeric nucleases derived from the fusion of minimal TAL (transcription activator-like) effector domains to the N-terminus of meganucleases derived from the LAGLIDADG family of homing endonucleases (Nucleic Acids Res. 2014 Feb;42(4):2591-601; Takeuchi et al, Methods Mol Biol 2015;1239:105-32 doi: 10.1007/978-1-4939-1862-1_6). MegaTALs thus consist of a site-specific meganuclease cleavage head with additional affinity and specificity provided by the TAL effector DNA binding domain. MegaTAL can be engineered to target TCR and/or HLA and thus create a double-strand break in the DNA that can create a frameshift mutation upon misrepair, resulting in decreased expression and amount of HLA in the cell. The I-OnuI (mn) meganuclease variant was used to construct TCRα-megaTAL to knock out the T-cell receptor alpha (TCRα) gene. The TCRα mn was fused to a TALE 10.5 repeat array designed to bind a DNA sequence upstream of the TCRα mn binding site. TCRα-targeted megaTAL was found to achieve extremely high gene disruption without appreciable off-target cleavage in primary human T cells. (Boissel et al., Nucleic Acids Res. 2014 Feb;42(4):2591-601).

Without wishing to be bound by any particular theory, it is believed that in some embodiments, the therapeutic T cell has a short persistence in the patient due to shortened telomeres in the T cell; accordingly, transfection with the telomerase genome can lengthen T cell telomeres and improve T cell resistance in a patient. See Carl June, "Adoptive T cell therapy for cancer in the clinic", Journal of Clinical Investigation, 117: 1466-1476 (2007). Thus, in one embodiment, the genetically modified immune cell of the invention ectopically expresses a telomerase subunit, eg a telomerase catalytic subunit, eg TERT, eg hTERT. In some aspects, this disclosure relates to a method for producing a CAR-expressing immune cell, comprising contacting the immune cell with a nucleic acid encoding a telomerase subunit, for example, a telomerase catalytic subunit, for example, TERT, for example, hTERT. The cell may contact the nucleic acid before, simultaneously with, or after contact with the CAR-encoding construct.

VII. THERAPEUTIC USE

The invention provides immune cells modified to express a CAR targeting the HLA-A2 antigen as a means to recruit said immune cells to sites of a specific immune or inflammatory response and to activate immune cells to suppress the immunological activity of immune effector cells at those sites. Thus, CAR-mediated recruitment and activation of immune cells provides a way to prevent or treat a number of immunological diseases or disorders for which such immunosuppressive activity is beneficial.

In one embodiment, the immune cell is a regulatory immune cell. The immune cell may be any regulatory immune cell suitable for use in cell therapy (see, for example, Wood, K.J. et al., 2012; Papp, G. et al., 2017). In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof.

In one embodiment, the immune cell may be a regulatory T cell. In another embodiment, the regulatory T cell may be a human regulatory T cell. Although the activation of regulatory T cells is antigen-dependent, the inhibitory effect of these cells is independent of antigen, TCR and MHC. Accordingly, CAR expression in regulatory T cells redirects these cells and their activation to the appropriate target tissue so that they are activated in an antigen-specific manner; however, their suppressive effects occur without the need for further recognition of disease-associated antigens. Therefore, as long as regulatory T cells are in the correct proximity to where the immune effector cells reside and mediate their unwanted effects, redirected regulatory T cells can be triggered or activated at that location to mediate their suppressive effects.

In one embodiment, the target HLA-A2 antigen may be present or expressed at the site or tissue target of an undesired immune or inflammatory response mediated by immune effector cells.

In one embodiment, the CAR-modified immune cells of the invention can be used to treat one or more diseases, disorders, symptoms, or conditions associated with organ or tissue transplantation (e.g., organ or tissue rejection/dysfunction, GVHD, and/or related conditions) . Graft rejection involves the destruction of the transplanted donor tissue by the recipient's immune cells through an immune response. The immune response is also involved in GVHD; however, in this case, the recipient's tissues are destroyed by the donor's immune cells transferred to the recipient via transplantation. Accordingly, CAR-mediated redirection and activation of immune cells provides a way to suppress the rejection of mismatched cells and/or tissues by immune effector cells in transplant recipients or to inhibit the pathogenic action of transplanted immunocompetent cells in the case of GVHD. In one embodiment, the mismatched cells and/or tissues contain cells and/or tissues that are mismatched for HLA-A2.

Thus, another embodiment of the present invention is a method of treating one or more diseases, disorders, symptoms, or conditions associated with organ or tissue transplantation (e.g., organ or tissue rejection/dysfunction, GVHD, and/or conditions associated therewith) to a subject, wherein said method includes administering to the subject an engineered CAR immune cell or composition as described herein.

In one embodiment, the method is a cell therapy method. In one embodiment, the cell therapy is autologous. In one embodiment, the cell therapy is heterologous. In one embodiment, the cell therapy is allogeneic. In one embodiment, the method is a gene therapy method.

Thus, another embodiment of the present invention is a CAR-engineered immune cell or composition as described herein for use in the treatment of one or more diseases, disorders, symptoms, or conditions associated with organ or tissue transplantation (e.g., rejection/ organ or tissue dysfunction, GVHD and/or related conditions) in the subject.

The CAR-modified immune cells of the invention can be used to stimulate immune tolerance, functional tolerance and/or immune accommodation in a subject. The CAR-modified immune cells of the present invention can be used to stimulate immune tolerance, functional tolerance and/or immune accommodation in an organ or tissue transplant subject. In one embodiment, a method is provided for inducing immune tolerance, functional tolerance, and/or immune accommodation in a subject, the method comprising administering to the subject a CAR-modified immune cell as described elsewhere herein. In another embodiment, a method is provided for inducing immune tolerance, functional tolerance, and/or immune accommodation in a subject, the method comprising administering to the subject a pharmaceutical composition described elsewhere herein. In one embodiment, the use may be to promote immune tolerance, functional tolerance, and/or immune accommodation to a transplanted organ or tissue in a subject. In one embodiment, the CAR-modified immune cell is administered simultaneously with, before or after organ or tissue transplantation. In one embodiment, the CAR-modified immune cell is administered at the same time as an organ or tissue transplant. In another embodiment, the CAR-modified immune cell is administered prior to organ or tissue transplantation. In one embodiment, the CAR-modified immune cell is administered following organ or tissue transplantation. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the immune cell is a regulatory T cell. In one embodiment, the immune cell is a regulatory CD4+ T cell. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In another embodiment, the immune cell is a human regulatory T cell. In one embodiment, the immune cell is a human CD4+ regulatory T cell.

The CAR-modified immune cells of the present invention can be used to prevent or treat rejection of a transplanted organ or tissue. In one embodiment, the CAR-modified immune (eg, CAR-engineered Tregs) of the present invention can be used to prevent or treat excessive rejection of a transplanted organ or tissue. In one embodiment, the CAR-modified immune cells (eg, CAR-engineered Treg cells) of the present invention can be used to prevent or treat antibody-mediated rejection of a transplanted organ or tissue. In one embodiment, the method comprises administering the CAR-engineered immune cells (eg, CAR-engineered Treg cells) of the present invention to a subject to be exposed to a transplanted organ or tissue. In one embodiment, a transplanted organ or tissue may include a bone marrow transplant, an organ transplant, a blood transfusion, or any other foreign tissue or cell that is purposefully introduced into the subject. In one embodiment, the CAR-modified immune cells (eg, CAR-engineered Treg cells) of the present invention can be used as a therapy to inhibit transplant rejection after transplantation. In one embodiment, graft rejection may be an allograft rejection. In one embodiment, transplant rejection may be a xenograft rejection. In one embodiment, a method is provided for preventing or treating organ or tissue transplant rejection in a subject, the method comprising administering to the subject a CAR-modified immune cell as described elsewhere herein. In one embodiment, a method is provided for preventing or treating organ or tissue transplant rejection in a subject, the method comprising administering to the subject a pharmaceutical composition described elsewhere herein. Thus, another embodiment of the present invention is a CAR-engineered immune cell (CAR-engineered Treg cell) of the invention, or a pharmaceutical composition containing said immune cells, for use in the prevention or treatment of organ or tissue transplant rejection in a subject. In one embodiment, the present invention relates to a method for increasing graft survival in a subject, comprising administering to the subject a CAR-modified immune cell (eg, a CAR-engineered Treg cell) described elsewhere herein. In one embodiment, the present invention relates to a method for increasing graft survival in a subject, comprising administering to the subject a pharmaceutical composition described elsewhere herein. In one embodiment, the method provides a graft survival period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, 100 years, or the entire lifetime of the object. In one embodiment, the subject is not undergoing any therapy with immunosuppressive agents. In one embodiment, administration of an immune cell or composition of the invention reduces the amount of immunosuppressant therapy received by the subject. In one embodiment, the graft may be an allograft. In one embodiment, the graft may be exposed to the CAR-modified immune cells of the present invention at the same time as, before, or after transplantation of the graft into a recipient. In one embodiment, the organ or tissue graft may be a heart, heart valve, lung, kidney, liver, pancreas, intestine, skin, blood vessels, bone marrow, stem cells, bones, or islet cells. However, the invention is not limited to a specific type of transplant. In one embodiment, the donor graft may be "preconditioned" or "pretreated" by treating the organ or tissue graft prior to transplantation into the recipient with the CAR-modified immune cells of the invention to reduce the immunogenicity of the graft against the recipient, thereby reducing or preventing graft rejection. In one embodiment, the host or recipient graft is HLA-A2 negative. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two different HLA-A subtypes selected from one or two of HLA-A*03, HLA-A*25, HLA-A*29 , HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one HLA-A subtype selected from one of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A* 30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one or two of HLA-A*03, HLA-A*25, HLA-A*29, HLA -A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A *30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from two of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A *30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two different HLA-A subtypes selected from one or two of HLA-A*25, HLA-A*29, HLA-A*30 . In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one HLA-A subtype selected from one of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one or two of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from two of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*03. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*25. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*29. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*31. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*33. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*36. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30: 01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30: 01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one of HLA-A*25:01, HLA-A*29:02 and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*25:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*29:02 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*30:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*03:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*31:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*33:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for HLA-A*36:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*68:01 positive. In one embodiment, the graft is HLA-A2 positive.

In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the immune cell is a regulatory T cell. In one embodiment, the immune cell is a regulatory CD4+ T cell. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In another embodiment, the immune cell is a human regulatory T cell. In one embodiment, the immune cell is a human CD4+ regulatory T cell.

The CAR-modified immune cells (eg, CAR-engineered Treg cells) of the present invention can be used to prevent or treat graft-versus-host disease (GVHD). In one embodiment, the method comprises administering a CAR-modified immune cell (eg, CAR-engineered Treg cells) of the present invention to a subject to be exposed to a transplanted organ or tissue. In one embodiment, the transplanted organ or tissue may include a bone marrow transplant, an organ transplant, a blood transfusion, or any other foreign tissue or cell that is purposefully introduced into the subject. For example, GVHD may occur following transplantation of the heart, heart valve, lung, kidney, liver, pancreas, intestine, skin, blood vessels, bone marrow, stem cells, bone, or islet cells. However, the invention is not limited to a specific type of transplant. In one embodiment, GVHD may occur following hematopoietic stem cell transplantation. In one embodiment, a method is provided for preventing or treating graft-versus-host disease (GVHD) in a subject, the method comprising administering to the subject a CAR-modified immune cell as described elsewhere herein. In one embodiment, a method is provided for preventing or treating graft-versus-host disease (GVHD) in a subject, comprising administering to the subject a pharmaceutical composition described elsewhere herein. In one embodiment, the invention relates to a method for contacting a donor graft, e.g., a biocompatible array or donor tissue, organ, or cell, with the CAR-modified immune cells of the present invention, simultaneously, before or after transplantation of the graft into a recipient. In one embodiment, the CAR-modified immune cells of the present invention can be used to attenuate, inhibit, or reduce the adverse response of a donor graft against a recipient, thereby preventing or treating GVHD. In one embodiment, the present invention relates to a method for preventing or delaying the onset of GVHD in a subject, comprising administering to the subject a CAR-modified immune cell as described elsewhere herein. In one embodiment, the present invention relates to a method for preventing or delaying the onset of GVHD in a subject, comprising administering to the subject a pharmaceutical composition described elsewhere herein. In one embodiment, the onset of GVHD is delayed by 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 1 year, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 20 years old, 30 years old, 40 years old, 50 years old, 60 years old, 70 years old, 80 years old, 90 years, 100 years, or for the entire lifetime of the object. In one embodiment, the subject is not undergoing any therapy with immunosuppressive agents. In one embodiment, the subject is subjected to immunosuppressive therapy. In one embodiment, the immune cells of the invention are administered to a subject in order to reduce the therapeutically effective amount of said immunosuppressive therapy. In one embodiment, GVHD may be acute GVHD or chronic GVHD. In one embodiment, a donor graft may be "preconditioned" or "pretreated" by treating the graft prior to transplantation into a recipient with the CAR-modified immune cells (e.g., CAR-engineered Treg cells) of the invention to reduce the immunogenicity of the graft against the recipient, thereby reducing or preventing GVHD. In one embodiment, the graft may be contacted with cells or tissue of the recipient prior to transplantation to activate T cells that may be associated with the graft. After the graft has been treated with the cells or tissue of the recipient, the cells or tissues may be removed from the graft. The treated graft can then be further contacted with the CAR-modified immune cells (e.g., CAR-engineered Treg cells) of the present invention to reduce, inhibit, or eliminate the activity of immune effector cells that have been activated upon treatment of cells or tissue from the recipient. . Following this treatment of the graft with the CAR-modified immune cells of the present invention, the CAR-modified immune cells can be removed from the graft prior to transplantation into a recipient. However, some CAR-modified immune cells can adhere to the graft and therefore can be administered to the recipient via the graft. In this situation, the CAR-modified immune cells administered to the recipient can suppress the immune response against the recipient caused by the graft-associated cell. In one embodiment, the host or recipient graft is HLA-A2 negative. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two different HLA-A subtypes selected from one or two of HLA-A*03, HLA-A*25, HLA-A*29 , HLA-A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one HLA-A subtype selected from one of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A* 30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one or two of HLA-A*03, HLA-A*25, HLA-A*29, HLA -A*30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A *30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from two of HLA-A*03, HLA-A*25, HLA-A*29, HLA-A *30, HLA-A*31, HLA-A*33, HLA-A*36, HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two different HLA-A subtypes selected from one or two of HLA-A*25, HLA-A*29, HLA-A*30 . In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one HLA-A subtype selected from one of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one or two of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from one of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two different HLA-A subtypes selected from two of HLA-A*25, HLA-A*29, HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*03. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*25. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*29. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*30. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*31. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*33. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*36. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for the HLA-A subtype of HLA-A*68. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A* 30:01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30: 01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two of HLA-A*03:01, HLA-A*25:01, HLA-A*29:02, HLA-A*30: 01, HLA-A*31:01, HLA-A*33:01, HLA-A*36:01 and HLA-A*68:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one or two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for one of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for two of HLA-A*25:01, HLA-A*29:02, and HLA-A*30:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*25:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*29:02 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*30:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*03:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*31:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is HLA-A*33:01 positive. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for HLA-A*36:01. In one embodiment, the host or recipient graft is HLA-A2 negative and is positive for HLA-A*68:01. In one embodiment, the graft is HLA-A2 positive. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the immune cell is a regulatory T cell. In one embodiment, the immune cell is a regulatory CD4+ T cell. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In another embodiment, the immune cell is a human regulatory T cell. In one embodiment, the immune cell is a human CD4+ regulatory T cell.

In one embodiment, the present invention provides a method for propagating a population of immune cells in a subject, wherein the immune cells are modified to express a chimeric antigen receptor (CAR), the method comprising administering to the subject an immune cell as described elsewhere herein, wherein the introduced modified immune cells produce the population of hereditary immune cells in the object. In one embodiment, the immune cell is a regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In one embodiment, the immune cells may be regulatory T cells. In one embodiment, the immune cells may be CD4+ regulatory T cells. In one embodiment, the immune cell is a human immune cell. In one embodiment, the regulatory immune cell is a human regulatory immune cell. In one embodiment, the regulatory immune cell is selected from the group consisting of regulatory T cell, CD4+ regulatory T cell, CD8+ regulatory T cell, regulatory γδ T cell, regulatory DN T cell, regulatory B cell, regulatory NK cell , regulatory macrophage, regulatory dendritic cell, and any combination thereof. In another embodiment, the immune cell is a human regulatory T cell. In one embodiment, the immune cell is a human CD4+ regulatory T cell. In one embodiment, the CAR-modified Treg cells of the present invention are capable of replicating in vivo, resulting in long-term persistence, which can lead to long-term suppression of the target cell's immune response and immune tolerance. In one embodiment, a method is provided for creating a persistent population of regulatory T cells in a subject, wherein the regulatory T cells are modified to express a chimeric antigen receptor (CAR), the method comprising administering to the subject the regulatory T cells described elsewhere herein, wherein the persistent population of modified regulatory T cells persists in the subject for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 200 days, 300 days, 400 days, 500 days, 600 days, 700 days, 800 days, 900 days or 1000 days after administration. In one embodiment, CAR Treg cells may be capable of self-renewal and re-activation in vivo to suppress the immune response of the target cell. In one embodiment, the CAR Tregs may be memory CAR Tregs that can be reactivated to suppress the immune response of the target cell.

Immune cells can be obtained from any source. For example, in one embodiment, immune cells can be obtained from a tissue donor, transplant recipient, or other unrelated source (another entity or species in general) to obtain the CAR-modified immune cells of the present invention. Accordingly, the CAR-modified immune cells of the present invention may be autologous, allogeneic, or xenogeneic to the transplant recipient, or otherwise unrelated source. In one embodiment, the CAR Treg cells of the present invention may be autologous, allogeneic, or xenogeneic for the transplant recipient. In one embodiment, the CAR Treg cells of the present invention may be autologous to a transplant recipient.

In one embodiment, the subject may be a mammal. In one embodiment, the subject may be a human.

In one embodiment, it may be desirable to administer activated T cells to a subject, and then subsequently rebleed (or perform apheresis), activate T cells from it in accordance with the present invention, and reinject those activated and expanded T cells to the subject. This process can be performed several times every few weeks. In some embodiments, T cells can be activated when blood is taken from 10 to 400 cm ³ . In some embodiments, T cells are activated when blood is drawn in volumes of 20 cm ³ , 30 cm ³ , 40 cm ³ , 50 cm ³ , 60 cm ³ , 70 cm ³ , 80 cm ³ , 90 cm ³ , or 100 cm ³ . Without wishing to be bound by theory, it is believed that the use of this multiple blood draw/multiple reinfusion protocol may serve to select specific T cell populations.

The CAR-modified immune cells of the present invention may be administered alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. In one embodiment, the pharmaceutical composition may contain a variety of modified immune cells described elsewhere in this document. In one embodiment, the pharmaceutical compositions of the present invention may contain a CAR-modified immune cell or cell population as described elsewhere herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. The pharmaceutical compositions of the present invention may be administered to a subject by any suitable means, including inhalation, injection, ingestion, transfusion, implantation, or transplantation. The pharmaceutical compositions described herein may be administered to a subject by parenteral administration. The pharmaceutical compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrasternally, intravenously (IV) by injection, infusion methods, or intraperitoneally. In one embodiment, the CAR-modified immune cell compositions of the present invention may be administered to a subject by intradermal or subcutaneous injection. In another embodiment, the CAR-modified immune cell compositions of the present invention may be administered by intravenous injection. In one embodiment, CAR-modified immune cell compositions can be injected directly into a lymph node, site of infection, site of inflammation, or site of tissue or organ rejection.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the prevention or treatment of a disease. The amount and frequency of administration will be determined by such factors as the condition of the subject and the type and severity of the subject's disease, although appropriate doses may be determined by clinical trials.

Where an "effective amount" or "therapeutic amount" is indicated, the amount of compositions of the present invention to be administered may be determined to account for individual differences in age, weight, antibody titer, and condition of the subject. In general, it can be argued that a pharmaceutical composition containing the CAR-modified immune cells described herein can be administered at a dosage of 1×10 ⁴ to 1×10 ⁹ cells/kg body weight. In one embodiment, CAR-modified immune cells can be administered at a dosage of 1×10 ⁵ to 100×10 ⁵ cells/kg body weight, including all integer values in these ranges. Compositions of CAR-modified immune cells can also be administered multiple times at these dosages. CAR-modified immune cells can be administered using infusion methods that are widely known in immunotherapy (see, for example, Rosenberg et al, 1988). The optimal dosage and treatment regimen for a particular subject can be readily determined by monitoring the subject for signs of disease and adjusting treatment accordingly.

In one embodiment, at least one CAR-engineered immune cell of the invention is administered to a subject in need thereof in combination with another active agent. According to one embodiment, at least one population of immune cells is administered before, simultaneously with, or after administration of another active agent.

In one embodiment, the CAR-modified immune cells of the present invention may be administered to a subject in combination with (e.g., before, concurrently, or after) any number of suitable treatments, including, but not limited to, treatment with agents such as antiviral therapy, chemotherapy, radiation, immunosuppressants, antibodies, immunoablative agents, cytokines, and radiation. In one embodiment, the CAR-modified immune cells of the present invention may be administered in combination with an immunosuppressive agent. Any immunosuppressive agent known in the art may be used. For example, the immunosuppressive agent may be calcineurin inhibitors such as cyclosporine, tacrolimus, azathioprine, methotrexate, methoxalene, rapamycin, mycophenolate mofetil, mycophenolic acid, sodium mycophenolate, 6-mercaptopurine, 6-thioguanine, rituximab, mTOR inhibitors such as sirolimus, everolimus, basiliximab, daclizumab, belatacept, alemtuzumab, muromonab-CD3, anti-thymocyte globulin, glucocorticosteroids or adrenocortical steroids such as prednisone and prednisolone, or any combination thereof. The CAR-modified immune cells of the present invention can be administered to a subject before, after, or simultaneously with an immunosuppressive agent. For example, the CAR-modified immune cells of the present invention may be administered after an immunosuppressive agent is administered to the subject, or the CAR-modified immune cells of the invention may be administered before the immunosuppressive agent is administered to the subject. Alternatively or in addition, the CAR-modified immune cells of the invention may be administered simultaneously with an immunosuppressive agent administered to the subject. The CAR-modified immune cells of the present invention and/or an immunosuppressive agent may be administered to a subject after transplantation. Alternatively or in addition, the CAR-modified immune cells of the present invention and/or an immunosuppressive agent may be administered to the subject prior to transplantation. The CAR-modified immune cells of the present invention and/or an immunosuppressive agent may be administered to a subject during transplant surgery. In one embodiment, the method of the invention for administering CAR-modified immune cells to a subject is performed after initiation of immunosuppressive therapy. In some embodiments, the method is performed more than once, for example, to monitor the transplant recipient over time and, if applicable, different immunosuppressive therapy regimens. In some embodiments, immunosuppressive therapy is reduced if the transplant recipient is predicted to be transplant tolerant. In some embodiments, no immunosuppressive therapy is administered, for example, immunosuppressive therapy is discontinued if the transplant recipient is predicted to be graft tolerant.

Also provided is a method for preventing or treating organ or tissue rejection in a subject, comprising administering to the subject (i) at least one immunosuppressive agent and (ii) CAR-engineered immune cells (e.g., CAR-engineered Treg cells) of the invention or a pharmaceutical a composition containing said immune cells.

Thus, another embodiment of the present invention is the combination of a CAR-engineered immune cell (preferably a CAR-engineered Treg cell) of the invention, or a pharmaceutical composition containing said immune cells, and at least one immunosuppressive agent for use in preventing or treating rejection. transplant of an organ or tissue in an object.

Also provided is a method for increasing graft survival in a subject, comprising administering to the subject at least one immunosuppressive agent and CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention, or a pharmaceutical composition containing them.

In one embodiment, the combination of at least one immunosuppressive agent with CAR engineered immune cells (CAR engineered Treg cells) of the present invention is used to prevent or treat graft versus host disease (GVHD). In one embodiment, GVHD may occur following hematopoietic stem cell transplantation.

Thus, another embodiment of the invention is a method of preventing or treating graft-versus-host disease (GVHD) in a subject, comprising administering to the subject at least one immunosuppressive agent and CAR-engineered immune cells (CAR-engineered Treg cells) or a pharmaceutical composition, as described in this document.

In one embodiment, the invention relates to a method of contacting a donor graft, for example, a biocompatible mesh or a donor tissue, organ or cell, with at least one immunosuppressive agent and with CAR-engineered immune cells (CAR-engineered Treg cells) of the present invention, simultaneously, before or after transplantation of the graft to the recipient.

In one embodiment, the combination of at least one immunosuppressive agent with CAR-engineered immune cells (CAR-engineered Treg cells) of the present invention can be used to attenuate, inhibit, or reduce an adverse response of a donor graft against a recipient, thereby preventing or exposing treatment for GVHD.

Thus, another embodiment of the present invention is a method of preventing or delaying the onset of GVHD in a subject, comprising administering to the subject at least one immunosuppressive agent and CAR engineered immune cells (preferably CAR engineered Treg cells), or a pharmaceutical composition as described in this document.

In one embodiment, the CAR engineered immune cells of the invention and at least one immunosuppressive agent are administered simultaneously or sequentially.

In one embodiment, the CAR-engineered immune cells of the present invention, and optionally at least one other active agent, preferably an immunosuppressive agent, are administered in combination with (eg, before, simultaneously or after) the graft.

The CAR-engineered immune cells of the present invention may be administered following the diagnosis of transplant organ or tissue rejection, followed by doses of both the CAR-engineered immune cells of the invention and an immunosuppressive agent until symptoms of organ or tissue rejection resolve. In a further embodiment, the CAR-engineered immune cell compositions of the present invention may be administered to a subject in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation. In another embodiment, the CAR-engineered immune cells of the present invention may be administered following ablative therapy with B cells, eg agents that react with CD20, eg rituximab. For example, in one embodiment, subjects may be treated with standard high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, subjects may receive an infusion of the expanded CAR-engineered immune cells of the present invention. In a further embodiment, expanded CAR-engineered immune cells can be administered before or after surgery.

In one embodiment, the subject (e.g., human) receives an initial administration of at least one immune cell or population of the invention and one or more subsequent administrations, with one or more subsequent administrations administered in less than 15 days, e.g., 14, 13 , 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after previous administration. In one embodiment, a therapeutically effective amount of the immune cells of the invention is or is to be administered to a subject. In one embodiment, the number of immune cells of at least one immune cell population of the invention administered to the subject is at least 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , or 10 ⁹ cells. In one embodiment, the number of immune cells of the invention administered to the subject is about ¹⁰² to about ¹⁰⁹ , about ¹⁰³ to about ¹⁰⁸ , about ¹⁰⁴ to about ¹⁰⁷ , or about ¹⁰⁵ to about ¹⁰⁶ cells. In another embodiment, the number of immune cells of the invention administered to the subject is about 10 ⁶ to about 10 ⁹ , about 10 ⁶ to 10 ⁷ , about 10 ⁶ to 10 ⁸ , about 10 ⁷ to 10 ⁹ , about 10 ⁷ up to 10 ⁸ , from about 10 ⁸ to 10 ⁹ . In another embodiment, the number of immune cells of the invention administered to the subject is about 10 ⁶ , about 10 ⁷ , about 10 ⁸ , or about 10 ⁹ . In one embodiment, the number of immune cells of at least one immune cell population of the invention administered to the subject is at least 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , or 10 ⁹ cells/kg body. In one embodiment, the number of immune cells of the invention administered to the subject is from about 10 ⁴ to 10 ⁹ cells/kg body weight, or 10 ⁵ to 10 ⁸ cells/kg body weight, including all integer values in these ranges. In one embodiment, the subject (e.g., human) is given more than one injection of at least one immune cell or population of the invention per week, such as 2, 3, or 4 injections of the genetically modified immune cell or population of the invention per week.

In some embodiments, it may be desirable to include a "suicide" gene in the CAR-modified immune cells of the present invention to allow selective destruction of the CAR-modified immune cells in vivo. In one embodiment, the CAR-modified immune cells of the present invention may further comprise a "suicide" gene system. In one embodiment, the CAR-modified immune cells of the present invention may further comprise a nucleic acid encoding a "suicide" gene system. The terms "suicide gene therapy", "suicide gene" and "suicide gene system", as described herein, refer to methods for selectively destroying a cell through apoptosis. Such methods use a "suicide" gene that will cause the cell to kill itself by apoptosis. The "suicide" gene encodes a product that causes cell death on its own or in the presence of other compounds. The "suicide" gene can be derived from any "suicide" gene system suitable for use in cell therapy (see e.g. Jones et al., 2014; Wang et al., 2017; Zhang et al., 2017; Casucci et al. ., 2011; Gargett et al., 2014; Khaleghi, 2012; Philip et al., 2014).

In one embodiment, the suicide gene system may include gene-targeted enzyme prodrug therapy (GDEPT), where a non-toxic drug (i.e., a prodrug of the suicide gene) is converted to a toxic compound in cells modified with the suicide gene. For example, in one embodiment, the suicide gene system may be a herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) thymidine kinase gene system that uses the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir. In other embodiments, the suicide gene system may be a cytosine deaminase/5-fluorocytosine-based suicide gene system. In some alternatives to the methods provided herein, where the CAR-modified immune cells comprise a "suicide" gene system, the methods further comprise introducing the "suicide" gene prodrug to the subject.

In other embodiments, the "suicide" gene system may include a dimerization induction mechanism in which apoptotic genes such as caspases destroy cells to induce apoptosis. Dimerization-inducing suicide gene systems can include chimeric proteins consisting of a drug-binding domain coupled to components of an apoptotic pathway allowing conditional dimerization and apoptosis of modified immune cells following administration of a chemical inducer of dimerization (CID). For example, in one embodiment, the suicide gene system may be a caspase-based suicide gene system. Non-limiting examples of an inducible caspase-based suicide gene system may include an inducible caspase-9 (iCasp9)-based suicide gene system and a caspase-8 (iCasp8)-based suicide gene system where caspase is activated by CID, FK506 or their equivalent. In some alternative methods provided herein, where the CAR-modified immune cells contain a "suicide" gene system, the methods further comprise administering a chemical dimerization inducer to the subject.

In other embodiments, the "suicide" gene system may be mediated by a therapeutic monoclonal antibody. Therapeutic mAb-mediated suicide gene systems involve the expression of cell surface proteins that render modified immune cells sensitive to therapeutic mAbs. Modified immune cells can be selectively destroyed after administration of a specific therapeutic mAb. Non-limiting examples of therapeutic mAb-mediated suicide gene systems can include the CD20-based suicide gene system and the RQR8-based suicide gene system, where the therapeutic mAb is an anti-CD20 mAb. In some embodiments, the anti-CD20 mAb may be rituximab. In some alternative methods provided herein, where the CAR-modified immune cells contain a "suicide" gene system, the methods further comprise administering a therapeutic monoclonal antibody to the subject.

VIII. FINISHED PRODUCTS AND SETS

In another embodiment, the invention provides an article of manufacture containing materials useful in the prevention and/or treatment of transplant rejection or GVHD. The product may include a container and a label or liner in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be made from various materials such as glass or plastic. The container includes a composition that is effective in preventing and/or treating an immunological condition such as transplant rejection or GVHD and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper suitable for piercing with a needle for subcutaneous injections). At least one active agent in the composition is a CAR-modified immune cell of the present invention. In one embodiment, the CAR-modified immune cell is a CAR-modified Treg. In one embodiment, the CAR-modified Treg is a CAR-modified human Treg. The label or package insert may indicate that the composition is used to prevent or treat transplant rejection or GVHD. The product, label, or package insert may further contain instructions for administering the CAR-engineered immune cell composition to a patient. In addition, the article may further comprise a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate saline, Ringer's solution and dextrose solution. In addition, it may include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

Kits are also provided that are useful for a variety of purposes (eg, to prevent or treat transplant rejection or GVHD). Kits may be offered that contain CAR-engineered immune cells. As with an article of manufacture, the kit may comprise a container and a label or liner in or associated with the container. The container contains a composition containing at least one CAR-modified immune cell of the present invention. Additional containers may be included which contain, for example, diluents and buffers. The label or package insert may contain a description of the composition as well as instructions for the intended use. In one embodiment, the CAR-modified immune cells are CAR-modified Tregs. In one embodiment, the CAR-modified Tregs are human CAR-modified Tregs.

Thus, the present invention further relates to a set of parts, including, in the first place, the immune cell or population of immune cells of the present invention, and in the second part, another active agent, including, but not limited to, antiviral therapy, chemotherapy, radiation therapy, immunosuppressants, antibodies, immunoablative agents, cytokines, and radiation. In some embodiments, a set of part of the present invention includes in the first part of the immune cell or population of immune cells of the present invention and in the second part one or more immunosuppressive agents. Examples of immunosuppressive agents include, but are not limited to, calcineurin inhibitors such as cyclosporine, tacrolimus, azathioprine, methotrexate, methoxsalen, rapamycin, mycophenolate mofetil, mycophenolic acid, sodium mycophenolate, 6-mercaptopurine, 6-thioguanine, rituximab, mTOR inhibitors such as sirolimus, everolimus, basiliximab, daclizumab, belatacept, alemtuzumab, muromonab-CD3, anti-thymocyte globulin, glucocorticosteroids or adrenocortical steroids such as pre-predolidone, or any combination thereof.

Table 1

Kabat Chothia VH CDR1 SYHIQ (SEQ ID NO: 1) GYTFTSY (SEQ ID NO: 2) VH CDR2 WIYPGDGSTQYNEKFKG (SEQ ID NO: 3) YPGDGS (SEQ ID NO: 4) VH CDR2 WIYPGDGSTKYSQKFQG (SEQ ID NO: 5) YPGDGS (SEQ ID NO: 4) VH CDR3 EGTYYAMDY (SEQ ID NO: 6) EGTYYAMDY (SEQ ID NO: 6)

table 2

Kabat Chothia VL CDR1 RSSQSIVHSNGNTYLE (SEQ ID NO: 7) RSSQSIVHSNGNTYLE (SEQ ID NO: 7) VL CDR2 KVSNRFS (SEQ ID NO: 8) KVSNRFS (SEQ ID NO: 8) VL CDR3 FQGSHVPRT (SEQ ID NO: 9) FQGSHVPRT (SEQ ID NO: 9)

Table 3

SEQ Name SEQ ID AA VH CDR2 (Kabat) ten WIYPGDGSTX ¹⁰ YX ¹² X ¹³ KFX ¹⁶ G VH FR1 (chothia) eleven QVQLVQSGAEVKKPGASVKVSCKAS VH FR1 (kabat) 12 QVQLVQSGAEVKKPGASVKVSCKASGYTFT VH FR2 (kabat/kabat variable) 13 WVRQAPGQX ⁹ LEWMGX ¹⁵ VH1/2/5/6 FR2 (kabat/kabat) fourteen WVRQAPGQRLEWMG VH3 FR2 (kabat/kabat) fifteen WVRQAPGQGLEWMGI VH4 FR2 (kabat/kabat) 16 WVRQAPGQGLEWMG VH FR2 (kabat/chothia variable) 17 WVRQAPGQX ⁹ LEWMGX ¹⁵ WI VH1/2/5/6 FR2 (kabat/chothia) eighteen WVRQAPGQRLEWMGWI VH3 FR2 (kabat/chothia) 19 WVRQAPGQGLEWMGIWI VH4 FR2 (kabat/chothia) twenty WVRQAPGQGLEWMGWI VH FR2 (chothia/chothia variable) 21 HIQWVRQAPGQX ¹² LEWMGX ¹⁸ WI VH1/2/5/6 FR2 (chothia/chothia) 22 HIQWVRQAPGQRLEWMGWI VH3 FR2 (chothia/chothia) 23 HIQWVRQAPGQGLEWMGIWI VH4 FR2 (chothia/chothia) 24 HIQWVRQAPGQGLEWMGWI VH FR2 (chothia/kabat variable) 25 HIQWVRQAPGQX ¹² LEWMGX ¹⁸ VH1/2/5/6 FR2 (chothia/kabat) 26 HIQWVRQAPGQRLEWMG VH3 FR2 (chothia/kabat) 27 HIQWVRQAPGQGLEWMGI VH4 FR2 (chothia/kabat) 28 HIQWVRQAPGQGLEWMG VH FR3 (kabat variable) 29 X ¹ VTX ⁴ TX ⁶ DTSX ¹⁰ STAYMX ¹⁶ LSX ¹⁹ LRSX ²³ DX ²⁵ AVYYCAR VH1/6 FR3 (kabat) thirty RVTITRDTSASTAYMELSSLRSEDTAVYYCAR VH2 FR3 (kabat) 31 RVTITADTSASTAYMLLSSLRSEDTAVYYCAR VH3 FR3 (kabat) 32 VTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VH4 FR3 (kabat) 33 RVMTRDTSISTAYMELSRLRSDDTAVYYCAR VH5 FR3 (kabat) 34 RVTITRDTSASTAYMELSSLRSEDMAVYYCAR VH FR3 (chothia variable) 35 TX ² YX ⁴ X ⁵ KFX ⁸ GX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR VH1/2/3/4/5 FR3 (chothia variable) 36 TQYNEKFKGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR VH6 FR3 (chothia variable) 37 TKYSQKFQGX ¹⁰ VTX ¹³ TX ¹⁵ DTSX ¹⁹ STAYMX ²⁵ LSX ²⁸ LRSX ³² DX ³⁴ AVYYCAR VH 1 FR3 (chothia) 38 TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR VH 2 FR3 (chothia) 39 TQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAR VH 3 FR3 (chothia) 40 TQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VH 4 FR3 (chothia) 41 TQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR VH 5 FR3 (chothia) 42 TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAR VH6FR3 (chothia) 43 TKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR VH FR4 44 WGQGTTVTVSS vh 45 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQX ⁴⁴ LEWMGX ⁵⁰ WIYPGDGSTX ⁶⁰ YX ⁶² X ⁶³ KFX ⁶⁶ GX ⁶⁸ VTX ⁷¹ TX ⁷³ DTSX ⁷⁷ STAYMX ⁸³ LSX ⁸⁶ LRSX ⁹⁰ DX ⁹² VL-FR1 46 DX ² VMTQX ⁷ PLSX ¹¹ X ¹² VTX ¹⁵ GQPASISX ²³ VL1/4 47 DVVMTQSPLSLPVTLGQPASISC VL2 FR1 48 DIVMTQTPLSLSVTPGQPASISC VL3FR1 49 DIVMTQTPLSSPVTLGQPASISC VL5 FR1 fifty DIVMTQTPLSSPVTLGQPASISF VL FR2 51 WX ² X ³ QX ⁵ PGQX ⁹ PX ¹¹ X ¹² LIY VL1 FR2 52 WFQQRPGQSPRRLIY VL2 FR2 53 WYLQKPGQSPQLLIY VL3/5FR2 54 WYQQRPGQPPRLLIY VL4FR2 55 WYQQRPGQSPRLLIY VL FR3 56 GVPDRFSGSGX ¹¹ GTDFTLKISRVEAEDVGVYYC VL1/2/4 FR3 57 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC VL3/5FR3 58 GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC VL FR4 59 FGGGTKVEIK VL 60 DX ² VMTQX ⁷ PLSX ¹¹ X ¹² VTX ¹⁵ GQPASISX ²³ RSSQSIVHSNGNTYLEWX ⁴¹ X ⁴² QX ⁴⁴ PGQX ⁴⁸ PX ⁵⁰ X ⁵¹ LIYKVSNRFSGVPDRFSGSGX ⁷² GTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1 (no leader) 61 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSS H2
(no leader) 62 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSS H3
(no leader) 63 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSS H4 (no leader) 64 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSS H5 (no leader) 65 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSS H6 (no leader) 66 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSS K1 67 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK K2 68 DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK K3 69 DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK K4 70 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK K5 71 DIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k1_AA (no leader) 72 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k2_AA
(no leader) 73 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k3_AA
(no leader) 74 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k4_AA
(no leader) 75 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k5_AA
(no leader) 76 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H2_k1_AA (no leader) 77 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H2_k2_AA (no leader) 78 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k2_AA (no leader) 79 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k3_AA (no leader) 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k4_AA (no leader) 81 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k5_AA (no leader) 82 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k2_AA (no leader) 83 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k3_AA (no leader) 84 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k4_AA (no leader) 85 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k5_AA (no leader) 86 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k2_AA (no leader) 87 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k3_AA (no leader) 88 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k4_AA (no leader) 89 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k5_AA (no leader) 90 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H6_k2_AA (no leader) 91 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H6 92 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQX ⁴⁴ LEWMGX ⁵⁰ WIYPGDGSTKYSQKFQGX ⁶⁸ VTX ⁷¹ TX ⁷³ DTSX ⁷⁷ STAYMX ⁸³ LSX ⁸⁶ LRSX ⁹⁰ DX ⁹² AVYYCAREGTYYAMDYWGQGTTVTVSS H1_k1_AA (with leader) 93 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k2_AA
(with leader) 94 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k3_AA
(with leader) 95 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k4_AA
(with leader) 96 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H1_k5_AA
(with leader) 97 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H2_k1_AA (with leader) 98 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H2_k2_AA (with leader) 99 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k2_AA (with leader) 100 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k3_AA (with leader) 101 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k4_AA (with leader) 102 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H3_k5_AA (with leader) 103 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k2_AA (with leader) 104 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k3_AA (with leader) 105 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k4_AA (with leader) 106 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H4_k5_AA (with leader) 107 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k2_AA (with leader) 108 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k3_AA (with leader) 109 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k4_AA (with leader) 110 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H5_k5_AA (with leader) 111 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK H6_k2_AA (with leader) 112 MDFQVQIFSFLLISASVIMSRASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK Leader 113 MDFQVQIFSFLLISASVIMSRAS linker 114 VDSSGGGGSGGGGSGGGGSTS Hinge CD8 115 SALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD CD28TM 116 FWVLVVVGGVLACYSLLVTVAFIFWV CD28IC 117 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD3z 118 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k1_AA (no leader) 119 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k2_AA
(no leader) 120 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k3_AA
(no leader) 121 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k4_AA
(no leader) 122 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k5_AA
(no leader) 123 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H2_k1_AA (no leader) 124 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H2_k2_AA (no leader) 125 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSASTAYMLLSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H3_k2_AA (no leader) 126 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H3_k3_AA (no leader) 127 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H3_k4_AA (no leader) 128 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H3_k5_AA (no leader) 129 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGIWIYPGDGSTQYNEKFKGVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H4_k2_AA (no leader) 130 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H4_k3_AA (no leader) 131 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H4_k4_AA (no leader) 132 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H4_k5_AA (no leader) 133 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H5_k2_AA (no leader) 134 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H5_k3_AA (no leader) 135 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H5_k4_AA (no leader) 136 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H5_k5_AA (no leader) 137 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDMAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSSPVTLGQPASISFRSSQSIVHSNGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H6_k2_AA (no leader) 138 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSVDSSGGGGSGGGGSGGGGSTSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR_H1_k2_AA 213 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4-1BB_AA
(SEQ ID NO: 23 of US 9,102,760) 216 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD8 AK hinge (SEQ ID NO:21 of US 9,102,760) 218 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY CD8 AK hinge 219 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 transmembrane_AA (SEQ ID NO:22 of US 9,102,760) 221 IWAPLAGTCGVLLLSLVITLYC CD8_transmembrane AAs 223 IYIWAPLAGTCGVLLLSLVITLYC

Table 4

SEQ Name SEQ ID NT H1_k1_NT 139 ATGGATTTCCAAGTTCAAATCTTCAGTTTCTTGCTTATCAGTGCTTCTGTTATTATGTCACGAGCAAGTCAAGTTCAACTCGTACAGTCTGGAGCCGAGGTGAAAAAACCGGGAGCGTCCGTGAAAGTGAGTTGCAAGGCGAGTGGATACACCTTCACTTCATACCATATACAATGGGTTCGGCAGGCGCCTGGTCAACGGCTGGAATGGATGGGCTGGATTTATCCCGGAGATGGTTCCACGCAGTACAATGAGAAATTCAAAGGGAGAGTGACAATCACCCGAGATACCAGTGCCTCTACGGCATATATGGAACTGAGTAGTCTGCGGTCTGAAGATACGGCGGTGTATTATTGTGCGAGAGAAGGGACGTACTACGCCATGGATTATTGGGGACAAGGAACAACAGTCACAGTCTCCAGCGTTGATTCCTCAGGCGGGGGCGGAAGTGGGGGCGGCGGATCTGGCGGCGGCGGGTCTACGTCTGACGTGGTCATGACCCAATCTCCATTGTCTTTGCCAGTTACTCTGGGACAGCCTGCAAGTATCAGTTGCCGATCCTCCCAATCTATCGTCCATTCAAACGGGAACACTTATTTGGAATGGTTTCAACAGAGACCTGGGCAAAGTCCGCGCCGACTGATATATAAGGTCAGTAACCGCTTTTCAGGCGTCCCCGATCGATTCAGTGGATCTGGGTCAGGGACTGACTTCACTCTGAAAATATCAAGAGTCGAAGCTGAAGATGTCGGAGTATATTACTGTTTCCAGGGGTCTCACGTCCCTCGGACGTTTGGAGGCGGAACTAAGGTTGAGATAAAA H1_k2_NT 140 ATGGATTTTCAGGTTCAAATCTTTAGCTTTCTCTTGATTTCCGCCTCCGTAATAATGAGTCGGGCCAGTCAGGTACAGCTCGTTCAATCTGGGGCTGAAGTAAAAAAGCCTGGAGCGTCTGTAAAGGTATCTTGCAAAGCGAGCGGCTACACATTCACAAGTTATCACATCCAATGGGTGAGACAGGCCCCAGGACAACGCTTGGAGTGGATGGGGTGGATTTACCCTGGCGACGGCAGCACACAGTACAATGAGAAATTTAAAGGCCGGGTGACTATCACTCGGGACACCTCCGCCAGCACGGCTTATATGGAGCTTAGCAGTTTGAGATCCGAAGATACAGCGGTATATTACTGCGCGAGAGAAGGAACGTACTACGCTATGGACTATTGGGGTCAGGGCACAACCGTTACAGTCTCCTCTGTGGACAGCTCCGGAGGTGGGGGTTCAGGAGGGGGTGGAAGCGGTGGTGGTGGCAGTACAAGCGATATAGTAATGACCCAAACCCCGCTCTCTCTGAGCGTCACGCCAGGACAACCAGCATCAATCTCTTGCCGCAGTAGTCAATCCATCGTTCACTCTAATGGAAACACATACCTTGAGTGGTATCTTCAGAAACCAGGTCAGAGCCCTCAGCTCCTCATCTATAAAGTCTCTAACCGGTTCTCAGGTGTTCCGGACCGGTTCAGTGGTTCCGGCTCAGGAACAGACTTCACCTTGAAGATCAGTCGAGTAGAAGCCGAAGACGTGGGTGTATATTATTGCTTTCAGGGTTCCCACGTTCCGCGCACCTTCGGCGGCGGGACCAAAGTTGAGATCAAA H2_k1_NT 141 ATGGACTTTCAAGTCCAAATCTTCTCATTCCTCCTGATCTCTGCGTCAGTAATCATGTCCAGAGCGTCACAAGTGCAACTCGTTCAATCCGGAGCTGAGGTAAAGAAGCCCGGCGCCAGCGTGAAAGTCTCCTGCAAAGCGAGCGGCTACACGTTCACCTCATATCACATTCAATGGGTAAGACAAGCACCTGGGCAACGACTCGAGTGGATGGGGTGGATCTACCCTGGGGACGGGAGCACGCAGTATAATGAGAAATTCAAAGGCAGGGTTACAATTACAGCCGATACCAGTGCATCTACGGCTTATATGCTCCTCTCCTCACTCCGGTCTGAGGACACAGCGGTTTATTATTGCGCACGGGAGGGAACGTACTACGCGATGGACTATTGGGGGCAAGGCACCACAGTTACAGTGAGCTCAGTTGACTCATCAGGAGGCGGAGGATCAGGGGGAGGTGGTAGTGGGGGCGGTGGGAGCACATCAGATGTTGTCATGACTCAGAGCCCACTTTCTTTGCCGGTGACGCTGGGGCAGCCCGCTTCAATCTCTTGCCGCTCATCACAGTCTATCGTTCATAGCAATGGTAACACTTACTTGGAATGGTTCCAACAAAGACCGGGTCAAAGTCCACGGCGCTTGATATATAAAGTATCAAATAGATTTTCAGGGGTGCCTGATCGGTTCAGCGGTTCTGGATCTGGCACCGACTTCACGCTTAAAATAAGTAGGGTAGAAGCCGAAGACGTGGGAGTGTATTATTGTTTCCAGGGGTCACACGTCCCTCGCACGTTCGGCGGAGGCACTAAAGTGGAGATCAAA H2_k2_NT 142 ATGGACTTTCAAGTCCAAATCTTCAGCTTTCTCCTTATATCTGCGTCTGTCATTATGAGTAGAGCAAGTCAAGTCCAGCTCGTACAAAGTGGAGCTGAGGTGAAAAAGCCGGGCGCGAGTGTGAAAGTCTCATGCAAGGCGAGTGGATACACCTTTACCTCTTACCACATTCAATGGGTGCGGCAGGCGCCTGGGCAGCGCTTGGAATGGATGGGCTGGATATATCCTGGCGACGGAAGTACCCAGTACAACGAAAAATTCAAAGGTAGGGTTACCATCACTGCTGATACCTCCGCGTCCACTGCTTATATGCTTCTTAGCTCCTTGCGAAGCGAGGATACAGCCGTGTATTATTGTGCCAGAGAGGGGACTTATTATGCCATGGACTATTGGGGTCAGGGTACAACGGTCACTGTCTCATCTGTTGACAGTAGCGGGGGAGGGGGGTCTGGAGGAGGGGGTTCCGGGGGGGGAGGTTCCACGAGCGATATAGTTATGACGCAAACGCCCTTGAGCCTCAGTGTTACACCCGGTCAACCTGCCTCTATTAGCTGTCGCTCCTCCCAATCAATTGTGCATAGCAATGGAAATACCTACCTTGAATGGTATCTCCAAAAGCCCGGGCAGAGTCCTCAACTTCTCATCTACAAAGTATCCAATCGATTCAGTGGCGTTCCTGACAGGTTCAGCGGAAGTGGGTCAGGGACCGATTTTACCCTCAAAATTAGTCGCGTCGAAGCTGAGGATGTTGGGGTGTATTACTGCTTCCAAGGGTCACACGTACCACGCACATTCGGGGGAGGCACGAAGGTTGAAATTAAG H1_k3_NT 143 ATGGATTTCCAAGTCCAAATATTCAGTTTCCTTTTGATAAGTGCTTCAGTTATCATGTCCCGAGCAAGCCAGGTACAGCTTGTGCAAAGCGGGGCGGAAGTTAAGAAACCGGGAGCCTCAGTTAAAGTATCTTGCAAAGCCAGCGGTTATACATTCACTTCATACCACATACAGTGGGTGCGCCAAGCACCGGGGCAGAGACTGGAATGGATGGGATGGATTTATCCCGGTGATGGTAGTACGCAATACAATGAAAAATTCAAAGGAAGGGTGACTATCACGCGAGACACAAGCGCGTCCACGGCCTATATGGAACTGAGCAGTCTGAGATCCGAGGACACCGCTGTGTATTATTGCGCCCGAGAGGGGACCTACTACGCCATGGATTATTGGGGTCAAGGGACCACTGTGACAGTCTCTTCTGTCGATTCCAGCGGCGGAGGAGGGAGTGGAGGGGGTGGGTCCGGGGGGGGAGGGTCTACGAGCGACATTGTCATGACACAGACGCCGCTTAGCTCCCCAGTTACACTCGGACAGCCCGCAAGTATTAGTTGCAGAAGTAGCCAGTCTATCGTACATTCAAATGGAAACACCTATTTGGAATGGTATCAACAACGGCCTGGACAGCCGCCCAGGCTGCTCATATACAAAGTCTCCAACCGCTTCAGCGGAGTACCCGACCGCTTTTCCGGCTCCGGAGCAGGAACTGACTTTACCTTGAAAATTAGTAGGGTCGAAGCAGAGGATGTCGGGGTATATTATTGTTTCCAAGGTAGTCATGTCCCACGGACGTTTGGTGGTGGGACGAAGGTTGAGATCAAA H1_k4_NT 144 ATGGACTTCCAGGTGCAGATATTTTCCTTTCTTCTCATATCTGCATCTGTAATAATGTCAAGGGCCAGCCAGGTCCAGCTCGTTCAAAGCGGAGCCGAGGTAAAAAAACCAGGCGCTTCCGTCAAGGTATCATGCAAAGCGTCCGGCTATACCTTCACAAGTTACCATATCCAATGGGTTCGACAGGCACCCGGACAAAGACTTGAATGGATGGGTTGGATATACCCCGGAGACGGCTCCACTCAGTATAACGAAAAGTTTAAGGGGAGAGTCACGATCACTAGGGACACATCAGCTTCTACGGCGTATATGGAACTCAGTTCTTTGCGATCCGAGGATACTGCCGTATATTACTGCGCCAGAGAAGGCACGTACTACGCAATGGATTACTGGGGGCAAGGGACAACTGTTACCGTCTCAAGCGTCGATTCATCAGGAGGCGGAGGGTCCGGAGGTGGGGGATCTGGCGGTGGGGGTTCTACGTCCGATGTTGTGATGACACAGTCCCCACTCTCTCTTCCAGTGACGCTGGGACAGCCCGCGAGCATCTCCTGTCGCAGCTCTCAGTCCATAGTACACAGTAATGGTAACACCTATCTTGAGTGGTATCAGCAACGACCCGGTCAGTCTCCCAGGTTGCTTATTTATAAGGTTAGTAACCGCTTTTCAGGTGTTCCAGACAGATTTAGCGGGAGTGGTTCCGGTACGGATTTCACATTGAAAATCAGCCGCGTCGAAGCCGAGGACGTGGGTGTTTACTACTGCTTCCAGGGATCTCACGTACCGAGGACCTTCGGCGGAGGAACGAAAGTAGAAATTAAG H1_k5_NT 145 ATGGATTTTCAAGTACAAATCTTTTCCTTCCTGCTTATTTCCGCAAGCGTTATTATGAGTAGAGCCAGCCAAGTACAACTGGTACAGTCCGGCGCGGAGGTGAAGAAGCCCGGAGCAAGTGTGAAGGTATCTTGCAAGGCGTCAGGCTATACCTTTACTTCATACCATATACAGTGGGTGCGACAGGCTCCCGGCCAGCGACTCGAATGGATGGGCTGGATTTATCCCGGAGATGGATCTACGCAGTATAATGAGAAATTCAAGGGTCGGGTCACGATTACACGAGACACGAGTGCTTCCACAGCTTATATGGAACTTTCTAGCCTGAGGTCTGAGGATACTGCCGTTTACTATTGTGCACGAGAAGGGACATACTATGCGATGGATTACTGGGGACAGGGCACCACTGTCACAGTTTCCAGCGTGGACTCAAGTGGAGGCGGTGGATCTGGTGGTGGCGGGTCCGGGGGGGGAGGCAGCACCAGTGACATTGTAATGACTCAAACACCTCTCAGTAGCCCAGTCACTCTCGGTCAGCCGGCGAGTATCTCTTTTAGGTCCTCACAATCTATAGTGCACTCTAACGGCAATACTTATCTTGAATGGTATCAACAAAGACCGGGGCAGCCACCTCGCCTTCTCATCTACAAAGTAAGCAATCGCTTCTCCGGTGTCCCCGATCGCTTCTCCGGTTCAGGAGCAGGAACTGACTTCACATTGAAGATTTCCAGAGTGGAGGCCGAAGACGTAGGGGTATATTATTGCTTTCAAGGGTCCCATGTGCCCAGAACCTTTGGGGGAGGAACGAAAGTTGAGATTAAA H3_k2_NT 146 ATGGATTTCCAAGTGCAGATTTTCTCTTTTCTCCTCATAAGCGCCTCCGTAATTATGTCTAGAGCTAGTCAAGTCCAATTGGTGCAATCCGGTGCCGAGGTTAAAAAGCCCGGCGCAAGTGTAAAAGTCTCCTGTAAGGCCAGTGGCTACACTTTCACCAGCTACCATATACAGTGGGTGCGGCAGGCGCCTGGTCAGGGTCTGGAGTGGATGGGTATTTGGATTTATCCCGGAGATGGAAGTACTCAATACAATGAGAAATTCAAGGGTGTCACTATGACAAGGGATACGAGCACTTCTACCGTATATATGGAGTTGTCATCTTTGCGATCAGAGGATACCGCTGTATATTATTGCGCACGGGAAGGTACATATTATGCCATGGACTACTGGGGCCAAGGAACCACCGTGACGGTAAGCTCTGTCGATTCTAGCGGTGGCGGGGGCTCTGGCGGTGGGGGTAGCGGGGGTGGCGGATCTACATCAGATATTGTAATGACACAGACCCCTCTTTCACTTTCCGTAACGCCAGGACAGCCGGCATCAATAAGTTGCCGATCAAGCCAGTCTATCGTACACTCCAATGGTAACACATACTTGGAATGGTATCTTCAAAAGCCCGGCCAGAGCCCGCAGCTTTTGATATATAAAGTGTCCAACAGATTCAGTGGGGTGCCGGACCGCTTTAGTGGATCTGGTTCAGGAACGGACTTCACATTGAAAATTAGTAGAGTTGAAGCGGAAGACGTGGGAGTCTACTACTGTTTCCAGGGTTCACATGTGCCTCGGACCTTTGGGGGAGGCACCAAGGTTGAGATAAAA H3_k3_NT 147 ATGGACTTCCAAGTCCAAATCTTTTCTTTTTTGTTGATAAGCGCATCAGTTATTATGTCTCGCGCCAGTCAAGTACAACTGGTGCAGTCCGGAGCTGAAGTGAAAAAACCAGGAGCAAGCGTGAAAGTAAGTTGTAAGGCAAGTGGTTACACTTTCACAAGCTACCATATTCAATGGGTCCGACAGGCTCCTGGACAGGGCTTGGAGTGGATGGGCATATGGATTTACCCTGGTGACGGGTCCACCCAGTATAATGAAAAGTTCAAGGGAGTCACGATGACCAGGGACACCTCTACATCTACCGTGTATATGGAGCTCTCTAGTTTGCGATCCGAAGACACTGCCGTTTATTACTGTGCAAGAGAAGGAACTTATTACGCGATGGACTACTGGGGTCAGGGGACAACAGTCACCGTTAGCTCCGTCGATTCCAGCGGGGGAGGTGGCTCAGGCGGGGGTGGTTCTGGGGGGGGCGGGAGCACTTCAGATATTGTAATGACCCAAACCCCACTGAGTAGTCCAGTCACGCTTGGTCAACCGGCAAGCATTTCTTGCAGGAGCTCTCAGAGTATTGTCCACTCTAACGGGAATACATATTTGGAGTGGTATCAGCAAAGACCGGGCCAACCACCACGCCTCTTGATTTATAAGGTGAGCAATAGGTTTTCAGGCGTGCCAGATAGGTTCTCAGGCTCCGGAGCGGGAACCGACTTCACCCTCAAGATAAGTCGGGTGGAAGCCGAAGACGTAGGAGTTTACTACTGCTTTCAAGGATCTCATGTTCCACGAACGTTTGGAGGAGGAACCAAGGTGGAAATAAAA H3_k4_NT 148 ATGGACTTTCAGGTCCAAATTTTTTCCTTCTTGCTCATATCCGCGAGTGTCATCATGTCAAGAGCAAGTCAAGTTCAACTCGTTCAATCAGGAGCTGAGGTGAAAAAACCAGGGGCGTCTGTCAAAGTAAGCTGCAAAGCATCAGGGTATACGTTCACGAGTTATCATATCCAGTGGGTTAGGCAGGCGCCAGGGCAGGGATTGGAATGGATGGGTATCTGGATTTACCCGGGTGACGGCAGCACTCAATACAATGAGAAATTCAAAGGCGTAACAATGACAAGGGACACGAGCACAAGCACAGTGTACATGGAGCTTAGCTCTTTGAGGTCAGAGGATACCGCTGTTTACTATTGTGCTCGGGAGGGTACTTACTATGCAATGGACTACTGGGGGCAAGGCACGACCGTTACAGTGAGTAGCGTAGATTCCTCCGGGGGTGGCGGTTCAGGCGGCGGAGGCTCAGGCGGAGGAGGGTCAACATCCGATGTCGTAATGACTCAGTCCCCTCTGTCATTGCCGGTGACTTTGGGACAGCCAGCGTCTATATCTTGTAGGTCCTCTCAATCAATAGTGCATTCCAACGGTAACACCTATCTGGAATGGTATCAGCAAAGGCCAGGACAAAGTCCACGCCTGCTTATATATAAGGTGTCTAATCGATTCAGTGGGGTTCCCGATAGGTTTTCCGGCTCTGGTAGCGGGACTGATTTCACGTTGAAAATATCACGCGTGGAAGCGGAAGATGTTGGGGTCTATTACTGCTTTCAGGGTAGTCATGTCCCTCGAACTTTTGGCGGTGGTACAAAGGTAGAAATCAAA H3_k5_NT 149 ATGGATTTTCAGGTACAGATATTCTCATTTCTCCTTATCTCAGCTAGTGTCATAATGTCCAGGGCGAGTCAAGTACAACTTGTCCAGTCAGGCGCAGAGGTCAAGAAGCCGGGCGCAAGCGTTAAGGTTTCCTGCAAAGCATCCGGCTATACATTCACGTCCTATCACATCCAATGGGTCAGGCAAGCACCCGGTCAAGGACTTGAGTGGATGGGCATCTGGATTTACCCTGGAGATGGCAGTACTCAGTACAACGAAAAATTCAAAGGTGTAACCATGACCCGCGACACATCTACTTCCACAGTTTATATGGAACTCAGCAGTTTGCGGAGCGAAGATACCGCTGTTTACTACTGTGCCCGAGAGGGAACTTACTACGCCATGGACTATTGGGGTCAAGGAACGACAGTAACAGTTAGTTCTGTAGATTCCAGTGGCGGCGGTGGGAGCGGGGGTGGGGGATCTGGCGGAGGCGGAAGTACAAGTGACATCGTTATGACTCAGACACCCCTTAGTAGTCCCGTTACGTTGGGCCAACCCGCGAGCATTTCCTTTCGATCCTCTCAGTCTATAGTTCACTCAAATGGGAATACTTATTTGGAGTGGTATCAACAGCGCCCCGGACAACCACCAAGGCTCCTGATATACAAGGTGTCCAATCGATTCTCTGGGGTGCCTGATAGATTTAGCGGAAGTGGAGCCGGTACAGATTTTACCCTGAAAATATCACGGGTAGAAGCCGAAGATGTCGGCGTCTACTACTGTTTCCAGGGTTCCCATGTACCGCGAACGTTCGGGGGCGGAACAAAAGTTGAGATCAAG H4_k2_NT 150 ATGGATTTCCAGGTTCAGATATTTAGTTTCCTCTTGATTTCTGCCAGTGTCATCATGAGCAGGGCTTCCCAAGTTCAGTTGGTGCAAAGTGGCGCTGAAGTCAAAAAACCTGGGGCTTCCGTTAAAGTATCTTGCAAGGCGTCCGGCTACACTTTCACATCCTACCACATTCAATGGGTCCGGCAAGCGCCCGGTCAGGGGCTCGAATGGATGGGGTGGATATACCCAGGAGATGGATCTACTCAGTACAACGAGAAATTTAAAGGACGGGTGACGATGACGCGCGACACTTCAATAAGCACTGCATACATGGAACTGTCCCGGCTTAGGTCAGATGACACCGCGGTCTACTATTGTGCGAGAGAGGGTACTTACTATGCTATGGACTACTGGGGGCAAGGCACGACGGTTACAGTTTCCTCAGTCGATAGTTCAGGCGGAGGCGGCTCCGGGGGCGGTGGTAGTGGAGGGGGTGGATCTACTTCCGACATTGTCATGACCCAGACCCCGTTGAGCCTTTCAGTGACGCCCGGTCAACCCGCCAGCATAAGTTGTCGATCAAGCCAGTCTATTGTACACTCCAATGGAAACACATATTTGGAGTGGTATCTCCAAAAACCCGGCCAAAGCCCTCAACTGCTCATCTACAAGGTCTCAAACAGGTTTAGCGGGGTCCCGGATCGCTTCTCAGGGTCAGGATCTGGTACGGACTTTACACTGAAAATTTCCCGAGTCGAAGCGGAAGACGTGGGTGTATATTACTGCTTCCAGGGGAGTCATGTTCCAAGAACCTTTGGGGGAGGTACAAAGGTCGAAATAAAA H4_k3_NT 151 ATGGATTTTCAGGTCCAAATTTTTTCCTTCTTGCTTATCAGCGCAAGTGTAATCATGTCCCGCGCGTCCCAAGTACAACTTGTGCAATCTGGCGCGGAGGTGAAAAAACCTGGAGCTTCCGTCAAGGTTTCTTGTAAGGCCTCTGGCTACACCTTCACGTCCTACCACATTCAGTGGGTTCGACAGGCGCCGGGCCAAGGACTGGAGTGGATGGGATGGATATATCCAGGAGATGGTTCTACTCAGTATAATGAGAAATTCAAGGGTCGCGTAACAATGACGAGGGATACATCAATCTCCACCGCGTACATGGAACTTTCAAGACTCCGGTCAGATGACACGGCGGTTTACTACTGTGCTCGGGAGGGCACTTACTATGCTATGGACTACTGGGGGCAAGGGACAACGGTAACGGTATCTAGTGTGGATTCTAGTGGCGGCGGCGGTTCAGGAGGAGGTGGTTCAGGCGGGGGGGGTAGTACAAGTGATATTGTGATGACCCAAACACCCCTTTCTAGCCCTGTTACTCTGGGTCAACCCGCGTCCATAAGTTGTCGAAGTAGTCAATCCATCGTGCATAGCAACGGCAACACTTACCTTGAATGGTATCAACAACGACCCGGACAGCCCCCGCGACTGCTTATCTATAAAGTATCAAACAGGTTCAGTGGCGTGCCAGATCGATTCTCCGGCTCTGGGGCAGGCACAGATTTCACGTTGAAAATTTCTCGGGTCGAGGCCGAGGACGTGGGCGTTTATTACTGTTTCCAGGGGAGTCACGTCCCCAGGACGTTCGGAGGAGGAACTAAAGTCGAAATAAAG H4_k4_NT 152 ATGGACTTCCAGGTCCAAATATTCAGCTTCCTCCTCATTTCCGCCAGTGTAATAATGTCCAGAGCCTCACAAGTACAGTTGGTTCAGAGCGGGGCTGAGGTTAAGAAACCAGGCGCGAGCGTCAAGGTATCCTGCAAGGCGAGTGGTTATACTTTCACTAGTTATCACATTCAGTGGGTCCGACAGGCCCCCGGTCAAGGCCTGGAGTGGATGGGGTGGATATATCCGGGAGATGGTTCTACCCAATATAATGAGAAGTTTAAGGGGAGAGTCACAATGACAAGGGACACCAGTATTAGCACCGCGTATATGGAGCTTTCCCGCCTGCGATCAGATGACACGGCCGTGTACTACTGTGCTAGGGAGGGAACCTATTATGCGATGGATTACTGGGGACAGGGTACTACAGTCACGGTCTCTAGCGTGGACAGTTCCGGGGGCGGTGGAAGCGGTGGTGGCGGTTCAGGTGGAGGAGGCTCTACGAGTGATGTTGTGATGACTCAGTCCCCGCTTTCACTTCCCGTCACCCTTGGGCAACCCGCAAGCATCTCATGCCGATCCTCCCAGTCTATAGTACATAGTAATGGCAACACATATCTTGAATGGTATCAGCAGAGGCCGGGTCAGTCTCCCCGACTCCTTATATATAAAGTGAGCAACAGATTCTCCGGAGTACCGGATAGATTTTCCGGCTCTGGGAGCGGCACCGACTTTACACTGAAAATTTCACGGGTTGAAGCTGAAGATGTTGGGGTATACTATTGTTTCCAGGGTTCTCACGTCCCGAGGACATTCGGGGGAGGAACGAAAGTCGAAATAAAG H4_k5_NT 153 ATGGATTTTCAGGTACAAATCTTCAGCTTCCTGCTCATCTCCGCGAGCGTAATCATGTCTAGGGCGTCCCAGGTGCAGTTGGTGCAATCAGGTGCAGAGGTGAAGAAGCCTGGTGCATCCGTTAAAGTAAGTTGTAAGGCAAGCGGATATACTTTTACATCCTATCATATTCAATGGGTCAGACAAGCACCTGGACAGGGTCTTGAGTGGATGGGCTGGATCTATCCAGGCGATGGCTCAACTCAATATAACGAGAAGTTCAAGGGGAGGGTTACTATGACCAGGGATACGTCTATTTCCACTGCGTACATGGAACTCTCCAGGTTGAGAAGTGATGATACCGCGGTTTACTACTGCGCTAGAGAAGGAACGTACTACGCTATGGATTACTGGGGGCAGGGTACAACTGTCACCGTCTCAAGTGTGGATTCTTCTGGGGGTGGGGGATCAGGAGGGGGAGGCTCCGGTGGGGGCGGGTCAACCAGCGACATTGTCATGACTCAAACCCCCCTGAGCAGCCCTGTCACCCTGGGTCAGCCTGCCTCAATATCCTTTAGAAGCTCCCAAAGCATCGTCCATTCAAATGGTAATACCTATCTGGAGTGGTATCAGCAAAGGCCTGGTCAACCCCCGCGCCTTCTCATTTACAAGGTGTCAAACAGGTTCTCCGGCGTACCGGATAGGTTTTCCGGAAGCGGTGCTGGAACCGACTTTACTCTCAAAATCTCTAGGGTGGAAGCTGAGGACGTCGGTGTATACTATTGTTTTCAAGGCTCCCATGTTCCCAGGACATTTGGTGGGGGAACGAAGGTAGAAATCAAG H5_k2_NT 154 ATGGACTTTCAGGTTCAGATTTTCTCTTTCTTGTTGATCTCCGCTAGTGTCATAATGTCACGGGCAAGTCAGGTACAACTCGTTCAGAGTGGTGCCGAAGTGAAGAAACCGGGTGCCTCCGTAAAGGTGTCATGTAAAGCTAGTGGCTATACATTCACAAGTTATCATATCCAATGGGTACGACAAGCACCGGGACAGCGACTGGAATGGATGGGATGGATCTATCCTGGGGACGGATCTACACAGTACAATGAGAAATTTAAGGGACGGGTCACGATAACCAGGGACACATCTGCTTCCACGGCTTACATGGAGCTTTCCTCCCTGCGGAGCGAGGACATGGCTGTTTACTATTGCGCTCGCGAAGGGACATACTACGCAATGGATTATTGGGGCCAAGGCACTACCGTGACGGTCTCTTCTGTCGATAGTTCCGGAGGAGGTGGTTCAGGGGGAGGCGGTTCAGGTGGGGGTGGATCTACCTCAGATATTGTCATGACACAGACACCTTTGTCCTTGAGTGTGACACCGGGTCAACCGGCGAGTATAAGCTGTCGCAGCTCACAATCTATTGTGCATAGCAACGGGAATACATATCTCGAATGGTATCTCCAAAAGCCGGGCCAATCCCCCCAACTTCTCATTTACAAAGTTTCTAATCGATTTTCAGGTGTACCAGATCGGTTTTCCGGGTCTGGCTCAGGTACTGACTTCACCTTGAAAATATCAAGGGTTGAAGCTGAGGATGTAGGTGTGTACTATTGCTTCCAGGGGTCTCACGTTCCTCGGACTTTTGGGGGGGGCACAAAAGTAGAGATTAAA H5_k3_NT 155 ATGGATTTCCAGGTGCAAATCTTCTCATTTCTTTTGATAAGTGCGTCAGTGATAATGTCTCGGGCCAGTCAAGTACAGCTTGTCCAAAGTGGCGCTGAAGTCAAGAAGCCGGGAGCCTCAGTTAAGGTTAGCTGCAAGGCCTCAGGGTATACTTTTACCTCCTATCATATACAGTGGGTACGACAAGCACCGGGACAGCGACTGGAGTGGATGGGTTGGATATATCCGGGAGATGGTTCAACCCAGTATAATGAGAAGTTCAAGGGGCGAGTTACGATAACCCGCGATACGAGTGCATCAACAGCGTACATGGAGTTGAGTTCCCTCCGCAGCGAGGACATGGCGGTATACTATTGTGCCAGGGAGGGGACTTATTATGCCATGGACTACTGGGGGCAGGGCACAACCGTAACAGTCTCTTCTGTAGACAGTTCAGGAGGGGGCGGAAGTGGAGGTGGCGGATCTGGTGGAGGTGGATCTACTTCCGACATCGTTATGACCCAAACACCACTTTCATCTCCCGTTACTCTCGGGCAACCTGCTAGTATTTCCTGTAGATCCTCACAATCTATAGTACATAGCAATGGCAATACCTACCTGGAGTGGTATCAACAACGCCCAGGCCAACCACCTCGCCTGCTTATCTATAAAGTAAGCAATAGATTCAGTGGTGTACCGGATAGGTTCTCTGGTTCCGGAGCAGGAACTGACTTTACACTCAAAATCAGTAGGGTGGAGGCGGAAGACGTGGGAGTATATTATTGCTTTCAAGGTTCACATGTACCTCGAACATTTGGCGGAGGAACTAAGGTTGAGATTAAA H5_k4_NT 156 ATGGATTTCCAAGTCCAGATATTCAGTTTTCTTTTGATAAGCGCTTCTGTAATCATGTCTCGGGCGTCCCAAGTACAACTGGTGCAATCAGGGGCAGAAGTGAAAAAACCAGGTGCATCCGTTAAGGTGAGTTGCAAGGCTTCCGGCTATACCTTTACATCATATCATATTCAATGGGTCAGGCAAGCACCTGGTCAGCGATTGGAATGGATGGGTTGGATATATCCTGGTGATGGGTCTACACAATATAACGAAAAATTCAAGGGGCGAGTGACCATCACAAGAGATACATCAGCGTCAACAGCGTATATGGAACTGTCATCCCTTAGATCAGAGGACATGGCGGTCTATTACTGTGCCAGAGAAGGCACTTATTATGCAATGGATTATTGGGGACAAGGAACCACTGTCACTGTTTCCAGCGTAGACTCCTCCGGTGGTGGTGGAAGTGGCGGCGGTGGGTCAGGAGGGGGTGGGTCAACTTCTGATGTAGTGATGACACAGAGCCCTCTGAGCTTGCCTGTGACCTTGGGTCAGCCGGCCTCAATAAGTTGTCGATCTAGTCAGTCAATCGTCCATAGTAATGGGAACACATACCTTGAATGGTATCAGCAAAGACCTGGACAATCTCCACGACTCCTTATATACAAAGTTAGCAACCGATTTAGCGGAGTGCCAGACCGCTTTTCTGGTTCCGGGTCTGGCACAGATTTTACCCTTAAGATCTCCCGCGTGGAGGCGGAAGACGTTGGTGTTTACTATTGCTTCCAGGGGTCACACGTTCCACGCACCTTTGGAGGAGGTACGAAGGTCGAGATTAAG H5_k5_NT 157 ATGGATTTTCAAGTACAGATCTTCTCTTTCTTGCTTATTTCAGCGAGCGTAATCATGAGTAGGGCATCTCAAGTTCAACTCGTTCAGTCAGGTGCTGAGGTAAAAAAACCAGGGGCTTCCGTTAAAGTTAGCTGTAAGGCATCTGGGTACACATTTACTAGCTACCATATCCAGTGGGTGCGACAAGCCCCGGGGCAGCGCTTGGAATGGATGGGCTGGATTTACCCAGGTGACGGCTCCACGCAATATAATGAGAAATTTAAGGGTAGAGTTACTATTACCAGGGACACAAGTGCTTCAACTGCCTATATGGAACTGAGCAGCCTTCGGAGTGAAGATATGGCCGTATATTACTGCGCAAGGGAGGGGACTTACTATGCAATGGACTACTGGGGTCAGGGAACGACTGTGACCGTGTCCTCAGTTGACTCCAGCGGTGGTGGCGGCTCTGGAGGTGGGGGTTCCGGCGGAGGCGGAAGCACATCTGATATAGTGATGACGCAAACGCCTCTTTCTTCCCCGGTAACTCTGGGACAGCCAGCGTCAATTTCATTTAGGTCCTCCCAGTCAATCGTACATAGTAATGGAAATACTTACCTGGAATGGTATCAACAACGACCAGGGCAACCGCCCCGATTGTTGATCTATAAAGTGAGCAATCGCTTTTCTGGCGTGCCCGATCGGTTCTCAGGGTCTGGAGCTGGGACTGACTTCACATTGAAAATTTCCAGGGTTGAGGCCGAGGATGTGGGGGTTTATTACTGCTTCCAAGGCTCCCACGTCCCCCGCACCTTCGGAGGGGGAACCAAAGTCGAAATAAAG H6_k2_NT 158 ATGGATTTTCAAGTTCAGATATTCTCATTTTTGCTTATATCAGCCTCCGTAATTATGTCACGGGCAAGTCAAGTTCAGTTGGTGCAGTCCGGAGCAGAAGTTAAGAAGCCCGGTGCTTCTGTGAAAGTCTCCTGCAAAGCGTCTGGGTACACCTTCACGAGCTACCATATACAGTGGGTCCGGCAAGCGCCTGGGCAGAGGCTGGAGTGGATGGGCTGGATTTACCCAGGAGATGGGAGTACAAAGTATAGTCAGAAGTTTCAAGGGCGAGTGACGATAACCAGAGATACGAGTGCAAGTACTGCATACATGGAACTGAGCTCCTTGAGGTCCGAGGATACAGCGGTGTACTATTGCGCTCGGGAAGGGACATATTATGCTATGGACTATTGGGGACAAGGGACAACGGTAACGGTGAGTTCCGTCGATTCCTCAGGTGGCGGAGGCAGTGGGGGCGGGGGTTCCGGCGGTGGCGGGTCCACGAGTGATATAGTTATGACACAGACCCCCCTCAGCCTTTCTGTGACCCCAGGACAACCCGCTAGTATCTCTTGCCGCAGTTCTCAGTCCATAGTACACAGTAACGGAAATACCTATCTTGAGTGGTATCTTCAAAAGCCCGGCCAGAGCCCTCAACTCTTGATATATAAAGTGTCAAATCGATTTTCAGGTGTGCCTGATCGATTCTCAGGGTCTGGTTCAGGGACAGATTTCACGCTTAAGATAAGCAGAGTAGAGGCTGAAGACGTGGGAGTCTACTATTGTTTTCAGGGGTCACACGTTCCCCGCACTTTTGGTGGGGGAACCAAGGTGGAAATCAAA Hinge CD8 159 TCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGAC CD28TM 160 TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG CD28IC 161 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGACTTCGCAGCCTATCGCTCC CD3z 162 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_k1_NT 163 ATGGATTTCCAAGTTCAAATCTTCAGTTTCTTGCTTATCAGTGCTTCTGTTATTATGTCACGAGCAAGTCAAGTTCAACTCGTACAGTCTGGAGCCGAGGTGAAAAAACCGGGAGCGTCCGTGAAAGTGAGTTGCAAGGCGAGTGGATACACCTTCACTTCATACCATATACAATGGGTTCGGCAGGCGCCTGGTCAACGGCTGGAATGGATGGGCTGGATTTATCCCGGAGATGGTTCCACGCAGTACAATGAGAAATTCAAAGGGAGAGTGACAATCACCCGAGATACCAGTGCCTCTACGGCATATATGGAACTGAGTAGTCTGCGGTCTGAAGATACGGCGGTGTATTATTGTGCGAGAGAAGGGACGTACTACGCCATGGATTATTGGGGACAAGGAACAACAGTCACAGTCTCCAGCGTTGATTCCTCAGGCGGGGGCGGAAGTGGGGGCGGCGGATCTGGCGGCGGCGGGTCTACGTCTGACGTGGTCATGACCCAATCTCCATTGTCTTTGCCAGTTACTCTGGGACAGCCTGCAAGTATCAGTTGCCGATCCTCCCAATCTATCGTCCATTCAAACGGGAACACTTATTTGGAATGGTTTCAACAGAGACCTGGGCAAAGTCCGCGCCGACTGATATATAAGGTCAGTAACCGCTTTTCAGGCGTCCCCGATCGATTCAGTGGATCTGGGTCAGGGACTGACTTCACTCTGAAAATATCAAGAGTCGAAGCTGAAGATGTCGGAGTATATTACTGTTTCCAGGGGTCTCACGTCCCTCGGACGTTTGGAGGCGGAACTAAGGTTGAGATAAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_k2_NT 164 ATGGATTTTCAGGTTCAAATCTTTAGCTTTCTCTTGATTTCCGCCTCCGTAATAATGAGTCGGGCCAGTCAGGTACAGCTCGTTCAATCTGGGGCTGAAGTAAAAAAGCCTGGAGCGTCTGTAAAGGTATCTTGCAAAGCGAGCGGCTACACATTCACAAGTTATCACATCCAATGGGTGAGACAGGCCCCAGGACAACGCTTGGAGTGGATGGGGTGGATTTACCCTGGCGACGGCAGCACACAGTACAATGAGAAATTTAAAGGCCGGGTGACTATCACTCGGGACACCTCCGCCAGCACGGCTTATATGGAGCTTAGCAGTTTGAGATCCGAAGATACAGCGGTATATTACTGCGCGAGAGAAGGAACGTACTACGCTATGGACTATTGGGGTCAGGGCACAACCGTTACAGTCTCCTCTGTGGACAGCTCCGGAGGTGGGGGTTCAGGAGGGGGTGGAAGCGGTGGTGGTGGCAGTACAAGCGATATAGTAATGACCCAAACCCCGCTCTCTCTGAGCGTCACGCCAGGACAACCAGCATCAATCTCTTGCCGCAGTAGTCAATCCATCGTTCACTCTAATGGAAACACATACCTTGAGTGGTATCTTCAGAAACCAGGTCAGAGCCCTCAGCTCCTCATCTATAAAGTCTCTAACCGGTTCTCAGGTGTTCCGGACCGGTTCAGTGGTTCCGGCTCAGGAACAGACTTCACCTTGAAGATCAGTCGAGTAGAAGCCGAAGACGTGGGTGTATATTATTGCTTTCAGGGTTCCCACGTTCCGCGCACCTTCGGCGGCGGGACCAAAGTTGAGATCAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H2_k1_NT 165 ATGGACTTTCAAGTCCAAATCTTCTCATTCCTCCTGATCTCTGCGTCAGTAATCATGTCCAGAGCGTCACAAGTGCAACTCGTTCAATCCGGAGCTGAGGTAAAGAAGCCCGGCGCCAGCGTGAAAGTCTCCTGCAAAGCGAGCGGCTACACGTTCACCTCATATCACATTCAATGGGTAAGACAAGCACCTGGGCAACGACTCGAGTGGATGGGGTGGATCTACCCTGGGGACGGGAGCACGCAGTATAATGAGAAATTCAAAGGCAGGGTTACAATTACAGCCGATACCAGTGCATCTACGGCTTATATGCTCCTCTCCTCACTCCGGTCTGAGGACACAGCGGTTTATTATTGCGCACGGGAGGGAACGTACTACGCGATGGACTATTGGGGGCAAGGCACCACAGTTACAGTGAGCTCAGTTGACTCATCAGGAGGCGGAGGATCAGGGGGAGGTGGTAGTGGGGGCGGTGGGAGCACATCAGATGTTGTCATGACTCAGAGCCCACTTTCTTTGCCGGTGACGCTGGGGCAGCCCGCTTCAATCTCTTGCCGCTCATCACAGTCTATCGTTCATAGCAATGGTAACACTTACTTGGAATGGTTCCAACAAAGACCGGGTCAAAGTCCACGGCGCTTGATATATAAAGTATCAAATAGATTTTCAGGGGTGCCTGATCGGTTCAGCGGTTCTGGATCTGGCACCGACTTCACGCTTAAAATAAGTAGGGTAGAAGCCGAAGACGTGGGAGTGTATTATTGTTTCCAGGGGTCACACGTCCCTCGCACGTTCGGCGGAGGCACTAAAGTGGAGATCAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H2_k2_NT 166 ATGGACTTTCAAGTCCAAATCTTCAGCTTTCTCCTTATATCTGCGTCTGTCATTATGAGTAGAGCAAGTCAAGTCCAGCTCGTACAAAGTGGAGCTGAGGTGAAAAAGCCGGGCGCGAGTGTGAAAGTCTCATGCAAGGCGAGTGGATACACCTTTACCTCTTACCACATTCAATGGGTGCGGCAGGCGCCTGGGCAGCGCTTGGAATGGATGGGCTGGATATATCCTGGCGACGGAAGTACCCAGTACAACGAAAAATTCAAAGGTAGGGTTACCATCACTGCTGATACCTCCGCGTCCACTGCTTATATGCTTCTTAGCTCCTTGCGAAGCGAGGATACAGCCGTGTATTATTGTGCCAGAGAGGGGACTTATTATGCCATGGACTATTGGGGTCAGGGTACAACGGTCACTGTCTCATCTGTTGACAGTAGCGGGGGAGGGGGGTCTGGAGGAGGGGGTTCCGGGGGGGGAGGTTCCACGAGCGATATAGTTATGACGCAAACGCCCTTGAGCCTCAGTGTTACACCCGGTCAACCTGCCTCTATTAGCTGTCGCTCCTCCCAATCAATTGTGCATAGCAATGGAAATACCTACCTTGAATGGTATCTCCAAAAGCCCGGGCAGAGTCCTCAACTTCTCATCTACAAAGTATCCAATCGATTCAGTGGCGTTCCTGACAGGTTCAGCGGAAGTGGGTCAGGGACCGATTTTACCCTCAAAATTAGTCGCGTCGAAGCTGAGGATGTTGGGGTGTATTACTGCTTCCAAGGGTCACACGTACCACGCACATTCGGGGGAGGCACGAAGGTTGAAATTAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_k3_NT 167 ATGGATTTCCAAGTCCAAATATTCAGTTTCCTTTTGATAAGTGCTTCAGTTATCATGTCCCGAGCAAGCCAGGTACAGCTTGTGCAAAGCGGGGCGGAAGTTAAGAAACCGGGAGCCTCAGTTAAAGTATCTTGCAAAGCCAGCGGTTATACATTCACTTCATACCACATACAGTGGGTGCGCCAAGCACCGGGGCAGAGACTGGAATGGATGGGATGGATTTATCCCGGTGATGGTAGTACGCAATACAATGAAAAATTCAAAGGAAGGGTGACTATCACGCGAGACACAAGCGCGTCCACGGCCTATATGGAACTGAGCAGTCTGAGATCCGAGGACACCGCTGTGTATTATTGCGCCCGAGAGGGGACCTACTACGCCATGGATTATTGGGGTCAAGGGACCACTGTGACAGTCTCTTCTGTCGATTCCAGCGGCGGAGGAGGGAGTGGAGGGGGTGGGTCCGGGGGGGGAGGGTCTACGAGCGACATTGTCATGACACAGACGCCGCTTAGCTCCCCAGTTACACTCGGACAGCCCGCAAGTATTAGTTGCAGAAGTAGCCAGTCTATCGTACATTCAAATGGAAACACCTATTTGGAATGGTATCAACAACGGCCTGGACAGCCGCCCAGGCTGCTCATATACAAAGTCTCCAACCGCTTCAGCGGAGTACCCGACCGCTTTTCCGGCTCCGGAGCAGGAACTGACTTTACCTTGAAAATTAGTAGGGTCGAAGCAGAGGATGTCGGGGTATATTATTGTTTCCAAGGTAGTCATGTCCCACGGACGTTTGGTGGTGGGACGAAGGTTGAGATCAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_k4_NT 168 ATGGACTTCCAGGTGCAGATATTTTCCTTTCTTCTCATATCTGCATCTGTAATAATGTCAAGGGCCAGCCAGGTCCAGCTCGTTCAAAGCGGAGCCGAGGTAAAAAAACCAGGCGCTTCCGTCAAGGTATCATGCAAAGCGTCCGGCTATACCTTCACAAGTTACCATATCCAATGGGTTCGACAGGCACCCGGACAAAGACTTGAATGGATGGGTTGGATATACCCCGGAGACGGCTCCACTCAGTATAACGAAAAGTTTAAGGGGAGAGTCACGATCACTAGGGACACATCAGCTTCTACGGCGTATATGGAACTCAGTTCTTTGCGATCCGAGGATACTGCCGTATATTACTGCGCCAGAGAAGGCACGTACTACGCAATGGATTACTGGGGGCAAGGGACAACTGTTACCGTCTCAAGCGTCGATTCATCAGGAGGCGGAGGGTCCGGAGGTGGGGGATCTGGCGGTGGGGGTTCTACGTCCGATGTTGTGATGACACAGTCCCCACTCTCTCTTCCAGTGACGCTGGGACAGCCCGCGAGCATCTCCTGTCGCAGCTCTCAGTCCATAGTACACAGTAATGGTAACACCTATCTTGAGTGGTATCAGCAACGACCCGGTCAGTCTCCCAGGTTGCTTATTTATAAGGTTAGTAACCGCTTTTCAGGTGTTCCAGACAGATTTAGCGGGAGTGGTTCCGGTACGGATTTCACATTGAAAATCAGCCGCGTCGAAGCCGAGGACGTGGGTGTTTACTACTGCTTCCAGGGATCTCACGTACCGAGGACCTTCGGCGGAGGAACGAAAGTAGAAATTAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_k5_NT 169 ATGGATTTTCAAGTACAAATCTTTTCCTTCCTGCTTATTTCCGCAAGCGTTATTATGAGTAGAGCCAGCCAAGTACAACTGGTACAGTCCGGCGCGGAGGTGAAGAAGCCCGGAGCAAGTGTGAAGGTATCTTGCAAGGCGTCAGGCTATACCTTTACTTCATACCATATACAGTGGGTGCGACAGGCTCCCGGCCAGCGACTCGAATGGATGGGCTGGATTTATCCCGGAGATGGATCTACGCAGTATAATGAGAAATTCAAGGGTCGGGTCACGATTACACGAGACACGAGTGCTTCCACAGCTTATATGGAACTTTCTAGCCTGAGGTCTGAGGATACTGCCGTTTACTATTGTGCACGAGAAGGGACATACTATGCGATGGATTACTGGGGACAGGGCACCACTGTCACAGTTTCCAGCGTGGACTCAAGTGGAGGCGGTGGATCTGGTGGTGGCGGGTCCGGGGGGGGAGGCAGCACCAGTGACATTGTAATGACTCAAACACCTCTCAGTAGCCCAGTCACTCTCGGTCAGCCGGCGAGTATCTCTTTTAGGTCCTCACAATCTATAGTGCACTCTAACGGCAATACTTATCTTGAATGGTATCAACAAAGACCGGGGCAGCCACCTCGCCTTCTCATCTACAAAGTAAGCAATCGCTTCTCCGGTGTCCCCGATCGCTTCTCCGGTTCAGGAGCAGGAACTGACTTCACATTGAAGATTTCCAGAGTGGAGGCCGAAGACGTAGGGGTATATTATTGCTTTCAAGGGTCCCATGTGCCCAGAACCTTTGGGGGAGGAACGAAAGTTGAGATTAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H3_k2_NT 170 ATGGATTTCCAAGTGCAGATTTTCTCTTTTCTCCTCATAAGCGCCTCCGTAATTATGTCTAGAGCTAGTCAAGTCCAATTGGTGCAATCCGGTGCCGAGGTTAAAAAGCCCGGCGCAAGTGTAAAAGTCTCCTGTAAGGCCAGTGGCTACACTTTCACCAGCTACCATATACAGTGGGTGCGGCAGGCGCCTGGTCAGGGTCTGGAGTGGATGGGTATTTGGATTTATCCCGGAGATGGAAGTACTCAATACAATGAGAAATTCAAGGGTGTCACTATGACAAGGGATACGAGCACTTCTACCGTATATATGGAGTTGTCATCTTTGCGATCAGAGGATACCGCTGTATATTATTGCGCACGGGAAGGTACATATTATGCCATGGACTACTGGGGCCAAGGAACCACCGTGACGGTAAGCTCTGTCGATTCTAGCGGTGGCGGGGGCTCTGGCGGTGGGGGTAGCGGGGGTGGCGGATCTACATCAGATATTGTAATGACACAGACCCCTCTTTCACTTTCCGTAACGCCAGGACAGCCGGCATCAATAAGTTGCCGATCAAGCCAGTCTATCGTACACTCCAATGGTAACACATACTTGGAATGGTATCTTCAAAAGCCCGGCCAGAGCCCGCAGCTTTTGATATATAAAGTGTCCAACAGATTCAGTGGGGTGCCGGACCGCTTTAGTGGATCTGGTTCAGGAACGGACTTCACATTGAAAATTAGTAGAGTTGAAGCGGAAGACGTGGGAGTCTACTACTGTTTCCAGGGTTCACATGTGCCTCGGACCTTTGGGGGAGGCACCAAGGTTGAGATAAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H3_k3_NT 171 ATGGACTTCCAAGTCCAAATCTTTTCTTTTTTGTTGATAAGCGCATCAGTTATTATGTCTCGCGCCAGTCAAGTACAACTGGTGCAGTCCGGAGCTGAAGTGAAAAAACCAGGAGCAAGCGTGAAAGTAAGTTGTAAGGCAAGTGGTTACACTTTCACAAGCTACCATATTCAATGGGTCCGACAGGCTCCTGGACAGGGCTTGGAGTGGATGGGCATATGGATTTACCCTGGTGACGGGTCCACCCAGTATAATGAAAAGTTCAAGGGAGTCACGATGACCAGGGACACCTCTACATCTACCGTGTATATGGAGCTCTCTAGTTTGCGATCCGAAGACACTGCCGTTTATTACTGTGCAAGAGAAGGAACTTATTACGCGATGGACTACTGGGGTCAGGGGACAACAGTCACCGTTAGCTCCGTCGATTCCAGCGGGGGAGGTGGCTCAGGCGGGGGTGGTTCTGGGGGGGGCGGGAGCACTTCAGATATTGTAATGACCCAAACCCCACTGAGTAGTCCAGTCACGCTTGGTCAACCGGCAAGCATTTCTTGCAGGAGCTCTCAGAGTATTGTCCACTCTAACGGGAATACATATTTGGAGTGGTATCAGCAAAGACCGGGCCAACCACCACGCCTCTTGATTTATAAGGTGAGCAATAGGTTTTCAGGCGTGCCAGATAGGTTCTCAGGCTCCGGAGCGGGAACCGACTTCACCCTCAAGATAAGTCGGGTGGAAGCCGAAGACGTAGGAGTTTACTACTGCTTTCAAGGATCTCATGTTCCACGAACGTTTGGAGGAGGAACCAAGGTGGAAATAAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H3_k4_NT 172 ATGGACTTTCAGGTCCAAATTTTTTCCTTCTTGCTCATATCCGCGAGTGTCATCATGTCAAGAGCAAGTCAAGTTCAACTCGTTCAATCAGGAGCTGAGGTGAAAAAACCAGGGGCGTCTGTCAAAGTAAGCTGCAAAGCATCAGGGTATACGTTCACGAGTTATCATATCCAGTGGGTTAGGCAGGCGCCAGGGCAGGGATTGGAATGGATGGGTATCTGGATTTACCCGGGTGACGGCAGCACTCAATACAATGAGAAATTCAAAGGCGTAACAATGACAAGGGACACGAGCACAAGCACAGTGTACATGGAGCTTAGCTCTTTGAGGTCAGAGGATACCGCTGTTTACTATTGTGCTCGGGAGGGTACTTACTATGCAATGGACTACTGGGGGCAAGGCACGACCGTTACAGTGAGTAGCGTAGATTCCTCCGGGGGTGGCGGTTCAGGCGGCGGAGGCTCAGGCGGAGGAGGGTCAACATCCGATGTCGTAATGACTCAGTCCCCTCTGTCATTGCCGGTGACTTTGGGACAGCCAGCGTCTATATCTTGTAGGTCCTCTCAATCAATAGTGCATTCCAACGGTAACACCTATCTGGAATGGTATCAGCAAAGGCCAGGACAAAGTCCACGCCTGCTTATATATAAGGTGTCTAATCGATTCAGTGGGGTTCCCGATAGGTTTTCCGGCTCTGGTAGCGGGACTGATTTCACGTTGAAAATATCACGCGTGGAAGCGGAAGATGTTGGGGTCTATTACTGCTTTCAGGGTAGTCATGTCCCTCGAACTTTTGGCGGTGGTACAAAGGTAGAAATCAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H3_k5_NT 173 ATGGATTTTCAGGTACAGATATTCTCATTTCTCCTTATCTCAGCTAGTGTCATAATGTCCAGGGCGAGTCAAGTACAACTTGTCCAGTCAGGCGCAGAGGTCAAGAAGCCGGGCGCAAGCGTTAAGGTTTCCTGCAAAGCATCCGGCTATACATTCACGTCCTATCACATCCAATGGGTCAGGCAAGCACCCGGTCAAGGACTTGAGTGGATGGGCATCTGGATTTACCCTGGAGATGGCAGTACTCAGTACAACGAAAAATTCAAAGGTGTAACCATGACCCGCGACACATCTACTTCCACAGTTTATATGGAACTCAGCAGTTTGCGGAGCGAAGATACCGCTGTTTACTACTGTGCCCGAGAGGGAACTTACTACGCCATGGACTATTGGGGTCAAGGAACGACAGTAACAGTTAGTTCTGTAGATTCCAGTGGCGGCGGTGGGAGCGGGGGTGGGGGATCTGGCGGAGGCGGAAGTACAAGTGACATCGTTATGACTCAGACACCCCTTAGTAGTCCCGTTACGTTGGGCCAACCCGCGAGCATTTCCTTTCGATCCTCTCAGTCTATAGTTCACTCAAATGGGAATACTTATTTGGAGTGGTATCAACAGCGCCCCGGACAACCACCAAGGCTCCTGATATACAAGGTGTCCAATCGATTCTCTGGGGTGCCTGATAGATTTAGCGGAAGTGGAGCCGGTACAGATTTTACCCTGAAAATATCACGGGTAGAAGCCGAAGATGTCGGCGTCTACTACTGTTTCCAGGGTTCCCATGTACCGCGAACGTTCGGGGGCGGAACAAAAGTTGAGATCAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H4_k2_NT 174 ATGGATTTCCAGGTTCAGATATTTAGTTTCCTCTTGATTTCTGCCAGTGTCATCATGAGCAGGGCTTCCCAAGTTCAGTTGGTGCAAAGTGGCGCTGAAGTCAAAAAACCTGGGGCTTCCGTTAAAGTATCTTGCAAGGCGTCCGGCTACACTTTCACATCCTACCACATTCAATGGGTCCGGCAAGCGCCCGGTCAGGGGCTCGAATGGATGGGGTGGATATACCCAGGAGATGGATCTACTCAGTACAACGAGAAATTTAAAGGACGGGTGACGATGACGCGCGACACTTCAATAAGCACTGCATACATGGAACTGTCCCGGCTTAGGTCAGATGACACCGCGGTCTACTATTGTGCGAGAGAGGGTACTTACTATGCTATGGACTACTGGGGGCAAGGCACGACGGTTACAGTTTCCTCAGTCGATAGTTCAGGCGGAGGCGGCTCCGGGGGCGGTGGTAGTGGAGGGGGTGGATCTACTTCCGACATTGTCATGACCCAGACCCCGTTGAGCCTTTCAGTGACGCCCGGTCAACCCGCCAGCATAAGTTGTCGATCAAGCCAGTCTATTGTACACTCCAATGGAAACACATATTTGGAGTGGTATCTCCAAAAACCCGGCCAAAGCCCTCAACTGCTCATCTACAAGGTCTCAAACAGGTTTAGCGGGGTCCCGGATCGCTTCTCAGGGTCAGGATCTGGTACGGACTTTACACTGAAAATTTCCCGAGTCGAAGCGGAAGACGTGGGTGTATATTACTGCTTCCAGGGGAGTCATGTTCCAAGAACCTTTGGGGGAGGTACAAAGGTCGAAATAAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H4_k3_NT 175 ATGGATTTTCAGGTCCAAATTTTTTCCTTCTTGCTTATCAGCGCAAGTGTAATCATGTCCCGCGCGTCCCAAGTACAACTTGTGCAATCTGGCGCGGAGGTGAAAAAACCTGGAGCTTCCGTCAAGGTTTCTTGTAAGGCCTCTGGCTACACCTTCACGTCCTACCACATTCAGTGGGTTCGACAGGCGCCGGGCCAAGGACTGGAGTGGATGGGATGGATATATCCAGGAGATGGTTCTACTCAGTATAATGAGAAATTCAAGGGTCGCGTAACAATGACGAGGGATACATCAATCTCCACCGCGTACATGGAACTTTCAAGACTCCGGTCAGATGACACGGCGGTTTACTACTGTGCTCGGGAGGGCACTTACTATGCTATGGACTACTGGGGGCAAGGGACAACGGTAACGGTATCTAGTGTGGATTCTAGTGGCGGCGGCGGTTCAGGAGGAGGTGGTTCAGGCGGGGGGGGTAGTACAAGTGATATTGTGATGACCCAAACACCCCTTTCTAGCCCTGTTACTCTGGGTCAACCCGCGTCCATAAGTTGTCGAAGTAGTCAATCCATCGTGCATAGCAACGGCAACACTTACCTTGAATGGTATCAACAACGACCCGGACAGCCCCCGCGACTGCTTATCTATAAAGTATCAAACAGGTTCAGTGGCGTGCCAGATCGATTCTCCGGCTCTGGGGCAGGCACAGATTTCACGTTGAAAATTTCTCGGGTCGAGGCCGAGGACGTGGGCGTTTATTACTGTTTCCAGGGGAGTCACGTCCCCAGGACGTTCGGAGGAGGAACTAAAGTCGAAATAAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H4_k4_NT 176 ATGGACTTCCAGGTCCAAATATTCAGCTTCCTCCTCATTTCCGCCAGTGTAATAATGTCCAGAGCCTCACAAGTACAGTTGGTTCAGAGCGGGGCTGAGGTTAAGAAACCAGGCGCGAGCGTCAAGGTATCCTGCAAGGCGAGTGGTTATACTTTCACTAGTTATCACATTCAGTGGGTCCGACAGGCCCCCGGTCAAGGCCTGGAGTGGATGGGGTGGATATATCCGGGAGATGGTTCTACCCAATATAATGAGAAGTTTAAGGGGAGAGTCACAATGACAAGGGACACCAGTATTAGCACCGCGTATATGGAGCTTTCCCGCCTGCGATCAGATGACACGGCCGTGTACTACTGTGCTAGGGAGGGAACCTATTATGCGATGGATTACTGGGGACAGGGTACTACAGTCACGGTCTCTAGCGTGGACAGTTCCGGGGGCGGTGGAAGCGGTGGTGGCGGTTCAGGTGGAGGAGGCTCTACGAGTGATGTTGTGATGACTCAGTCCCCGCTTTCACTTCCCGTCACCCTTGGGCAACCCGCAAGCATCTCATGCCGATCCTCCCAGTCTATAGTACATAGTAATGGCAACACATATCTTGAATGGTATCAGCAGAGGCCGGGTCAGTCTCCCCGACTCCTTATATATAAAGTGAGCAACAGATTCTCCGGAGTACCGGATAGATTTTCCGGCTCTGGGAGCGGCACCGACTTTACACTGAAAATTTCACGGGTTGAAGCTGAAGATGTTGGGGTATACTATTGTTTCCAGGGTTCTCACGTCCCGAGGACATTCGGGGGAGGAACGAAAGTCGAAATAAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H4_k5_NT 177 ATGGATTTTCAGGTACAAATCTTCAGCTTCCTGCTCATCTCCGCGAGCGTAATCATGTCTAGGGCGTCCCAGGTGCAGTTGGTGCAATCAGGTGCAGAGGTGAAGAAGCCTGGTGCATCCGTTAAAGTAAGTTGTAAGGCAAGCGGATATACTTTTACATCCTATCATATTCAATGGGTCAGACAAGCACCTGGACAGGGTCTTGAGTGGATGGGCTGGATCTATCCAGGCGATGGCTCAACTCAATATAACGAGAAGTTCAAGGGGAGGGTTACTATGACCAGGGATACGTCTATTTCCACTGCGTACATGGAACTCTCCAGGTTGAGAAGTGATGATACCGCGGTTTACTACTGCGCTAGAGAAGGAACGTACTACGCTATGGATTACTGGGGGCAGGGTACAACTGTCACCGTCTCAAGTGTGGATTCTTCTGGGGGTGGGGGATCAGGAGGGGGAGGCTCCGGTGGGGGCGGGTCAACCAGCGACATTGTCATGACTCAAACCCCCCTGAGCAGCCCTGTCACCCTGGGTCAGCCTGCCTCAATATCCTTTAGAAGCTCCCAAAGCATCGTCCATTCAAATGGTAATACCTATCTGGAGTGGTATCAGCAAAGGCCTGGTCAACCCCCGCGCCTTCTCATTTACAAGGTGTCAAACAGGTTCTCCGGCGTACCGGATAGGTTTTCCGGAAGCGGTGCTGGAACCGACTTTACTCTCAAAATCTCTAGGGTGGAAGCTGAGGACGTCGGTGTATACTATTGTTTTCAAGGCTCCCATGTTCCCAGGACATTTGGTGGGGGAACGAAGGTAGAAATCAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H5_k2_NT 178 ATGGACTTTCAGGTTCAGATTTTCTCTTTCTTGTTGATCTCCGCTAGTGTCATAATGTCACGGGCAAGTCAGGTACAACTCGTTCAGAGTGGTGCCGAAGTGAAGAAACCGGGTGCCTCCGTAAAGGTGTCATGTAAAGCTAGTGGCTATACATTCACAAGTTATCATATCCAATGGGTACGACAAGCACCGGGACAGCGACTGGAATGGATGGGATGGATCTATCCTGGGGACGGATCTACACAGTACAATGAGAAATTTAAGGGACGGGTCACGATAACCAGGGACACATCTGCTTCCACGGCTTACATGGAGCTTTCCTCCCTGCGGAGCGAGGACATGGCTGTTTACTATTGCGCTCGCGAAGGGACATACTACGCAATGGATTATTGGGGCCAAGGCACTACCGTGACGGTCTCTTCTGTCGATAGTTCCGGAGGAGGTGGTTCAGGGGGAGGCGGTTCAGGTGGGGGTGGATCTACCTCAGATATTGTCATGACACAGACACCTTTGTCCTTGAGTGTGACACCGGGTCAACCGGCGAGTATAAGCTGTCGCAGCTCACAATCTATTGTGCATAGCAACGGGAATACATATCTCGAATGGTATCTCCAAAAGCCGGGCCAATCCCCCCAACTTCTCATTTACAAAGTTTCTAATCGATTTTCAGGTGTACCAGATCGGTTTTCCGGGTCTGGCTCAGGTACTGACTTCACCTTGAAAATATCAAGGGTTGAAGCTGAGGATGTAGGTGTGTACTATTGCTTCCAGGGGTCTCACGTTCCTCGGACTTTTGGGGGGGGCACAAAAGTAGAGATTAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H5_k3_NT 179 ATGGATTTCCAGGTGCAAATCTTCTCATTTCTTTTGATAAGTGCGTCAGTGATAATGTCTCGGGCCAGTCAAGTACAGCTTGTCCAAAGTGGCGCTGAAGTCAAGAAGCCGGGAGCCTCAGTTAAGGTTAGCTGCAAGGCCTCAGGGTATACTTTTACCTCCTATCATATACAGTGGGTACGACAAGCACCGGGACAGCGACTGGAGTGGATGGGTTGGATATATCCGGGAGATGGTTCAACCCAGTATAATGAGAAGTTCAAGGGGCGAGTTACGATAACCCGCGATACGAGTGCATCAACAGCGTACATGGAGTTGAGTTCCCTCCGCAGCGAGGACATGGCGGTATACTATTGTGCCAGGGAGGGGACTTATTATGCCATGGACTACTGGGGGCAGGGCACAACCGTAACAGTCTCTTCTGTAGACAGTTCAGGAGGGGGCGGAAGTGGAGGTGGCGGATCTGGTGGAGGTGGATCTACTTCCGACATCGTTATGACCCAAACACCACTTTCATCTCCCGTTACTCTCGGGCAACCTGCTAGTATTTCCTGTAGATCCTCACAATCTATAGTACATAGCAATGGCAATACCTACCTGGAGTGGTATCAACAACGCCCAGGCCAACCACCTCGCCTGCTTATCTATAAAGTAAGCAATAGATTCAGTGGTGTACCGGATAGGTTCTCTGGTTCCGGAGCAGGAACTGACTTTACACTCAAAATCAGTAGGGTGGAGGCGGAAGACGTGGGAGTATATTATTGCTTTCAAGGTTCACATGTACCTCGAACATTTGGCGGAGGAACTAAGGTTGAGATTAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H5_k4_NT 180 ATGGATTTCCAAGTCCAGATATTCAGTTTTCTTTTGATAAGCGCTTCTGTAATCATGTCTCGGGCGTCCCAAGTACAACTGGTGCAATCAGGGGCAGAAGTGAAAAAACCAGGTGCATCCGTTAAGGTGAGTTGCAAGGCTTCCGGCTATACCTTTACATCATATCATATTCAATGGGTCAGGCAAGCACCTGGTCAGCGATTGGAATGGATGGGTTGGATATATCCTGGTGATGGGTCTACACAATATAACGAAAAATTCAAGGGGCGAGTGACCATCACAAGAGATACATCAGCGTCAACAGCGTATATGGAACTGTCATCCCTTAGATCAGAGGACATGGCGGTCTATTACTGTGCCAGAGAAGGCACTTATTATGCAATGGATTATTGGGGACAAGGAACCACTGTCACTGTTTCCAGCGTAGACTCCTCCGGTGGTGGTGGAAGTGGCGGCGGTGGGTCAGGAGGGGGTGGGTCAACTTCTGATGTAGTGATGACACAGAGCCCTCTGAGCTTGCCTGTGACCTTGGGTCAGCCGGCCTCAATAAGTTGTCGATCTAGTCAGTCAATCGTCCATAGTAATGGGAACACATACCTTGAATGGTATCAGCAAAGACCTGGACAATCTCCACGACTCCTTATATACAAAGTTAGCAACCGATTTAGCGGAGTGCCAGACCGCTTTTCTGGTTCCGGGTCTGGCACAGATTTTACCCTTAAGATCTCCCGCGTGGAGGCGGAAGACGTTGGTGTTTACTATTGCTTCCAGGGGTCACACGTTCCACGCACCTTTGGAGGAGGTACGAAGGTCGAGATTAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H5_k5_NT 181 ATGGATTTTCAAGTACAGATCTTCTCTTTCTTGCTTATTTCAGCGAGCGTAATCATGAGTAGGGCATCTCAAGTTCAACTCGTTCAGTCAGGTGCTGAGGTAAAAAAACCAGGGGCTTCCGTTAAAGTTAGCTGTAAGGCATCTGGGTACACATTTACTAGCTACCATATCCAGTGGGTGCGACAAGCCCCGGGGCAGCGCTTGGAATGGATGGGCTGGATTTACCCAGGTGACGGCTCCACGCAATATAATGAGAAATTTAAGGGTAGAGTTACTATTACCAGGGACACAAGTGCTTCAACTGCCTATATGGAACTGAGCAGCCTTCGGAGTGAAGATATGGCCGTATATTACTGCGCAAGGGAGGGGACTTACTATGCAATGGACTACTGGGGTCAGGGAACGACTGTGACCGTGTCCTCAGTTGACTCCAGCGGTGGTGGCGGCTCTGGAGGTGGGGGTTCCGGCGGAGGCGGAAGCACATCTGATATAGTGATGACGCAAACGCCTCTTTCTTCCCCGGTAACTCTGGGACAGCCAGCGTCAATTTCATTTAGGTCCTCCCAGTCAATCGTACATAGTAATGGAAATACTTACCTGGAATGGTATCAACAACGACCAGGGCAACCGCCCCGATTGTTGATCTATAAAGTGAGCAATCGCTTTTCTGGCGTGCCCGATCGGTTCTCAGGGTCTGGAGCTGGGACTGACTTCACATTGAAAATTTCCAGGGTTGAGGCCGAGGATGTGGGGGTTTATTACTGCTTCCAAGGCTCCCACGTCCCCCGCACCTTCGGAGGGGGAACCAAAGTCGAAATAAAGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H6_k2_NT 182 ATGGATTTTCAAGTTCAGATATTCTCATTTTTGCTTATATCAGCCTCCGTAATTATGTCACGGGCAAGTCAAGTTCAGTTGGTGCAGTCCGGAGCAGAAGTTAAGAAGCCCGGTGCTTCTGTGAAAGTCTCCTGCAAAGCGTCTGGGTACACCTTCACGAGCTACCATATACAGTGGGTCCGGCAAGCGCCTGGGCAGAGGCTGGAGTGGATGGGCTGGATTTACCCAGGAGATGGGAGTACAAAGTATAGTCAGAAGTTTCAAGGGCGAGTGACGATAACCAGAGATACGAGTGCAAGTACTGCATACATGGAACTGAGCTCCTTGAGGTCCGAGGATACAGCGGTGTACTATTGCGCTCGGGAAGGGACATATTATGCTATGGACTATTGGGGACAAGGGACAACGGTAACGGTGAGTTCCGTCGATTCCTCAGGTGGCGGAGGCAGTGGGGGCGGGGGTTCCGGCGGTGGCGGGTCCACGAGTGATATAGTTATGACACAGACCCCCCTCAGCCTTTCTGTGACCCCAGGACAACCCGCTAGTATCTCTTGCCGCAGTTCTCAGTCCATAGTACACAGTAACGGAAATACCTATCTTGAGTGGTATCTTCAAAAGCCCGGCCAGAGCCCTCAACTCTTGATATATAAAGTGTCAAATCGATTTTCAGGTGTGCCTGATCGATTCTCAGGGTCTGGTTCAGGGACAGATTTCACGCTTAAGATAAGCAGAGTAGAGGCTGAAGACGTGGGAGTCTACTATTGTTTTCAGGGGTCACACGTTCCCCGCACTTTTGGTGGGGGAACCAAGGTGGAAATCAAAAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CAR_H1_K2_NT 214 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTTCATGCCGCCAGACCATCTCAGGTCCAGCTAGTACAAAGCGGCGCCGAAGTAAAGAAACCTGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACCACATCCAGTGGGTTCGACAGGCCCCTGGACAGAGACTAGAGTGGATGGGCTGGATCTATCCTGGCGACGGCAGCACCCAGTACAACGAGAAGTTCAAGGGCAGAGTTACCATCACCCGAGACACCAGCGCCAGCACAGCCTATATGGAGCTGAGCAGCCTGCGAAGCGAGGACACAGCTGTTTACTATTGTGCCAGAGAGGGCACCTACTACGCAATGGATTATTGGGGCCAGGGGACCACCGTGACCGTTTCTTCTGGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCAGATATTGTAATGACCCAGACACCTCTGTCCCTGTCTGTGACACCTGGACAGCCTGCAAGCATCAGCTGTCGGAGCAGCCAGAGCATCGTTCACAGCAACGGCAACACCTACCTGGAATGGTATCTGCAGAAGCCCGGACAGTCCCCCCAGCTGCTGATCTACAAGGTGTCCAACCGCTTCAGTGGAGTACCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTAGAAGCAGAGGACGTTGGAGTGTACTACTGCTTCCAAGGCAGCCATGTGCCAAGAACCTTTGGTGGAGGCACAAAGGTGGAAATCAAGCGGACAACAACACCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTGAGGCCCGAAGCTTGCAGGCCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCACCTCTGGCTGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCA CCCTGTATTGCCGGAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGATCCAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAGCTCTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTACAGAAAGACAAGATGGCAGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA 4-1BB NT
(SEQ ID NO: 17 or US9,102,760) 215 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG NT Hinge CD8 (SEQ ID NO: 15 or US9,102,760) 217 ACCAGACGCCAGCGCCGCGACCACCAACACCGGCCCACCATCGCGTCGCAGCCCCTGTCCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT NT hinge CD8 220 ACAACAACACCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTGAGGCCCGAAGCTTTGCAGGCCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGCGAC NT transmembrane CD8 (SEQ ID NO: 16 or US9,102,760) 222 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC NT transmembrane CD8 224 ATCTACATCTGGGCACCTTGGCTGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGC

Table 5

Kabat Chothia BB7.2
VH CDR1 SYHIQ (SEQ ID NO: 183) GYTFTSY (SEQ ID NO: 184) BB7.2
VH CDR2 WIYPGDGSTQYNEKFKG (SEQ ID NO: 185) YPGDGS (SEQ ID NO: 186) BB7.2
VH CDR3 EGTYYAMDY (SEQ ID NO: 187) EGTYYAMDY (SEQ ID NO: 187)

Table 6

Kabat Chothia BB7.2
VL CDR1 RSSQSIVHSNGNTYLE (SEQ ID NO: 188) RSSQSIVHSNGNTYLE (SEQ ID NO: 188) BB7.2
VL CDR2 KVSNRFS (SEQ ID NO: 189) KVSNRFS (SEQ ID NO:189) BB7.2
VL CDR3 FQGSHVPRT (SEQ ID NO: 190) FQGSHVPRT (SEQ ID NO: 190)

Table 7

SEQ Name SEQ ID AA BB7.2_AA
vh 191 QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSS BB7.2_AA
VL 192 DVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK BB7.2_AA
scFv
(no leader) 193 QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSSVDSSGGGGSGGGGSGGGGSTSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK BB7.2CAR
(no leader) 194 QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSSVDSSGGGGSGGGGSGGGGSTSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKLEQKLISEEDLNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR BB7.2CAR
(with leader) 195 MDFQVQIFSFLLISASVIMSRASQVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSSVDSSGGGGSGGGGSGGGGSTSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKLEQKLISEEDLNRIRGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ Name SEQ ID NT BB7.2CAR
(with leader) 196 ATGGACTTCCAGGTGCAGATCTTCAGCTTCCTGCTGATCAGCGCCAGCGTGATCATGAGCCGCGCTAGCCAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACCACATCCAGTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTGGATCTACCCCGGCGACGGCAGCACCCAGTACAACGAGAAGTTCAAGGGCAAGACCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGCGAGGACAGCGCCATCTACTTCTGCGCCCGCGAGGGCACCTACTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGTCGACAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCACTAGTGACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGTGAGCATCAGCTGCCGCAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACCTGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACCGCTTCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTTCCAGGGCAGCCACGTGCCCCGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCTCGAGCAGAAGCTGATCAGCGAGGAGGACCTGAACCGGATCCGTGGGGTCACCGTCTCTTCAGCGCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC AGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACCCCTTTGGGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTCGAGAGAGTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

EXAMPLES

Ways

Obtaining humanized HLA-A*02-specific scFv. The humanized genes were codon optimized using the Invitrogen GeneArt Gene Synthesis codon optimizer using Homo sapiens settings. The gBlocks® gene fragments were ordered from Integrated DNA Technologies (Coralville, Iowa) such that the chimeric antigen receptor 5' region contained the Kozak sequence and a 38 nucleotide overlap with the pcDNA3 plasmid. The 3' end contains a BamHI site and an overlap with the CD8 hinge sequence to facilitate Gibson assembly into a plasmid in frame with the CD8 hinge and intracellular signaling domains of the chimeric antigen receptor.

Obtaining hA2-CAR. The scFv variants were fused to the stem region of human CD8α, human CD28 transmembrane and intracellular domains, and human CD3ζ as described (Sadelain et al., 2013). The resulting cDNAs were cloned into a lentiviral vector that encodes cell surface expression of a truncated nerve growth factor receptor (TrkA) (ΔNGFR) as a marker (Figure 1). Surface expression was determined by flow cytometry of transiently transfected HEK 293T cells (jetPRIME®, Polyplus Transfection). Virus particles were obtained as described (Allan et al., 2008).

Obtaining HLA-expressing cell lines K562. K562 cells expressing CD64 (K562.64) were donated by James Riley, University of Pennsylvania, Philadelphia, USA. cDNA for HLA-A*02:01 and A*24:02 was isolated from mRNA of peripheral blood mononuclear cells from a donor homozygous for A*02:01 or A*24:02, respectively, at the HLA-A locus using primer sequences : 5'-TTTTCTAGACGCGTGCCACCATGGCCGTCATGGCGCC-3' (forward) (SEQ ID NO: 197) and 5'-AAGTCGACGCTAGCTCACACTTTACAAGCTGTGAGAGACA-3' (reverse) (SEQ ID NO: 198). The resulting sequence was confirmed by Sanger sequencing, aligned with expected sequences from the IPD and IMGT/HLA database (Robinson et al., 2015), and transduced into K562 cells, respectively, using a lentiviral expression vector. To obtain A25- and A68-K562 cells, HLA sequences for A*25:01, A*68:01 were extracted from the IPD and IMGT/HLA database (Robinson et al., 2015), optimized for codons using the codon optimization tool ( for Homo sapiens) of the ThermoFisher Invitrogen GeneArt gene synthesis service, then cloned into a lentiviral expression vector and transduced into K562 cells. The resulting K562 cell lines were then sorted on FACSAria II (BD Biosciences) using anti-HLA-ABC (ThermoFisher, 12-9983-41) to ensure equivalent surface expression of transduced HLA, and anti-HLA-A2 (BB7.2) (ThermoFisher , 17-9876-42) to ensure cleanliness.

Sorting, transduction and propagation of Treg. CD4+ T cells were isolated from HLA-A2- donors via RosetteSep (Stemcell) and enriched for CD25+ cells (Miltenyi) before sorting live CD4+CD25hiCD127lo Treg (for in vitro and luciferase experiments) or CD4+CD127loCD25hi CD45RA+ Treg (for xenogeneic GvHD and skin graft experiments) using MoFlo® Astrios (Beckman Coulter) or FACSAria II (BD Biosciences), respectively. Sorted Tregs were stimulated with L cells and αCD3 mAb (OKT3; 100 ng/ml) in immunocult-XF T cell expansion medium (STEMCELL Technologies) with 1000 U/ml IL-2 (Proleukin). One day later, the cells were transduced with lentivirus in a 10 viral particle:1 cell multiple infection. On day 7, NGFR+ cells were purified by magnetic selection (Miltenyi), then used in assays or restimulated with L cells as described above, and expanded for 5 days for in vivo experiments. To test the effects of HLA-A2-mediated stimulation, Tregs were cultured with IL-2 restriction (100 U/mL) for 24 hours and then restimulated with irradiated anti-CD3/anti-CD28 loaded K562.64 cells or K562.64 cells. expressing HLA-A*02:01, A*24:02, A*25:01, or A*68:01 at a ratio of 1:2 (K562:T cells) for 24 hours.

flow cytometry. For phenotypic analysis, cells were stained with fixable viability dye (FVD, 65-0865-14, ThermoFisher; 423102, Biolegend) and surface markers prior to fixation/permaneabilization with the FOXP3/Transcription Factor Staining Buffer Kit (eBioscience) and intracellular protein staining. Samples were read on Cytoflex (Beckman-Coulter) and the results analyzed using FlowJo software versions 9.9.4 and 10.3 (Tree Star).

Surface staining was performed for ΔNGFR (130-091-885, Miltenyi), CD3 (564465, BD Biosciences), CD4 (317410, Biolegend), CD25 (130-091-024, Miltenyi), LAP (25-9829-42, ThermoFisher ), CD69 (310946, Biolegend), CD154 (555702, BD Biosciences), CD71 (BD Biosciences, 563768) and CD127 (48-1278-42, ThermoFisher). Tetramer staining was performed with HLA-A*02:01 monomers converted to tetramers with streptavidin-allophycocyanin (PJ27S, Prozyme). Intracellular staining was performed according to CTLA-4 (369606, Biolegend).

For in vivo experiments, 50 µl of blood was collected weekly and at the endpoints. Ammonium chloride was used to lyse erythrocytes. Cells were resuspended in PBS with mouse anti-CD16/32 (ThermoFisher, 14-0161-82) and stained for extracellular markers using fixed viability dye (FVD; ThermoFisher, 65-0865-14), with mouse anti-CD45 ( ThermoFisher, 25-0451-82) and human anti-CD45 (BD Biosciences, 560777), anti-CD4 (Biolegend, 300554, 317434), anti-CD8 (ThermoFisher, 48-0087-42), human anti-CD271 (NGFR ; BD Biosciences, 557196) anti-HLA-A2 (BD Biosciences, 551285). Intracellular staining for FOXP3 (ThermoFisher, 12-4777-42) was performed using the FOXP3/Transcription Factor Staining Buffer kit (ThermoFisher, 00-5523-00). 10,000 counted pellets were added to each sample (ThermoFisher, 01-1234-42). Gating strategies in xenogeneic GvHD, luciferase and skin graft experiments are shown in Figures 8D, 10A and 12A, respectively.

Analysis of cross-reactivity of HLA alleles. 0.25×10 ⁶ CAR Treg (prepared as described above after 7 days of culture) were incubated with a panel of individual antigen beads for detection of FlowPRA antibodies (FL1HD01, FL1HD02, FL1HD03, FL1HD04, FL1HD06 and FL1HD08, One Lambda) and fixed viability dye (FVD, 65-0865-14, ThermoFisher) for 30 minutes at room temperature, then washed, fixed with 0.5% formaldehyde and analyzed by flow cytometry. Two hundred negative control beads were used per sample. For analysis, dead cells were first removed using a fixed dye to determine viability. Single beads were then gated after dead cells and doublets were excluded. The number of beads for each HLA antigen was then determined by their respective PE intensity. The data were normalized by dividing the number of negative beads in the sample by the number of negative beads in the Treg-NGFR sample times the number of negative beads in the Treg-NGFR sample. Relative percent binding is the number of beads in the NGFR sample for HLA-specific beads minus the normalized number of beads in the sample for those HLAs, divided by the number of beads in the NGFR sample, multiplied by 100.

, затем нормализовали полученные значения от 0 до 100%, согласно формуле для каждого независимого эксперимента:

Suppression of reactions of mixed lymphocytes. Adherent cells from PBMCs from healthy HLA-A2+ donors were differentiated into monocytic dendritic cells as described (Dijke et al., 2015). For mixed lymphocyte reactions, HLA-A2-PBMC responder cells were labeled with cell proliferation dye eF450 (65-0842-85, ThermoFisher), then seeded with 5x104 HLA-A2+ monocytic dendritic cells and increased the ratio of propagated ΔNGFR- or hA2-CAR-expressing Tregs labeled with cell proliferation dye e670 (65-0840-90, ThermoFisher). After 6 days, division of HLA-A2-CD4+ T responder cells was measured by flow cytometry. Percent suppression was calculated based on the cell combination's proliferation index and ratio compared to a positive control (HLA-A2+ monocytic dendritic cells with HLA-A2-CD4+ T responder cells only) as described (McMurchy and Levings, 2012). The data were normalized, first by calculating the percent suppression as follows:

, then normalized the obtained values from 0 to 100%, according to the formula for each independent experiment:

Suppression of xenogeneic graft-versus-host disease in vivo. Female NSG mice aged 8 to 12 weeks (Jackson lab, self-bred) received whole body irradiation (150 cGy, RS-2000 Pro Biological System) 1 day prior to administration of 8×10 ⁶ HLA-A2+ PBMC with or without 4× 10 ⁶ hA2-CAR Treg intravenously in the tail vein. Mice with saline injection served as controls. hA2-CAR Treg was obtained from four different healthy donors. GVHD was assessed based on mass, fur texture, posture, activity level, and skin integrity, from 0 to 2 points per category, as described (Cooke, et al., 1996; Hill et al., 1997). GVHD was estimated by two investigators with no specific information about the experiment. Peripheral blood from the saphenous vein was centrifuged; then the erythrocytes were lysed and the leukocytes were measured by flow cytometry.

Study approval. For human PBMCs, healthy volunteers provided written informed consent in accordance with protocols approved by the University of British Columbia Clinical Research Ethics Board and Canadian Blood Services. Human skin samples discarded from plastic surgery were obtained from the Harvard Skin Resource Center, Skin Works, or Cambie Surgical Clinic following protocols approved by the University of British Columbia Clinical Research Ethics Board. Animal testing protocols have been approved by the UBC Animal Care Committee.

Skin transplant. To assess A2-CAR Treg homing and ability to inhibit skin rejection, female NSG mice aged 8 to 12 weeks (Jackson Lab, self-bred) were transplanted with skin from HLA-A2+ NSG transgenic mice (Jackson Lab, self-bred) and NSG skin (HLA -A2-negative). For mouse skin grafts, the skin was cut into round pieces using an 8 mm biopsy punch and the skin was placed on fresh PBS plates and stored at low temperature (4-8°C) until grafting (~1-4 hours). Human skin HLA-A2 expression was assessed by flow cytometry and qPCR. Cleaved explants were obtained by trimming the fat from the sample. The explants were washed with sterile PBS and stored in RPMI. Quality skin explants were cut into 1 cm ² pieces, placed on fresh PBS plates and stored at low temperature (4-8°C) until transplantation (~1-4 hours). For mouse and human skin grafting, preshaved mice were anesthetized, the skin on the back was cut near the shoulder, and the skin of a similar size mouse was removed, then the grafts were placed on the open area and stabilized with sterile strips (3M, Nexcare). The grafts were covered with Vaseline gauze and wrapped with a 2 cm wide CoFlex bandage (3M, Nexcare) to secure the graft for 14 days prior to cell injection.

Histology

Human skin graft and surrounding mouse skin were harvested 27 days after cell injection, fixed overnight at 4°C in 10% formalin (tissue to formalin ratio 1:10 v/v) and stored in 70% ethanol before paraffin embedding. Paraffin sections (5 μm thick) were prepared and stained with hematoxylin-eosin at the Histology Service of BC Children's Hospital (Vancouver, British Columbia, Canada). For immunostaining, sections were deparaffinized and rehydrated with a series of washes with xylene (×3), a varying strength alcohol solution set (2×100% ethanol, 1×95% ethanol and 1×70% ethanol), and 1×PBS. High temperature antigen unmasking (HIER) was performed on glass slides using a microwave oven to reach 93-95°C (5 minutes high power followed by 20 minutes low power) in 10 mM sodium citrate buffer (0.5% Tween-20, pH 6 ,0). After HIER, the slides were washed with running tap water, deionized water and PBS. Sections were incubated with serum-free DAKO® protein blocker (Dako, Burlington, Canada, X0909) to limit non-specific antibody staining. Sections were then incubated at 4°C overnight with the following primary antibodies: FOXP3 (Invitrogen, clone PCH101, 14-4776-82), CD45 (eBioscience, clone H130, 17-0459), Ki67 (eBioscience, clone 20Raji, 17- 5699), Involucrine (Abcam, ab53112). The next day, sections were washed gently with PBS several times, then stained for 1 hour at room temperature with the following secondary antibodies: donkey anti-rat 488 (Life Tech, A11006), goat anti-mouse APC (Invitrogen, 1834696), donkey against rabbit antibodies 488 (Life Tech, A21206). Finally, sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to identify cell nuclei and embedded in VECTASHIELD® DAPI Embedding Medium (Vector Laboratories, Burlingame, CA, USA, H-1200). All antibodies were diluted in antibody diluent (Dako, Burlington, Canada, S3022). The images were taken with an Olympus BX61 fluorescence microscope and an automatic brightfield upright microscope with a QImaging Retiga Exi camera and an Olympus DP71 color camera. Quantitative analysis of fluorescent images was performed using Fiji using the Olympus Viewer plugin (Schneider et al., 2012; Eliceiri et al., 2012).

Slides stained with hematoxylin-eosin were evaluated by a clinical pathologist with no specific information about the experiment, using a scoring system determined by 8 factors, each of which was rated from 0 to 3-4; Lerner grade (0, 1 - focal or diffuse vacuolar degeneration, 2 - dyskeratosis, 3 - cracks in the basal or superficial layers, 4 - obvious loss of the epidermis), spongiosis (0, 1 - only the basal layer, 2 - up to half way, 3 - full thickness), necrotic keratinocytes (0, 1 - rare (1/hpf), 2 - random (2-3/hpf), 3 - many (> 4/hpf)), location of necrotic keratinocytes (0, 1 - only basal, 2 - to the upper half, 3 - full thickness), satertosis (only 0, 1 - 1, 2 - 2-3/hpf, 3 -> 4/hpf), exocytosis (0, 1 - focal, 2 - < 50% biopsy, 3 -> 50% biopsy), adjacent organ involvement (0, 1 - slight involvement of any adjacent organs, 2 - marked involvement of <50% of adjacent organs, 3 - marked involvement of >50% of adjacent organs) and lymphoid cuffs in dermis (0, 1 - slight, 2 - abundant, 3-band) (Massi et al., 1999; Fischer et al., 2015, Kanitakis et al., 2008).

qPCR. RNA was collected from human skin samples according to the manufacturer's instructions (RNeasy Plus Mini Kit; Qiagen) and converted into complementary DNA (cDNA). qPCR was performed using SYBR green (Biorad) and primers for IL17, IL6, IL1B, DEFB4, IFNg, TNFa, ribosomal 18S RNA (Table 8). Melt curve data and SYBR green emission data were collected. Relative concentrations were calculated using a standard curve and the values were normalized to the 18S ribosomal RNA amplification products. Log2 (RQ) values for each sample were obtained using the double delta Ct (∆∆Ct) method (Schmittgen et al., 2008). The ∆Ct value of each sample was obtained by calculating the average Ct (gene of interest) - Ct (housekeeping gene). To obtain ∆∆Ct, the ∆Ct of the control sample was subtracted from the ∆Ct of the treated sample. Then the fold expression of the gene was calculated by 2-(∆∆Ct).

Table 8: Primers used in the qPCR assay

Name
oligonucleotide Direction
Forward (FWD) or Reverse (RVS) Oligonucleotide sequence (5'-3') human IL17 FWD TCA ACC CGA TTG TCC ACC AT (SEQ ID NO: 199) human IL17 RVS GAG TTT AGT CCG AAA TGA GGC TG (SEQ ID NO: 200) human IL6 FWD TCC AAA GAT GTA GCC GCC CCA (SEQ ID NO: 201) human IL6 RVS CCA GTG CCT CTT TGC TGC TTT CA (SEQ ID NO: 202) human IL1B FWD CTG AGC TCG CCA GTG AAA TGA TG (SEQ ID NO: 203) human IL1B RVS TGC TGT AGT GGT GGT CGG AGA (SEQ ID NO: 204) human DEFB4 FWD ACC TGC CTT AAG AGT GGA GCC A (SEQ ID NO: 205) human DEFB4 RVS ACA TGT CGC ACG TCT CTG ATG A (SEQ ID NO: 206) human IFNG FWD TGC CCA GAG CAT CCA AAA GA (SEQ ID NO:207) human IFNG RVS TGT ATT GCT TTG CGT TGG AC (SEQ ID NO: 208) human TNFA FWD AGG CGC TCC CCA AGA AGA CA (SEQ ID NO: 209) human TNFA RVS GGG CTG ATT AGA GAG AGG TCC CT (SEQ ID NO: 210) 18S ribosomal RNA FWD CAA GAC GGA CCA GAG CGA AA (SEQ ID NO: 211) 18S ribosomal RNA RVS GGC GGG TCA TGG GAA TAA C (SEQ ID NO: 212)

Luciferase. To assess Treg homing to HLA-A2 expressing mouse skin grafts in vivo, sorted Tregs (CD4+CD25hiCD127lo) were stimulated with L cells as described above. The next day, cells were transduced with lentivirus HER2-CAR, mA2-CAR or h1k2 hA2-CAR at MOI 10 and 8 h later with lentivirus with luciferase and GFP at MOI 5. Lentiviral plasmid encoding beetle luciferase-GFP fusion protein (pELNS.CBG -T2A-GFP (CBR)), was used as previously described (Barrett et al., 2014). After 7 days of culture, double transduced GFP+ΔNGFR+ Tregs expressing CAR and luciferase were sorted prior to restimulation with L cells as described above. On day 12 of culture, 1-3×10 ⁶ luciferase-CAR Treg and 6×10 ⁶ human allogeneic HLA-A2-PBMC were intravenously injected into skin-grafted NSG mice. For bioluminescent imaging, potassium salt of D-luciferin (150 mg/kg, Gold Bio) was injected intraperitoneally just before anesthesia with isoflurane and images were acquired within 15-20 minutes on Ami-X (Spectral Instruments Imaging). The data were analyzed with AmiView software (Spectral Instruments Imaging, version 1.7.06) and the luminescent signal was quantified as photons/sec/cm ² /steradian in HLA-A2+ versus HLA-A2- skin graft. At the end point of the experiment, axillary lymph nodes draining the skin and spleen were harvested, placed in a 70 µm cell sieve (BD Falcon), then fragmented and filtered using a 1 cc syringe plunger. The cells were then stained for flow cytometry.

Statistical analysis All statistics were made using Prism 7.0b. For Figure 9, IBM*SPSS Statistics version 24.0.0.0 was used.

results

Obtaining humanized HLA-A2-specific CARs

The amino acid sequence of the heavy and light chain variable regions of mouse mAb BB7.2 was aligned with human immunoglobulin sequences obtained from the ImMunoGeneTics® (IMGT®) international database information system using the IgBLAST tool available from the National Center for Biotechnology Information (NCBI). The V-gene demarcation system was set up on the Kabat sequence to obtain Kabat-defined CDRs (Kabat et al., 1991). In addition, a Chothia definition has been identified (Chothia et al., 1987).

A number of different human germline genes were tested as framework sequences for CDR grafting. In addition to identifying human germline sequences that were most consistent with that of the mouse, CDR lengths were also considered to be structurally supportive as much as possible. The human CDRs (Kabat numbering) were replaced with mouse CDR analogs from the BB7.2 antibody. In addition, additional amino acids of the heavy chain CDRs were replaced by combining Chothia and Kabat numbering. 6 different humanized heavy chains and 5 different humanized light chains were made, and a total of 20 different chimeric antigen receptors were made by combining humanized heavy and light chains.

Expression and antigenic specificity of humanized HLA-A2-specific CARs

To test for antigen specificity, various hA2-CAR constructs were transiently transfected into 293T cells, stained with HLA-A*02:01 tetramers, and analyzed by flow cytometry. Eleven hA2-CAR constructs were expressed and linked to the A*02:01 tetramer (Figure 2).

We then compared the binding strength of different hA2-CAR constructs. Transiently transfected 293T cells were stained with increasing dilutions of A*02:01 tetramer, then analyzed by flow cytometry. As shown in Figure 3 (A, B and C), eleven hA2-CAR constructs highly bind the A*02:01 tetramer depending on tetramer concentration.

hA2-CAR-mediated stimulation activates Treg

To test A2-CAR function in Treg, CD4+CD25hiCD127lo cells were sorted from peripheral blood. As shown in Figure 4 (A, B and C), Tregs were stimulated, transduced and cultured for an additional 6-7 days. At the end of 7-8 days of culture, cells were purified as NGFR+ cells and cell surface expression of the hA2-CAR variant was tested by flow cytometry. As shown in Figure 4, Tregs transduced with hA2-CAR constructs showed positive HLA-A2 tetramer staining compared to Tregs transduced with a control ΔNGFR construct, indicating retention of antigen specificity for Tregs expressing hA2-CAR. There were technical difficulties in obtaining a lentivirus with a high titer for (H1k3) and this CAR was not tested in Treg in this experiment. CAR cell surface expression and specificity was tested by A*02:01 tetramer staining, showing that H2k2, H4k4 and H5k4 had a bimodal Treg expression pattern with lower mean fluorescence intensity (MFI) of A2-tetramer+ cells (Figure 4D and 4E) .

Next, we examined whether stimulation with hA2-CAR-activated Tregs (hA2-CAR-Treg) compared to Tregs expressing only a truncated NGFR transduction marker (NGFR Treg). NGFR Tregs and hA2-CAR Tregs were left unstimulated, stimulated via CAR with K562 cells expressing HLA-A*02:01, or stimulated via TCR with K562 cells expressing CD64, a high affinity Fc receptor that was preloaded anti-CD3/anti-CD28 antibodies. A comparison between NGFR Tregs and hA2-CAR Tregs showed significantly higher CAR-stimulated expression of CD69 and CD71 in most hA2-CAR Tregs over several time points. In contrast, when stimulated through the TCR, both NGFR Treg and hA2-CAR Treg were able to positively regulate CD69, indicating that they retain their ability to signal through the TCR after CAR DNA transduction (as shown in Figure 13). hA2-CAR mediated Treg stimulation also caused significantly stronger upregulation of proteins associated with Treg function, demonstrating that hA2-CAR stimulation retains the expected high expression of CTLA-4 and LAP (Figures 5A-F).

Cross-reactivity of hA2-CAR constructs on other HLA class alleles

There are many different HLA alleles that have evolved over time from a smaller number of ancestral alleles. Therefore, there are families of alleles that can only differ by a few amino acids, and a single anti-HLA antibody can recognize multiple alleles in an evolutionarily related family. A mouse monoclonal antibody (BB7.2) is known to be cross-reactive to additional HLA-A alleles (Hilton & Parham, 2013.). Specifically, when tested in a solid phase assay, BB7.2 was found to recognize five HLA-A2 subtypes (*02:01, *02:02, *02:03, *02:05, *02:06) and is cross-functional. reactive with HLA-A*69:01, and when tested at high concentrations also with HLA-A*23:01, A*24:02, A*24:03, A*68:01 and A*68:02 ( Hilton & Parham, 2013). In the context of transplantation, knowledge of the specificity of alloAg is necessary to ensure specific targeting of allogeneic cells, tissues and/or organs. The traditional way to measure T cell alloreactivity is inaccurate and non-quantitative as it involves functional MLRs with large banks of haplotyped PBMCs. In order to evaluate the cross-reactivity of the humanized anti-HLA-A2 antibodies of the invention and compare with the BB7.2 antibody, we adapted the ONE Lambda solid-phase assay, which is designed to measure anti-HLA antibodies in serum, by measuring the binding of HLA-coated beads to interested in humanized CARs. BB7.2 CAR was created as described in MacDonald, K.G. (2016). NGFR+ Tregs expressing these humanized CARs were incubated with Flow Panel Reactive Single Antigen beads and quantified binding to one bead class as signal loss at the bead gates on a forward scatter/side scatter plot (see methods for details). The data were normalized to the number of negative control beads in the sample, and the amount of relative binding to hCAR-expressing Tregs was calculated relative to the amount of binding to NGFR-control Tregs by the number of negative beads in the NGFR sample times the number of negative beads in the NGFR sample. To validate the methodology, the relative binding of each m/hA2-CAR construct to HLA-A*02:01 as determined by FlowPRT cell assay (Figure 6B) was compared to tetramer binding MFI (Figure 6C). This analysis revealed a strong direct correlation between the two detection methods for A*02:01 binding. We also asked if the amount of A*02:01 binding determined in the FlowPRT cell assay correlated with the biological effect of A*02:01 exposure. In fact, we found that there was a direct correlation between the amount of A*02:01 binding quantified by the FlowPRT cell assay and stimulation of Treg activation as determined by the upregulation of CD69 after exposure to A*02:01-expressing APCs (Figure 6D ). These data demonstrate the utility of the cellular FlowPRT method for measuring the ability of alloAg-specific CARs to bind to various HLA alleles.

As shown in Figure 6 and Table 9, all hA2-CAR constructs significantly bind HLA-A*02:01, confirming tetramer binding assays. When we tested BB7.2 antibody binding in one FlowPRA single antigen assay, we confirmed high binding to A*69:01 but could not confirm cross-reactivity to A*23:01 or A*24:02 (Figure 6). When testing the relative ability of m/hA2-CAR Tregs to bind to various HLA-A alleles, we found that mA2-CAR-Tregs are significantly associated with HLA-A*03:01, A*25:01, A*29:02, A*30:01. A*31:01, A*33:01, A*36:01, A*68:01 and A*69:01 (Figure 6, Table 9). In contrast, all hA2-CAR Treg variants unexpectedly showed reduced cross-reactivity compared to mA2-CAR Treg (Figure 6, Table 9). As expected, all CAR constructs bind to HLA-A*69:01, an A*02:01 variant that differs by only 6 amino acids. None of the CAR constructs showed any significant binding to HLA-B (Figure 6).

The relationship between the degree of CAR-Treg Ag binding and biological activity is unknown. To determine the biological significance of HLA cross-reactivity, we generated APCs expressing HLA-A*24:02, A*25:01, or A*68:01; all cells had comparable levels of HLA-A expression (data not shown). We found that only co-culture with HLA-A*02:01-expressing cells resulted in significant m/hCAR-Treg activation as evidenced by increased expression of CD69, CD71, LAP, CTLA-4 (Figure 6I) or CD40L (data not shown). These data suggest that efficient CAR-mediated Treg activation requires high affinity and/or avidity of interactions. Accordingly, although some hA2-CARs show binding of A*25:01 and A*68:01 in the FlowPRT assay, the binding strength is insufficient for cellular activation.

Table 9. Binding interactions between m/hA2-CAR and HLA-A/B protein were found to be statistically significant (two-way analysis of variance, Dunnett's post hoc test).

HLA allele CAR p-value A*02:01 mA2-CAR 0.0001 H1k2 0.0001 H2K2 0.0001 H4k2 0.0001 H5k2 0.0001 H6k2 0.0001 H4k3 0.0001 H5k3 0.0001 H3k4 0.0001 H4k4 0.0006 H5k4 0.0001 A*25:01 mA2-CAR 0.0001 H1K2 0.0061 A*69:01 mA2-CAR 0.0001 H4k2 0.0001 H5k2 0.0001 H1k2 0.0001 H3k4 0.0013 H2k2 0.0084 H5k3 0.0001 H4k3 0.0001 H6k2 0.0060 A*03:01 mA2-CAR 0.0315 A*29:02 mA2-CAR 0.0001 A*30:01 mA2-CAR 0.0001 A*31:01 mA2-CAR 0.0001 H1k2 0.0077 A*33:01 mA2-CAR 0.0019 A*36:01 mA2-CAR 0.0035 H1k2 0.0082 A*68:01 mA2-CAR 0.0001 H1k2 0.0001 H5k2 0.0337 H6k2 0.0009

We have developed a new way to systematically test the specificity of CARs for alloantigens, creating a new platform for the comprehensive identification of constructs with specific allelic specificity. Surprisingly, we found that compared to mA2-CAR, CAR humanization reduced cross-reactivity to several HLA-A allelic variants.

hA2-CAR Tregs mediate HLA-A2-specific suppression in vitro

To test the ability of hA2-CAR to stimulate antigen-specific suppressive activity in Treg, we used mixed lymphocyte reactions (MLR) according to the experimental design shown in Figure 7A. In particular, the proliferation of HLA-A*02:01-negative T cells was stimulated by co-culture with mature HLA-A*02:01-positive dendritic cells in the absence or presence of increasing ratios of Treg that are either not transduced or transduced with control NGFR. -lentivirus or lentivirus encoding H1k2 hA2-CAR. As shown in Figures 7B-E, hA2-CAR expressing Tregs were significantly better able to suppress alloantigen-stimulated CD4+ T cell proliferation compared to control Tregs transduced with a control lentiviral NGFR construct.

hA2-CAR Tregs mediate HLA-A2-specific suppression of xenogeneic GVHD in vivo

To test the functional capacity of hA2-CAR Treg in vivo, we used a mouse model in which human PBMCs implanted in immunodeficient NSG mice induced xenogeneic GVHD. 8x10 ⁶ PBMC from an HLA-A2+ donor were injected into irradiated NSG mice with or without Treg expressing h12-CAR H1k2. The ratio of tested PBMC and hA2-CAR Tregs was 1:2 (ie 8×10 ⁶ PBMC to 4×10 ⁶ Tregs). Mice were followed for 7 weeks by the clinical assessment described in the methods. Consistent with in vitro data in Figure 7, mice treated with hA2-CAR expressing Tregs had improved survival, reduced weight loss, and delayed onset of xenoGVHD compared to mice that did not receive hA2-CAR Tregs (solid gray line) (Figure 8A, B and C). Although there was a biological effect of Tregs, we did not detect circulating m/hA2-CAR Tregs measured 14 days after administration (Figure 8D and 8E).

Movement of hA2-CAR Treg to HLA-A2+ skin grafts in vivo

To test how CAR targeting specificity affects Treg movement, we performed parallel skin grafting from NSG or NSG-A*02:01 transgenic mice to NSG mice (Figure 9A). After graft retrieval, PBMCs were injected in the absence or presence of m/hA2-CAR Tregs or with HER2-CAR Tregs as non-specific polyclonal control Tregs (MacDonald et al., 2016). In addition to CAR, Tregs were co-transduced with a lentivirus encoding a luciferase-GFP fusion protein. Bioluminescence imaging was performed after D-luciferin injection for 21 days after Treg injection (Barrett et al., 2014). Polyclonal HER2-CAR Tregs showed a trend towards allografts but were equally distributed between A2-positive and A2-negative grafts. These polyclonal Tregs can migrate in response to inflammatory signals from postoperative skin because, in untreated, immunodeficient mice, human T cells are usually found in the lungs (Nervi et al., 2007). In contrast to the polyclonal HER2-CAR Tregs, which migrated equally to A2-negative and A2-positive skin, both m/hA2-CAR Tregs rapidly migrate to A2-expressing skin. In addition, m/hA2-CAR Tregs persisted longer than non-specific HER2-CAR Tregs. While HER2-CAR Tregs were not detected by day 21, a strong m/hA2-CAR Treg signal remained (Figure 9B). Quantification of the ratio of luminescence in the A2-positive and A2-negative graft revealed a significant Ag-driven movement of both H1k2- and mA2-CAR Treg into the A2-expressing graft (Figure 9C and 9D).

In addition to the m/hA2-CAR Treg localized in the graft, on day 21 we noted an adjacent signal consistent with the location of the local draining lymph node. After sacrifice, lymph nodes draining the graft were harvested and analysis by flow cytometry showed a significant proportion of hCD4+FOXP3+ΔNGFR+A2-tetramer+ CAR Treg. In contrast, very few of these cells were found in the spleen (Figure 10B and C). It has previously been reported that this two-stage migration process from graft to lymph node is required for the induction of tolerance (Zhang et al., 2009). Importantly, CAR Tregs located in the lymph nodes retained FOXP3 and CAR expression, illustrating their stable phenotype.

hA2-CAR Tregs prevent human skin allograft rejection

To evaluate the immunoregulatory potential of hA2-CAR Treg in a solid organ transplant model, we used a humanized skin transplant model in which human HLA-A2pos skin grafts were transplanted into NSG mice. After 6 weeks, mice were injected with HLA-A2neg PBMC with or without autologous H1k2 hA2-CAR Tregs. Four weeks after cell injection, mice were sacrificed and skin grafts were harvested to evaluate pathology and expression of inflammatory cytokines. All mice maintained a stable body weight, indicating the absence of xenogenic GvHD (Figure 11A), with similar engraftment levels of human leukocytes in the blood and spleen (Figure 11B). Sections stained with hematoxylin-eosin were evaluated using a 25-point scale, revealing a significant reduction in the cumulative score of pathological rejection in mice that received H1k2 hA2-CAR Treg compared to PBMC (Figure 11C). Immunostaining showed that compared to mice treated with PBMC alone, mice treated with PBMC and H1k2 hA2-CAR Treg had a significant reduction in Ki67+ keratinocytes and had reduced involucrin destruction (Figure 11D). Quantitative qPCR analysis also showed an overall reduction in inflammatory cytokines in the H1k2 hA2-CAR graft of Treg-treated mice (Figure 11E).

With regard to the xenogeneic GVHD model, while PBMCs were detected in the blood, CAR Tregs were not detected (Figure 12). However, immunostaining showed that compared to PBMC treated mice, mice treated with PBMC and H1k2 hA2-CAR Treg had a significantly higher proportion of FOXP3+ cells in the graft (Figure 11F). The presence of FOXP3+ cells was unique to the transplanted skin graft as they were not found in the intestine, liver, or lungs (Figure 11G). These data indicate that, as in the case of the A2 transgenic NSG skin model, H1k2 hA2-CAR Tregs specifically bind to the A2+ human skin allograft, where they persist indefinitely.

This application includes a sequence listing in electronic form in the form of a text file "CDRD-003WO_SEQ LISTING_ST25", created on September 18, 2018 and having a size of 320 KB. The contents of the text file are incorporated into this document by reference in their entirety.

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Tarbell, K.V., L. Petit, X. Zuo, P. Toy, X. Luo, A. Mqadmi, H. Yang, M. Suthanthiran, S. Mojsov, and R.M. Steinman. 2007. Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. The Journal of experimental medicine 204:191-201.

Tarbell, K.V., S. Yamazaki, K. Olson, P. Toy, and R.M. Steinman. 2004. CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. The Journal of experimental medicine 199:1467-1477.

Trenado, A., M. Sudres, Q. Tang, S. Maury, F. Charlotte, S. Gregoire, M. Bonyhadi, D. Klatzmann, B.L. Salomon, and J.L. Cohen. 2006. Ex vivo-expanded CD4+CD25+ immunoregulatory T cells prevent graft-versus-host-disease by inhibiting activation/differentiation of pathogenic T cells. Journal of immunology 176:1266-1273.

Trzonkowski, P., M. Bieniaszewska, J. Juscinska, A. Dobyszuk, A. Krzystyniak, N. Marek, J. Mysliwska, and A. Hellmann. 2009. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells. Clinical immunology 133:22-26.

Tsang, J.Y., Y. Tanriver, S. Jiang, S.A. Xue, K. Ratnasothy, D. Chen, H.J. Stauss, R.P. Bucy, G. Lombardi, and R. Lechler. 2008. Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice. The Journal of Clinical Investigation 118:3619-3628.

Verginis, P., K.A. McLaughlin, K.W. Wucherpfennig, H. von Boehmer, and I. Apostolou. 2008. Induction of antigen-specific regulatory T cells in wild-type mice: visualization and targets of suppression. Proceedings of the National Academy of Sciences of the United States of America 105:3479-3484.

Zhenguang Wang, Zhiqiang Wu, Yang Liu and Weidong Han. 2017. New development in CAR-T cell therapy. Journal of Hematology & Oncology, 10:53.

Watanabe, N., Bajgain, P., Sukumaran, S., Ansari, S., Heslop, H. E., Rooney, C. M., Brenner, M. K., Leen, A. M., Vera, J. F. Fine-tuning the CAR spacer improves T-cell potency. Onco Immunology. 2016, 5(12): e1253656.

Wesch, D., Peters, C., and G.M. Siegers. Human gamma delta T regulatory cells in cancer: fact or fiction? Front Immunol. 2014 Nov 20; 5:598.

Wood, K.J., Bushell, A., and J. Hester. Regulatory immune cells in transplantation. Nat Rev Immunol. 2012, 12(6):417-30.

Zhang, E. and Xu, H. J. 2017. A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy. Hematol Oncol 10:1.

Zhang, N., Schroppel, B., Lal, G., Jakubzick, C., Mao, X., Chen, D., Yin, N., Jessberger, R., Ochando, J.C., Ding, Y., Bromberg , J.S. 2009. Regulatory T cells sequentially migrate from inflamed tissues to draining lymph nodes to suppress the alloimmune response. immunity. 30:458-469.

While the foregoing invention has been described in some detail for clarity and understanding, one skilled in the art will appreciate from reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the methods and devices described above may be used in various combinations. All publications, patents, patent applications and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application and/or other document were cited separately for inclusion link for all purposes.

--->

SEQUENCE LIST

<110> LEVINGS, Megan

ORBAN, Paul

DAWSON, Nicholas

LAMARCHE, Caroline

BERGQVIST, Jan P

<120> ANTI-HLA-A2 ANTIBODIES AND METHODS OF USING THE SAME

<130> CDRD-003WO

<150> US 62/560,574

<151> 2017-09-19

<150> US 62/692,386

<151> 2018-06-29

<160> 224

<170>PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

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<400> 1

Ser Tyr His Ile Gln

fifteen

<210> 2

<211> 7

<212> PRT

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<400> 2

Gly Tyr Thr Phe Thr Ser Tyr

fifteen

<210> 3

<211> 17

<212> PRT

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<223> Synthetic sequence

<400> 3

Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

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<211> 6

<212> PRT

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Tyr Pro Gly Asp Gly Ser

fifteen

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<211> 17

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Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

gly

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Glu Gly Thr Tyr Tyr Ala Met Asp Tyr

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Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

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<212> PRT

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Lys Val Ser Asn Arg Phe Ser

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<212> PRT

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Phe Gln Gly Ser His Val Pro Arg Thr

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<221> misc_feature

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<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa is E or Q

<220>

<221> misc_feature

<222> (16)..(16)

<223> Xaa is K or Q

<400> 10

Trp Ile Tyr Pro Gly Asp Gly Ser Thr Xaa Tyr Xaa Xaa Lys Phe Xaa

1 5 10 15

gly

<210> 11

<211> 25

<212> PRT

<213> Artificial Sequence

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<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 12

<211> 30

<212> PRT

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<400> 12

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

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<211> 15

<212> PRT

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<221> misc_feature

<222>(9)..(9)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is I or absent

<400> 13

Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met Gly Xaa

1 5 10 15

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<212> PRT

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Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly

1 5 10

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Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile

1 5 10 15

<210> 16

<211> 14

<212> PRT

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<400> 16

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 17

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<212> PRT

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<221> misc_feature

<222>(9)..(9)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is I or absent

<400> 17

Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met Gly Xaa Trp

1 5 10 15

ile

<210> 18

<211> 16

<212> PRT

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<400> 18

Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ile

1 5 10 15

<210> 19

<211> 17

<212> PRT

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<400> 19

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Trp

1 5 10 15

ile

<210> 20

<211> 16

<212> PRT

<213> Artificial Sequence

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<400> 20

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile

1 5 10 15

<210> 21

<211> 20

<212> PRT

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<221> misc_feature

<222> (12)..(12)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (18)..(18)

<223> Xaa is I or absent

<400> 21

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met

1 5 10 15

Gly Xaa Trp Ile

twenty

<210> 22

<211> 19

<212> PRT

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<400> 22

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

1 5 10 15

Gly Trp Ile

<210> 23

<211> 20

<212> PRT

<213> Artificial Sequence

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<400> 23

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

1 5 10 15

Gly Ile Trp Ile

twenty

<210> 24

<211> 19

<212> PRT

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<400> 24

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

1 5 10 15

Gly Trp Ile

<210> 25

<211> 18

<212> PRT

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<221> misc_feature

<222> (12)..(12)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (18)..(18)

<223> Xaa is I or absent

<400> 25

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met

1 5 10 15

Gly Xaa

<210> 26

<211> 17

<212> PRT

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<400> 26

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

1 5 10 15

gly

<210> 27

<211> 18

<212> PRT

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<400> 27

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

1 5 10 15

Gly Ile

<210> 28

<211> 17

<212> PRT

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<400> 28

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

1 5 10 15

gly

<210> 29

<211> 32

<212> PRT

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<221> misc_feature

<222> (1)..(1)

<223> Xaa is R or absent

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<221> misc_feature

<222> (4)..(4)

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<221> misc_feature

<222>(6)..(6)

<223> Xaa is R or A

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<221> misc_feature

<222> (10)..(10)

<223> Xaa is A, T or I

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<221> misc_feature

<222> (16)..(16)

<223> Xaa is E or L

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<221> misc_feature

<222> (19)..(19)

<223> Xaa is S or R

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<221> misc_feature

<222> (23)..(23)

<223> Xaa is E or D

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<221> misc_feature

<222> (25)..(25)

<223> Xaa is T or M

<400> 29

Xaa Val Thr Xaa Thr Xaa Asp Thr Ser Xaa Ser Thr Ala Tyr Met Xaa

1 5 10 15

Leu Ser Xaa Leu Arg Ser Xaa Asp Xaa Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 30

<211> 32

<212> PRT

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<400> 30

Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu

1 5 10 15

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

20 25 30

<210> 31

<211> 32

<212> PRT

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<400> 31

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

1 5 10 15

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

20 25 30

<210> 32

<211> 31

<212> PRT

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<400> 32

Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu

1 5 10 15

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

20 25 30

<210> 33

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

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<400> 33

Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu

1 5 10 15

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

20 25 30

<210> 34

<211> 32

<212> PRT

<213> Artificial Sequence

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<400> 34

Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 35

<211> 41

<212> PRT

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<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa is Q or K

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa is N or S

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa is E or Q

<220>

<221> misc_feature

<222>(8)..(8)

<223> Xaa is K or Q

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<221> misc_feature

<222> (10)..(10)

<223> Xaa is R or absent

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa is I or M

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is R or A

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<221> misc_feature

<222> (19)..(19)

<223> Xaa is A, T or I

<220>

<221> misc_feature

<222> (25)..(25)

<223> Xaa is E or L

<220>

<221> misc_feature

<222> (28)..(28)

<223> Xaa is S or R

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<221> misc_feature

<222> (32)..(32)

<223> Xaa is E or D

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa is T or M

<400> 35

Thr Xaa Tyr Xaa Xaa Lys Phe Xaa Gly Xaa Val Thr Xaa Thr Xaa Asp

1 5 10 15

Thr Ser Xaa Ser Thr Ala Tyr Met Xaa Leu Ser Xaa Leu Arg Ser Xaa

20 25 30

Asp Xaa Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 36

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa is R or absent

<220>

<221> misc_feature

<222> (13)..(13)

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<221> misc_feature

<222> (15)..(15)

<223> Xaa is R or A

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<221> misc_feature

<222> (19)..(19)

<223> Xaa is A, T or I

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<221> misc_feature

<222> (25)..(25)

<223> Xaa is E or L

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<221> misc_feature

<222> (28)..(28)

<223> Xaa is S or R

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<221> misc_feature

<222> (32)..(32)

<223> Xaa is E or D

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<221> misc_feature

<222> (34)..(34)

<223> Xaa is T or M

<400> 36

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Xaa Val Thr Xaa Thr Xaa Asp

1 5 10 15

Thr Ser Xaa Ser Thr Ala Tyr Met Xaa Leu Ser Xaa Leu Arg Ser Xaa

20 25 30

Asp Xaa Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 37

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa is R or absent

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa is I or M

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is R or A

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<221> misc_feature

<222> (19)..(19)

<223> Xaa is A, T or I

<220>

<221> misc_feature

<222> (25)..(25)

<223> Xaa is E or L

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<221> misc_feature

<222> (28)..(28)

<223> Xaa is S or R

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<221> misc_feature

<222> (32)..(32)

<223> Xaa is E or D

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa is T or M

<400> 37

Thr Lys Tyr Ser Gln Lys Phe Gln Gly Xaa Val Thr Xaa Thr Xaa Asp

1 5 10 15

Thr Ser Xaa Ser Thr Ala Tyr Met Xaa Leu Ser Xaa Leu Arg Ser Xaa

20 25 30

Asp Xaa Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 38

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 38

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

1 5 10 15

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

20 25 30

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 39

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 39

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

1 5 10 15

Thr Ser Ala Ser Thr Ala Tyr Met Leu Leu Ser Ser Leu Arg Ser Glu

20 25 30

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 40

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 40

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Val Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 41

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 41

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp

1 5 10 15

Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp

20 25 30

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 42

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 42

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

1 5 10 15

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

20 25 30

Asp Met Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 43

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 43

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

1 5 10 15

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

20 25 30

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

35 40

<210> 44

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 44

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 45

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (50)..(50)

<223> Xaa is I or absent

<220>

<221> misc_feature

<222> (60)..(60)

<223> Xaa is Q or K

<220>

<221> misc_feature

<222> (62)..(62)

<223> Xaa is N or S

<220>

<221> misc_feature

<222> (63)..(63)

<223> Xaa is E or Q

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa is K or Q

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa is R or absent

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa is I or M

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<221> misc_feature

<222> (73)..(73)

<223> Xaa is R or A

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<221> misc_feature

<222> (77)..(77)

<223> Xaa is A, T or I

<220>

<221> misc_feature

<222> (83)..(83)

<223> Xaa is E or L

<220>

<221> misc_feature

<222> (86)..(86)

<223> Xaa is S or R

<220>

<221> misc_feature

<222> (90)..(90)

<223> Xaa is E or D

<220>

<221> misc_feature

<222> (92)..(92)

<223> Xaa is T or M

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met

35 40 45

Gly Xaa Trp Ile Tyr Pro Gly Asp Gly Ser Thr Xaa Tyr Xaa Xaa Lys

50 55 60

Phe Xaa Gly Xaa Val Thr Xaa Thr Xaa Asp Thr Ser Xaa Ser Thr Ala

65 70 75 80

Tyr Met Xaa Leu Ser Xaa Leu Arg Ser Xaa Asp Xaa Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 46

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa is V or I

<220>

<221> misc_feature

<222>(7)..(7)

<223> Xaa is S or T

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa is L or S

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa is P or S

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is L or P

<220>

<221> misc_feature

<222> (23)..(23)

<223> Xaa is C or F

<400> 46

Asp Xaa Val Met Thr Gln Xaa Pro Leu Ser Xaa Xaa Val Thr Xaa Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Xaa

twenty

<210> 47

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 47

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

twenty

<210> 48

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 48

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

twenty

<210> 49

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 49

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

twenty

<210> 50

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 50

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Phe

twenty

<210> 51

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa is F or Y

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa is Q or L

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa is R or K

<220>

<221> misc_feature

<222>(9)..(9)

<223> Xaa is S or P

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa is R or Q

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa is R or L

<400> 51

Trp Xaa Xaa Gln Xaa Pro Gly Gln Xaa Pro Xaa Xaa Leu Ile Tyr

1 5 10 15

<210> 52

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 52

Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr

1 5 10 15

<210> 53

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 53

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

1 5 10 15

<210> 54

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 54

Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 55

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 55

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

1 5 10 15

<210> 56

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa is S or A

<400> 56

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Xaa Gly Thr Asp Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 57

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 57

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 58

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 58

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 59

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 59

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 60

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa is V or I

<220>

<221> misc_feature

<222>(7)..(7)

<223> Xaa is S or T

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa is L or S

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa is P or S

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa is L or P

<220>

<221> misc_feature

<222> (23)..(23)

<223> Xaa is C or F

<220>

<221> misc_feature

<222> (41)..(41)

<223> Xaa is F or Y

<220>

<221> misc_feature

<222> (42)..(42)

<223> Xaa is Q or L

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa is R or K

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa is S or P

<220>

<221> misc_feature

<222> (50)..(50)

<223> Xaa is R or Q

<220>

<221> misc_feature

<222> (51)..(51)

<223> Xaa is R or L

<220>

<221> misc_feature

<222> (72)..(72)

<223> Xaa is S or A

<400> 60

Asp Xaa Val Met Thr Gln Xaa Pro Leu Ser Xaa Xaa Val Thr Xaa Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Xaa Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Xaa Xaa Gln Xaa Pro Gly Gln Xaa

35 40 45

Pro Xaa Xaa Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Xaa Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 61

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 62

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 63

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 64

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 65

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 66

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 66

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 67

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 67

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 68

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 68

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 69

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 69

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 70

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 70

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 71

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 71

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 72

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 73

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 73

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 74

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 75

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 76

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 76

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 77

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 77

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 78

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 78

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 79

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 79

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 80

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 81

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 81

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 82

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 82

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 83

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 83

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 84

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 84

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 85

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 86

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 87

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 88

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 89

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 89

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 90

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 91

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 92

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa is R or G

<220>

<221> misc_feature

<222> (50)..(50)

<223> Xaa is I or absent

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa is R or absent

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa is I or M

<220>

<221> misc_feature

<222> (73)..(73)

<223> Xaa is R or A

<220>

<221> misc_feature

<222> (77)..(77)

<223> Xaa is A, T or I

<220>

<221> misc_feature

<222> (83)..(83)

<223> Xaa is E or L

<220>

<221> misc_feature

<222> (86)..(86)

<223> Xaa is S or R

<220>

<221> misc_feature

<222> (90)..(90)

<223> Xaa is E or D

<220>

<221> misc_feature

<222> (92)..(92)

<223> Xaa is T or M

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met

35 40 45

Gly Xaa Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys

50 55 60

Phe Gln Gly Xaa Val Thr Xaa Thr Xaa Asp Thr Ser Xaa Ser Thr Ala

65 70 75 80

Tyr Met Xaa Leu Ser Xaa Leu Arg Ser Xaa Asp Xaa Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 93

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 93

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 94

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 94

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 95

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 95

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 96

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 96

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 97

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 97

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Phe Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 98

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 98

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Leu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 99

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 99

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Leu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 100

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 100

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

Gly Gln Gly Leu Glu Trp Met Gly Ile Trp Ile Tyr Pro Gly Asp Gly

65 70 75 80

Ser Thr Gln Tyr Asn Glu Lys Phe Lys Gly Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 101

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 101

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

Gly Gln Gly Leu Glu Trp Met Gly Ile Trp Ile Tyr Pro Gly Asp Gly

65 70 75 80

Ser Thr Gln Tyr Asn Glu Lys Phe Lys Gly Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 102

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 102

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

Gly Gln Gly Leu Glu Trp Met Gly Ile Trp Ile Tyr Pro Gly Asp Gly

65 70 75 80

Ser Thr Gln Tyr Asn Glu Lys Phe Lys Gly Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 103

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 103

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

Gly Gln Gly Leu Glu Trp Met Gly Ile Trp Ile Tyr Pro Gly Asp Gly

65 70 75 80

Ser Thr Gln Tyr Asn Glu Lys Phe Lys Gly Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Phe Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 104

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 104

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 105

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 105

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 106

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 106

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 107

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 107

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp

85 90 95

Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Phe Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 108

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 108

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Met Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 109

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 109

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Met Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 110

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 110

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Met Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 111

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 111

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Met Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr

165 170 175

Leu Gly Gln Pro Ala Ser Ile Ser Phe Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly

195 200 205

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

210 215 220

Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 112

<211> 274

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 112

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser Gln Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr

165 170 175

Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

245 250 255

Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

260 265 270

Ile Lys

<210> 113

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 113

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Ala Ser

twenty

<210> 114

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 114

Val Asp Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Thr Ser

twenty

<210> 115

<211> 63

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 115

Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val

1 5 10 15

Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

20 25 30

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

35 40 45

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

50 55 60

<210> 116

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 116

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 117

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 117

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 118

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 118

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

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 119

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 119

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 120

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 120

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 121

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 122

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 122

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 123

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 123

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 124

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 124

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 125

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 126

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 127

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 127

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 128

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 128

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 129

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 130

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 130

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 131

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 132

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 132

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 133

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 134

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 135

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 135

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 136

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 136

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Val Met Thr

130 135 140

Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 137

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 137

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Phe Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 138

<211> 510

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 138

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Asn Arg Ile Arg Gly

245 250 255

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

260 265 270

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

275 280 285

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

290 295 300

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

305 310 315 320

Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 139

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 139

atggatttcc aagttcaaat cttcagtttc ttgcttatca gtgcttctgt tattatgtca 60

cgagcaagtc aagttcaact cgtacagtct ggagccgagg tgaaaaaacc gggagcgtcc 120

gtgaaagtga gttgcaaggc gagtggatac accttcactt cataccatat acaatgggtt 180

cggcaggcgc ctggtcaacg gctggaatgg atgggctgga tttatcccgg agatggttcc 240

acgcagtaca atgagaaatt caaagggaga gtgacaatca cccgagatac cagtgcctct 300

acggcatata tggaactgag tagtctgcgg tctgaagata cggcggtgta ttattgtgcg 360

agagaaggga cgtactacgc catggattat tggggacaag gaacaacagt cacagtctcc 420

agcgttgatt cctcaggcgg gggcggaagt gggggcggcg gatctggcgg cggcgggtct 480

acgtctgacg tggtcatgac ccaatctcca ttgtctttgc cagttactct gggacagcct 540

gcaagtatca gttgccgatc ctcccaatct atcgtccatt caaacgggaa cacttatttg 600

gaatggtttc aacagagacc tgggcaaagt ccgcgccgac tgatatataa ggtcagtaac 660

cgcttttcag gcgtccccga tcgattcagt ggatctgggt cagggactga cttcactctg 720

aaaatatcaa gagtcgaagc tgaagatgtc ggagtatatt actgtttcca ggggtctcac 780

gtccctcgga cgtttgggagg cggaactaag gttgagataa aa 822

<210> 140

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 140

atggattttc aggttcaaat ctttagcttt ctcttgattt ccgcctccgt aataatgagt 60

cgggccagtc aggtacagct cgttcaatct ggggctgaag taaaaaagcc tggagcgtct 120

gtaaaggtat cttgcaaagc gagcggctac acattcacaa gttatcacat ccaatgggtg 180

agacaggccc caggacaacg cttggagtgg atggggtgga tttaccctgg cgacggcagc 240

acacagtaca atgagaaatt taaaggccgg gtgactatca ctcgggacac ctccgccagc 300

360

agagaaggaa cgtactacgc tatggactat tggggtcagg gcacaaccgt tacagtctcc 420

480

acaagcgata tagtaatgac ccaaaccccg ctctctctga gcgtcacgcc aggacaacca 540

gcatcaatct cttgccgcag tagtcaatcc atcgttcact ctaatggaaa cacatacctt 600

gagtggtatc ttcagaaacc aggtcagagc cctcagctcc tcatctataa agtctctaac 660

cggttctcag gtgttccgga ccggttcagt ggttccggct caggaacaga cttcaccttg 720

aagatcagtc gagtagaagc cgaagacgtg ggtgtatatt attgctttca gggttcccac 780

gttccgcgca ccttcggcgg cgggaccaaa gttgagatca aa 822

<210> 141

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 141

atggactttc aagtccaaat cttctcattc ctcctgatct ctgcgtcagt aatcatgtcc 60

agagcgtcac aagtgcaact cgttcaatcc ggagctgagg taaagaagcc cggcgccagc 120

gtgaaagtct cctgcaaagc gagcggctac acgttcacct catatcacat tcaatgggta 180

agacaagcac ctgggcaacg actcgagtgg atggggtgga tctaccctgg ggacggggagc 240

acgcagtata atgagaaatt caaaggcagg gttacaatta cagccgatac cagtgcatct 300

360

cgggagggaa cgtactacgc gatggactat tgggggcaag gcaccacagt tacagtgagc 420

tcagttgact catcaggagg cggaggatca gggggaggtg gtagtggggg cggtgggagc 480

acatcagatg ttgtcatgac tcagagccca ctttctttgc cggtgacgct ggggcagccc 540

gcttcaatct cttgccgctc atcacagtct atcgttcata gcaatggtaa cacttacttg 600

gaatggttcc aacaaagacc gggtcaaagt ccacggcgct tgatatataa agtatcaaat 660

agattttcag gggtgcctga tcggttcagc ggttctggat ctggcaccga cttcacgctt 720

aaaataagta gggtagaagc cgaagacgtg ggagtgtatt attgtttcca ggggtcacac 780

gtccctcgca cgttcggcgg aggcactaaa gtggagatca aa 822

<210> 142

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 142

atggactttc aagtccaaat cttcagcttt ctccttatat ctgcgtctgt cattatgagt 60

agagcaagtc aagtccagct cgtacaaagt ggagctgagg tgaaaaagcc gggcgcgagt 120

gtgaaagtct catgcaaggc gagtggatac acctttacct cttaccacat tcaatgggtg 180

240

acccagtaca acgaaaaatt caaaggtagg gttaccatca ctgctgatac ctccgcgtcc 300

actgcttata tgcttcttag ctccttgcga agcgaggata cagccgtgta ttattgtgcc 360

agagaggggga cttattatgc catggactat tggggtcagg gtacaacggt cactgtctca 420

tctgttgaca gtagcggggg aggggggtct ggaggagggg gttccgggggg gggaggttcc 480

acgagcgata tagttatgac gcaaacgccc ttgagcctca gtgttacacc cggtcaacct 540

gcctctatta gctgtcgctc ctcccaatca attgtgcata gcaatggaaa tacctacctt 600

gaatggtatc tccaaaagcc cgggcagagt cctcaacttc tcatctacaa agtatccaat 660

cgattcagtg gcgttcctga caggttcagc ggaagtgggt cagggaccga ttttaccctc 720

aaaattagtc gcgtcgaagc tgaggatgtt ggggtgtatt actgcttcca agggtcacac 780

gtaccacgca cattcggggg aggcacgaag gttgaaatta ag 822

<210> 143

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 143

atggatttcc aagtccaaat attcagtttc cttttgataa gtgcttcagt tatcatgtcc 60

cgagcaagcc aggtacagct tgtgcaaagc ggggcggaag ttaagaaacc gggagcctca 120

gttaaagtat cttgcaaagc cagcggttat acattcactt cataccacat acagtgggtg 180

cgccaagcac cggggcagag actggaatgg atgggatgga tttatcccgg tgatggtagt 240

acgcaataca atgaaaaatt caaaggaagg gtgactatca cgcgagacac aagcgcgtcc 300

360

cgaggggga cctactacgc catggattat tggggtcaag ggaccactgt gacagtctct 420

tctgtcgatt ccagcggcgg aggagggagt ggagggggtg ggtccggggg gggagggtct 480

acgagcgaca ttgtcatgac acagacgccg cttagctccc cagttacact cggacagccc 540

gcaagtatta gttgcagaag tagccagtct atcgtacatt caaatggaaa cacctatttg 600

gaatggtatc aacaacggcc tggacagccg cccaggctgc tcatatacaa agtctccaac 660

cgcttcagcg gagtacccga ccgcttttcc ggctccggag caggaactga ctttaccttg 720

aaaattagta gggtcgaagc agaggatgtc ggggtatatt attgtttcca aggtagtcat 780

gtcccacgga cgtttggtgg tgggacgaag gttgagatca aa 822

<210> 144

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 144

atggacttcc aggtgcagat attttccttt cttctcatat ctgcatctgt aataatgtca 60

agggccagcc aggtccagct cgttcaaagc ggagccgagg taaaaaaacc aggcgcttcc 120

gtcaaggtat catgcaaagc gtccggctat accttcacaa gttaccatat ccaatgggtt 180

cgacaggcac ccggacaaag acttgaatgg atgggttgga tataccccgg agacggctcc 240

actcagtata acgaaaagtt taaggggaga gtcacgatca ctagggacac atcagcttct 300

acggcgtata tggaactcag ttctttgcga tccgaggata ctgccgtata ttactgcgcc 360

agagaaggca cgtactacgc aatggattac tgggggcaag ggacaactgt taccgtctca 420

agcgtcgatt catcaggagg cggagggtcc ggaggtgggg gatctggcgg tgggggttct 480

acgtccgatg ttgtgatgac acagtcccca ctctctcttc cagtgacgct gggacagccc 540

gcgagcatct cctgtcgcag ctctcagtcc atagtacaca gtaatggtaa cacctatctt 600

gagtggtatc agcaacgacc cggtcagtct cccaggttgc ttatttataa ggttagtaac 660

cgcttttcag gtgttccaga cagatttagc gggagtggtt ccggtacgga tttcacattg 720

aaaatcagcc gcgtcgaagc cgaggacgtg ggtgtttact actgcttcca gggatctcac 780

gtaccgagga ccttcggcgg aggaacgaaa gtagaaatta ag 822

<210> 145

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 145

atggattttc aagtacaaat cttttccttc ctgcttattt ccgcaagcgt tattatgagt 60

agagccagcc aagtacaact ggtacagtcc ggcgcggagg tgaagaagcc cggagcaagt 120

gtgaaggtat cttgcaaggc gtcaggctat acctttactt cataccatat acagtgggtg 180

cgacaggctc ccggccagcg actcgaatgg atgggctgga tttatcccgg agatggatct 240

acgcagtata atgagaaatt caagggtcgg gtcacgatta cacgagacac gagtgcttcc 300

acagcttata tggaactttc tagcctgagg tctgaggata ctgccgttta ctattgtgca 360

cgagaaggga catactatgc gatggattac tggggacagg gcaccactgt cacagtttcc 420

agcgtggact caagtggagg cggtggatct ggtggtggcg ggtccggggg gggaggcagc 480

accagtgaca ttgtaatgac tcaaacacct ctcagtagcc cagtcactct cggtcagccg 540

gcgagtatct cttttaggtc ctcacaatct atagtgcact ctaacggcaa tacttatctt 600

gaatggtatc aacaaagacc ggggcagcca cctcgccttc tcatctacaa agtaagcaat 660

cgcttctccg gtgtccccga tcgcttctcc ggttcaggag caggaactga cttcacattg 720

aagatttcca gagtggaggc cgaagacgta ggggtatatt attgctttca agggtcccat 780

gtgccgagaa cctttgggggg aggaacgaaa gttgagatta aa 822

<210> 146

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 146

atggatttcc aagtgcagat ttttctctttt ctcctcataa gcgcctccgt aattatgtct 60

agagctagtc aagtccaatt ggtgcaatcc ggtgccgagg ttaaaaagcc cggcgcaagt 120

gtaaaagtct cctgtaaggc cagtggctac actttcacca gctaccatat acagtgggtg 180

cggcaggcgc ctggtcaggg tctggagtgg atgggtattt ggatttatcc cggagatgga 240

agtactcaat acaatgagaa attcaagggt gtcactatga caagggatac gagcacttct 300

accgtatata tggagttgtc atctttgcga tcagaggata ccgctgtata ttattgcgca 360

cgggaaggta catattatgc catggactac tggggccaag gaaccaccgt gacggtaagc 420

tctgtcgatt ctagcggtgg cggggggctct ggcggtgggg gtagcggggg tggcggatct 480

acatcagata ttgtaatgac acagacccct ctttcacttt ccgtaacgcc aggacagccg 540

gcatcaataa gttgccgatc aagccagtct atcgtacact ccaatggtaa cacatacttg 600

gaatggtatc ttcaaaagcc cggccagagc ccgcagcttt tgatatataa agtgtccaac 660

agattcagtg gggtgccgga ccgctttagt ggatctggtt caggaacgga cttcacattg 720

aaaattagta gagttgaagc ggaagacgtg ggagtctact actgtttcca gggttcacat 780

gtgcctcgga cctttggggg aggcaccaag gttgagataa aa 822

<210> 147

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 147

atggacttcc aagtccaaat cttttctttt ttgttgataa gcgcatcagt tattatgtct 60

cgcgccagtc aagtacaact ggtgcagtcc ggagctgaag tgaaaaaacc aggagcaagc 120

gtgaaagtaa gttgtaaggc aagtggttac actttcacaa gctaccatat tcaatgggtc 180

cgacaggctc ctggacaggg cttggagtgg atgggcatat ggatttaccc tggtgacggg 240

tccacccagt ataatgaaaa gttcaaggga gtcacgatga ccagggacac ctctacatct 300

accgtgtata tggagctctc tagtttgcga tccgaagaca ctgccgttta ttactgtgca 360

agagaaggaa cttattacgc gatggactac tggggtcagg ggacaacagt caccgttagc 420

tccgtcgatt ccagcggggg aggtggctca ggcggggggtg gttctggggg gggcgggggc 480

acttcagata ttgtaatgac ccaaacccca ctgagtagtc cagtcacgct tggtcaaccg 540

gcaagcattt cttgcaggag ctctcagagt attgtccact ctaacgggaa tacatatttg 600

660

aggttttcag gcgtgccaga taggttctca ggctccggag cgggaaccga cttcaccctc 720

aagataagtc gggtggaagc cgaagacgta ggagtttact actgctttca aggatctcat 780

gttccacgaa cgtttggagg aggaaccaag gtggaaataa aa 822

<210> 148

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 148

atggactttc aggtccaaat ttttccttc ttgctcatat ccgcgagtgt catcatgtca 60

agagcaagtc aagttcaact cgttcaatca ggagctgagg tgaaaaaacc aggggcgtct 120

gtcaaagtaa gctgcaaagc atcagggtat acgttcacga gttatcatat ccagtgggtt 180

aggcaggcgc cagggcaggg attggaatgg atgggtatct ggatttaccc gggtgacggc 240

agcactcaat acaatgagaa attcaaaggc gtaacaatga caagggacac gagcacaagc 300

acagtgtaca tggagcttag ctctttgagg tcagaggata ccgctgttta ctattgtgct 360

cgggagggta cttactatgc aatggactac tgggggcaag gcacgaccgt tacagtgagt 420

agcgtagatt cctccggggg tggcggttca ggcggcggag gctcaggcgg aggagggtca 480

acatccgatg tcgtaatgac tcagtcccct ctgtcattgc cggtgacttt gggacagcca 540

gcgtctatat cttgtaggtc ctctcaatca atagtgcatt ccaacggtaa cacctatctg 600

gaatggtatc agcaaaggcc aggacaaagt ccacgcctgc ttatatataa ggtgtctaat 660

cgattcagtg gggttcccga taggttttcc ggctctggta gcgggactga tttcacgttg 720

aaaatatcac gcgtggaagc ggaagatgtt ggggtctatt actgctttca gggtagtcat 780

gtccctcgaa cttttggcgg tggtacaaag gtagaaatca aa 822

<210> 149

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 149

atggattttc aggtacagat attctcattt ctccttatct cagctagtgt cataatgtcc 60

agggcgagtc aagtacaact tgtccagtca ggcgcagagg tcaagaagcc gggcgcaagc 120

gttaaggttt cctgcaaagc atccggctat acattcacgt cctatcacat ccaatgggtc 180

aggcaagcac ccggtcaagg acttgagtgg atgggcatct ggatttaccc tggagatggc 240

agtactcagt acaacgaaaa attcaaaggt gtaaccatga cccgcgacac atctacttcc 300

acagtttata tggaactcag cagtttgcgg agcgaagata ccgctgttta ctactgtgcc 360

cgagagggaa cttactacgc catggactat tggggtcaag gaacgacagt aacagttagt 420

tctgtagatt ccagtggcgg cggtgggagc gggggtgggg gatctggcgg aggcggaagt 480

acaagtgaca tcgttatgac tcagacaccc cttagtagtc ccgttacgtt gggccaaccc 540

gcgagcattt cctttcgatc ctctcagtct atagttcact caaatgggaa tacttatttg 600

gagtggtatc aacagcgccc cggacaacca ccaaggctcc tgatatacaa ggtgtccaat 660

cgattctctg gggtgcctga tagatttagc ggaagtggag ccggtacaga ttttaccctg 720

aaaatatcac gggtagaagc cgaagatgtc ggcgtctact actgtttcca gggttcccat 780

gtaccgcgaa cgttcggggg cggaacaaaa gttgagatca ag 822

<210> 150

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 150

atggatttcc aggttcagat atttagtttc ctcttgattt ctgccagtgt catcatgagc 60

agggcttccc aagttcagtt ggtgcaaagt ggcgctgaag tcaaaaaacc tggggcttcc 120

gttaaagtat cttgcaaggc gtccggctac actttcacat cctaccacat tcaatgggtc 180

cggcaagcgc ccggtcaggg gctcgaatgg atggggtgga tatacccagg agatggatct 240

actcagtaca acgagaaatt taaaggacgg gtgacgatga cgcgcgacac ttcaataagc 300

actgcataca tggaactgtc ccggcttagg tcagatgaca ccgcggtcta ctattgtgcg 360

agagagggta cttactatgc tatggactac tgggggcaag gcacgacggt tacagtttcc 420

tcagtcgata gttcaggcgg aggcggctcc gggggcggtg gtagtggagg gggtggatct 480

acttccgaca ttgtcatgac ccagaccccg ttgagccttt cagtgacgcc cggtcaaccc 540

gccagcataa gttgtcgatc aagccagtct attgtacact ccaatggaaa cacatatttg 600

gagtggtatc tccaaaaacc cggccaaagc cctcaactgc tcatctacaa ggtctcaaac 660

aggtttagcg gggtcccggga tcgcttctca gggtcaggat ctggtacgga ctttacactg 720

aaaatttccc gagtcgaagc ggaagacgtg ggtgtatatt actgcttcca ggggagtcat 780

gttccaagaa ccttttggggg aggtacaaag gtcgaaataa aa 822

<210> 151

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 151

atggattttc aggtccaaat ttttccttc ttgcttatca gcgcaagtgt aatcatgtcc 60

cgcgcgtccc aagtacaact tgtgcaatct ggcgcggagg tgaaaaaacc tggagcttcc 120

gtcaaggttt cttgtaaggc ctctggctac accttcacgt cctaccacat tcagtgggtt 180

cgacaggcgc cgggccaagg actggagtgg atgggatgga tatatccagg agatggttct 240

actcagtata atgagaaatt caagggtcgc gtaacaatga cgagggatac atcaatctcc 300

accgcgtaca tggaactttc aagactccgg tcagatgaca cggcggttta ctactgtgct 360

cgggaggca cttactatgc tatggactac tgggggcaag ggacaacggt aacggtatct 420

agtgtggatt ctagtggcgg cggcggttca ggaggaggtg gttcaggcgg ggggggtagt 480

acaagtgata ttgtgatgac ccaaacaccc ctttctagcc ctgttactct gggtcaaccc 540

gcgtccataa gttgtcgaag tagtcaatcc atcgtgcata gcaacggcaa cacttacctt 600

gaatggtatc aacaacgacc cggacagccc ccgcgactgc ttatctataa agtatcaaac 660

aggttcagtg gcgtgccaga tcgattctcc ggctctgggg caggcacaga tttcacgttg 720

aaaatttctc gggtcgaggc cgaggacgtg ggcgtttatt actgtttcca ggggagtcac 780

gtccccagga cgttcgggagg aggaactaaa gtcgaaataa ag 822

<210> 152

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 152

atggacttcc aggtccaaat attcagcttc ctcctcattt ccgccagtgt aataatgtcc 60

agagcctcac aagtacagtt ggttcagagc ggggctgagg ttaagaaacc aggcgcgagc 120

gtcaaggtat cctgcaaggc gagtggttat actttcacta gttatcacat tcagtgggtc 180

cgacaggccc ccggtcaagg cctggagtgg atggggtgga tatatccggg agatggttct 240

acccaatata atgagaagtt taaggggaga gtcacaatga caagggacac cagtattagc 300

accgcgtata tggagctttc ccgcctgcga tcagatgaca cggccgtgta ctactgtgct 360

agggagggaa cctattatgc gatggattac tggggacagg gtactacagt cacggtctct 420

agcgtggaca gttccggggg cggtggaagc ggtggtggcg gttcaggtgg aggaggctct 480

acgagtgatg ttgtgatgac tcagtccccg ctttcacttc ccgtcaccct tgggcaaccc 540

gcaagcatct catgccgatc ctcccagtct atagtacata gtaatggcaa cacatatctt 600

gaatggtatc agcagaggcc gggtcagtct ccccgactcc ttatatataa agtgagcaac 660

agattctccg gagtaccggga tagattttcc ggctctggga gcggcaccga ctttacactg 720

aaaatttcac gggttgaagc tgaagatgtt ggggtatact attgtttcca gggttctcac 780

gtcccgagga cattcggggg aggaacgaaa gtcgaaataa ag 822

<210> 153

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 153

atggattttc aggtacaaat cttcagcttc ctgctcatct ccgcgagcgt aatcatgtct 60

agggcgtccc aggtgcagtt ggtgcaatca ggtgcagagg tgaagaagcc tggtgcatcc 120

gttaaagtaa gttgtaaggc aagcggatat acttttacat cctatcatat tcaatgggtc 180

agacaagcac ctggacaggg tcttgagtgg atgggctgga tctatccagg cgatggctca 240

actcaatata acgagaagtt caaggggagg gttactatga ccagggatac gtctatttcc 300

actgcgtaca tggaactctc caggttgaga agtgatgata ccgcggttta ctactgcgct 360

agagaaggaa cgtactacgc tatggattac tgggggcagg gtacaactgt caccgtctca 420

agtgtggatt cttctggggg tgggggatca ggagggggag gctccggtgg gggcgggtca 480

accagcgaca ttgtcatgac tcaaaccccc ctgagcagcc ctgtcaccct gggtcagcct 540

gcctcaatat cctttagaag ctcccaaagc atcgtccatt caaatggtaa tacctatctg 600

gagtggtatc agcaaaggcc tggtcaaccc ccgcgccttc tcatttacaa ggtgtcaaac660

aggttctccg gcgtaccggga taggttttcc ggaagcggtg ctggaaccga ctttactctc 720

aaaatctcta gggtggaagc tgaggacgtc ggtgtatact attgttttca aggctcccat 780

gttccgga catttggtgg gggaacgaag gtagaaatca ag 822

<210> 154

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 154

atggactttc aggttcagat tttctctttc ttgttgatct ccgctagtgt cataatgtca 60

cgggcaagtc aggtacaact cgttcagagt ggtgccgaag tgaagaaacc gggtgcctcc 120

gtaaaggtgt catgtaaagc tagtggctat acattcacaa gttatcatat ccaatgggta 180

cgacaagcac cgggacagcg actggaatgg atgggatgga tctatcctgg ggacggatct 240

acacagtaca atgagaaatt taagggacgg gtcacgataa ccagggacac atctgcttcc 300

acggcttaca tggagctttc ctccctgcgg agcgaggaca tggctgttta ctattgcgct 360

cgcgaaggga catactacgc aatggattat tggggccaag gcactaccgt gacggtctct 420

tctgtcgata gttccgggagg aggtggttca gggggaggcg gttcaggtgg gggtggatct 480

acctcagata ttgtcatgac acagacacct ttgtccttga gtgtgacacc gggtcaaccg 540

gcgagtataa gctgtcgcag ctcacaatct attgtgcata gcaacgggaa tacatatctc 600

gaatggtatc tccaaaagcc gggccaatcc ccccaacttc tcatttacaa agtttctaat 660

cgattttcag gtgtaccaga tcggttttcc gggtctggct caggtactga cttcaccttg 720

aaaatatcaa gggttgaagc tgaggatgta ggtgtgtact attgcttcca ggggtctcac 780

gttcctcgga cttttggggg gggcacaaaa gtagagatta aa 822

<210> 155

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 155

atggatttcc aggtgcaaat cttctcattt cttttgataa gtgcgtcagt gataatgtct 60

cgggccagtc aagtacagct tgtccaaagt ggcgctgaag tcaagaagcc gggagcctca 120

gttaaggtta gctgcaaggc ctcagggtat acttttacct cctatcatat acagtgggta 180

cgacaagcac cgggacagcg actggagtgg atgggttgga tatatccggg agatggttca 240

acccagtata atgagaagtt caaggggcga gttacgataa cccgcgatac gagtgcatca 300

acagcgtaca tggagttgag ttccctccgc agcgaggaca tggcggtata ctattgtgcc 360

agggagggga cttattatgc catggactac tgggggcagg gcacaaccgt aacagtctct 420

tctgtagaca gttcaggagg gggcggaagt ggaggtggcg gatctggtgg aggtggatct 480

acttccgaca tcgttatgac ccaaacacca ctttcatctc ccgttactct cgggcaacct 540

gctagtattt cctgtagatc ctcacaatct atagtacata gcaatggcaa tacctacctg 600

gagtggtatc aacaacgccc aggccaacca cctcgcctgc ttatctataa agtaagcaat 660

agattcagtg gtgtaccgga taggttctct ggttccggag caggaactga ctttacactc 720

aaaatcagta gggtggaggc ggaagacgtg ggagtatatt attgctttca aggttcacat 780

gtacctcgaa catttggcgg aggaactaag gttgagatta aa 822

<210> 156

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 156

atggatttcc aagtccagat attcagtttt cttttgataa gcgcttctgt aatcatgtct 60

cgggcgtccc aagtacaact ggtgcaatca ggggcagaag tgaaaaaacc aggtgcatcc 120

gttaaggtga gttgcaaggc ttccggctat acctttacat catatcatat tcaatgggtc 180

aggcaagcac ctggtcagcg attggaatgg atgggttgga tatatcctgg tgatgggtct 240

acacaatata acgaaaaatt caaggggcga gtgaccatca caagagatac atcagcgtca 300

acagcgtata tggaactgtc atcccttaga tcagaggaca tggcggtcta ttactgtgcc 360

agagaaggca cttattatgc aatggattat tggggacaag gaaccactgt cactgtttcc 420

agcgtagact cctccggtgg tggtggaagt ggcggcggtg ggtcaggagg gggtgggtca 480

acttctgatg tagtgatgac acagagccct ctgagcttgc ctgtgacctt gggtcagccg 540

gcctcaataa gttgtcgatc tagtcagtca atcgtccata gtaatgggaa cacatacctt 600

660

cgatttagcg gagtgccaga ccgcttttct ggttccgggt ctggcacaga ttttaccctt 720

aagatctccc gcgtggaggc ggaagacgtt ggtgtttact attgcttcca ggggtcacac 780

gttccacgca cctttggagg aggtacgaag gtcgagatta ag 822

<210> 157

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 157

atggattttc aagtacagat cttctctttc ttgcttattt cagcgagcgt aatcatgagt 60

agggcatctc aagttcaact cgttcagtca ggtgctgagg taaaaaaacc aggggcttcc 120

180

cgacaagccc cggggcagcg cttggaatgg atgggctgga tttacccagg tgacggctcc 240

acgcaatata atgagaaatt taagggtaga gttactatta ccagggacac aagtgcttca 300

actgcctata tggaactgag cagccttcgg agtgaagata tggccgtata ttactgcgca 360

agggagggga cttactatgc aatggactac tggggtcagg gaacgactgt gaccgtgtcc 420

tcagttgact ccagcggtgg tggcggctct ggaggtgggg gttccggcgg aggcggaagc 480

acatctgata tagtgatgac gcaaacgcct ctttcttccc cggtaactct gggacagcca 540

gcgtcaattt catttaggtc ctcccagtca atcgtacata gtaatggaaa tacttacctg 600

gaatggtatc aacaacgacc agggcaaccg ccccgattgt tgatctataa agtgagcaat 660

cgcttttctg gcgtgcccga tcggttctca gggtctggag ctgggactga cttcacattg 720

aaaatttcca gggttgaggc cgaggatgtg ggggtttatt actgcttcca aggctcccac 780

gtcccccgca ccttcgggagg gggaaccaaa gtcgaaataa ag 822

<210> 158

<211> 822

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 158

atggattttc aagttcagat attctcattt ttgcttatat cagcctccgt aattatgtca 60

cgggcaagtc aagttcagtt ggtgcagtcc ggagcagaag ttaagaagcc cggtgcttct 120

gtgaaagtct cctgcaaagc gtctgggtac accttcacga gctaccatat acagtgggtc 180

240

acaaagtata gtcagaagtt tcaagggcga gtgacgataa ccagagatac gagtgcaagt 300

actgcataca tggaactgag ctccttgagg tccgaggata cagcggtgta ctattgcgct 360

cgggaaggga catattatgc tatggactat tggggacaag ggacaacggt aacggtgagt 420

tccgtcgatt cctcaggtgg cggaggcagt gggggcgggg gttccggcgg tggcgggtcc 480

acgagtgata tagttatgac acagaccccc ctcagccttt ctgtgacccc aggacaaccc 540

gctagtatct cttgccgcag ttctcagtcc atagtacaca gtaacggaaa tacctatctt 600

gagtggtatc ttcaaaagcc cggccagagc cctcaactct tgatatataa agtgtcaaat 660

cgattttcag gtgtgcctga tcgattctca gggtctggtt cagggacaga tttcacgctt 720

aagataagca gagtagaggc tgaagacgtg ggagtctact attgttttca ggggtcacac 780

gttccccgca cttttggtgg gggaaccaag gtggaaatca aa 822

<210> 159

<211> 192

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 159

tcttcagcgc tgagcaactc catcatgtac ttcagccact tcgtgccggt cttcctgcca 60

gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 120

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg

agggggctgg ac 192

<210> 160

<211> 81

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 160

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gccttttatta ttttctggggt g 81

<210> 161

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 161

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggccccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 162

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 162

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 163

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 163

atggatttcc aagttcaaat cttcagtttc ttgcttatca gtgcttctgt tattatgtca 60

cgagcaagtc aagttcaact cgtacagtct ggagccgagg tgaaaaaacc gggagcgtcc 120

gtgaaagtga gttgcaaggc gagtggatac accttcactt cataccatat acaatgggtt 180

cggcaggcgc ctggtcaacg gctggaatgg atgggctgga tttatcccgg agatggttcc 240

acgcagtaca atgagaaatt caaagggaga gtgacaatca cccgagatac cagtgcctct 300

acggcatata tggaactgag tagtctgcgg tctgaagata cggcggtgta ttattgtgcg 360

agagaaggga cgtactacgc catggattat tggggacaag gaacaacagt cacagtctcc 420

agcgttgatt cctcaggcgg gggcggaagt gggggcggcg gatctggcgg cggcgggtct 480

acgtctgacg tggtcatgac ccaatctcca ttgtctttgc cagttactct gggacagcct 540

gcaagtatca gttgccgatc ctcccaatct atcgtccatt caaacgggaa cacttatttg 600

gaatggtttc aacagagacc tgggcaaagt ccgcgccgac tgatatataa ggtcagtaac 660

cgcttttcag gcgtccccga tcgattcagt ggatctgggt cagggactga cttcactctg 720

aaaatatcaa gagtcgaagc tgaagatgtc ggagtatatt actgtttcca ggggtctcac 780

gtccctcgga cgtttgggagg cggaactaag gttgagataa aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 164

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 164

atggattttc aggttcaaat ctttagcttt ctcttgattt ccgcctccgt aataatgagt 60

cgggccagtc aggtacagct cgttcaatct ggggctgaag taaaaaagcc tggagcgtct 120

gtaaaggtat cttgcaaagc gagcggctac acattcacaa gttatcacat ccaatgggtg 180

agacaggccc caggacaacg cttggagtgg atggggtgga tttaccctgg cgacggcagc 240

acacagtaca atgagaaatt taaaggccgg gtgactatca ctcgggacac ctccgccagc 300

360

agagaaggaa cgtactacgc tatggactat tggggtcagg gcacaaccgt tacagtctcc 420

480

acaagcgata tagtaatgac ccaaaccccg ctctctctga gcgtcacgcc aggacaacca 540

gcatcaatct cttgccgcag tagtcaatcc atcgttcact ctaatggaaa cacatacctt 600

gagtggtatc ttcagaaacc aggtcagagc cctcagctcc tcatctataa agtctctaac 660

cggttctcag gtgttccgga ccggttcagt ggttccggct caggaacaga cttcaccttg 720

aagatcagtc gagtagaagc cgaagacgtg ggtgtatatt attgctttca gggttcccac 780

gttccgcgca ccttcggcgg cgggaccaaa gttgagatca aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 165

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 165

atggactttc aagtccaaat cttctcattc ctcctgatct ctgcgtcagt aatcatgtcc 60

agagcgtcac aagtgcaact cgttcaatcc ggagctgagg taaagaagcc cggcgccagc 120

gtgaaagtct cctgcaaagc gagcggctac acgttcacct catatcacat tcaatgggta 180

agacaagcac ctgggcaacg actcgagtgg atggggtgga tctaccctgg ggacggggagc 240

acgcagtata atgagaaatt caaaggcagg gttacaatta cagccgatac cagtgcatct 300

360

cgggagggaa cgtactacgc gatggactat tgggggcaag gcaccacagt tacagtgagc 420

tcagttgact catcaggagg cggaggatca gggggaggtg gtagtggggg cggtgggagc 480

acatcagatg ttgtcatgac tcagagccca ctttctttgc cggtgacgct ggggcagccc 540

gcttcaatct cttgccgctc atcacagtct atcgttcata gcaatggtaa cacttacttg 600

gaatggttcc aacaaagacc gggtcaaagt ccacggcgct tgatatataa agtatcaaat 660

agattttcag gggtgcctga tcggttcagc ggttctggat ctggcaccga cttcacgctt 720

aaaataagta gggtagaagc cgaagacgtg ggagtgtatt attgtttcca ggggtcacac 780

gtccctcgca cgttcggcgg aggcactaaa gtggagatca aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 166

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 166

atggactttc aagtccaaat cttcagcttt ctccttatat ctgcgtctgt cattatgagt 60

agagcaagtc aagtccagct cgtacaaagt ggagctgagg tgaaaaagcc gggcgcgagt 120

gtgaaagtct catgcaaggc gagtggatac acctttacct cttaccacat tcaatgggtg 180

240

acccagtaca acgaaaaatt caaaggtagg gttaccatca ctgctgatac ctccgcgtcc 300

actgcttata tgcttcttag ctccttgcga agcgaggata cagccgtgta ttattgtgcc 360

agagaggggga cttattatgc catggactat tggggtcagg gtacaacggt cactgtctca 420

tctgttgaca gtagcggggg aggggggtct ggaggagggg gttccgggggg gggaggttcc 480

acgagcgata tagttatgac gcaaacgccc ttgagcctca gtgttacacc cggtcaacct 540

gcctctatta gctgtcgctc ctcccaatca attgtgcata gcaatggaaa tacctacctt 600

gaatggtatc tccaaaagcc cgggcagagt cctcaacttc tcatctacaa agtatccaat 660

cgattcagtg gcgttcctga caggttcagc ggaagtgggt cagggaccga ttttaccctc 720

aaaattagtc gcgtcgaagc tgaggatgtt ggggtgtatt actgcttcca agggtcacac 780

gtaccacgca cattcggggg aggcacgaag gttgaaatta agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 167

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 167

atggatttcc aagtccaaat attcagtttc cttttgataa gtgcttcagt tatcatgtcc 60

cgagcaagcc aggtacagct tgtgcaaagc ggggcggaag ttaagaaacc gggagcctca 120

gttaaagtat cttgcaaagc cagcggttat acattcactt cataccacat acagtgggtg 180

cgccaagcac cggggcagag actggaatgg atgggatgga tttatcccgg tgatggtagt 240

acgcaataca atgaaaaatt caaaggaagg gtgactatca cgcgagacac aagcgcgtcc 300

360

cgaggggga cctactacgc catggattat tggggtcaag ggaccactgt gacagtctct 420

tctgtcgatt ccagcggcgg aggagggagt ggagggggtg ggtccggggg gggagggtct 480

acgagcgaca ttgtcatgac acagacgccg cttagctccc cagttacact cggacagccc 540

gcaagtatta gttgcagaag tagccagtct atcgtacatt caaatggaaa cacctatttg 600

gaatggtatc aacaacggcc tggacagccg cccaggctgc tcatatacaa agtctccaac 660

cgcttcagcg gagtacccga ccgcttttcc ggctccggag caggaactga ctttaccttg 720

aaaattagta gggtcgaagc agaggatgtc ggggtatatt attgtttcca aggtagtcat 780

gtccccggga cgtttggtgg tgggacgaag gttgagatca aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 168

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 168

atggacttcc aggtgcagat attttccttt cttctcatat ctgcatctgt aataatgtca 60

agggccagcc aggtccagct cgttcaaagc ggagccgagg taaaaaaacc aggcgcttcc 120

gtcaaggtat catgcaaagc gtccggctat accttcacaa gttaccatat ccaatgggtt 180

cgacaggcac ccggacaaag acttgaatgg atgggttgga tataccccgg agacggctcc 240

actcagtata acgaaaagtt taaggggaga gtcacgatca ctagggacac atcagcttct 300

acggcgtata tggaactcag ttctttgcga tccgaggata ctgccgtata ttactgcgcc 360

agagaaggca cgtactacgc aatggattac tgggggcaag ggacaactgt taccgtctca 420

agcgtcgatt catcaggagg cggagggtcc ggaggtgggg gatctggcgg tgggggttct 480

acgtccgatg ttgtgatgac acagtcccca ctctctcttc cagtgacgct gggacagccc 540

gcgagcatct cctgtcgcag ctctcagtcc atagtacaca gtaatggtaa cacctatctt 600

gagtggtatc agcaacgacc cggtcagtct cccaggttgc ttatttataa ggttagtaac 660

cgcttttcag gtgttccaga cagatttagc gggagtggtt ccggtacgga tttcacattg 720

aaaatcagcc gcgtcgaagc cgaggacgtg ggtgtttact actgcttcca gggatctcac 780

gtaccgagga ccttcggcgg aggaacgaaa gtagaaatta agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 169

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 169

atggattttc aagtacaaat cttttccttc ctgcttattt ccgcaagcgt tattatgagt 60

agagccagcc aagtacaact ggtacagtcc ggcgcggagg tgaagaagcc cggagcaagt 120

gtgaaggtat cttgcaaggc gtcaggctat acctttactt cataccatat acagtgggtg 180

cgacaggctc ccggccagcg actcgaatgg atgggctgga tttatcccgg agatggatct 240

acgcagtata atgagaaatt caagggtcgg gtcacgatta cacgagacac gagtgcttcc 300

acagcttata tggaactttc tagcctgagg tctgaggata ctgccgttta ctattgtgca 360

cgagaaggga catactatgc gatggattac tggggacagg gcaccactgt cacagtttcc 420

agcgtggact caagtggagg cggtggatct ggtggtggcg ggtccggggg gggaggcagc 480

accagtgaca ttgtaatgac tcaaacacct ctcagtagcc cagtcactct cggtcagccg 540

gcgagtatct cttttaggtc ctcacaatct atagtgcact ctaacggcaa tacttatctt 600

gaatggtatc aacaaagacc ggggcagcca cctcgccttc tcatctacaa agtaagcaat 660

cgcttctccg gtgtccccga tcgcttctcc ggttcaggag caggaactga cttcacattg 720

aagatttcca gagtggaggc cgaagacgta ggggtatatt attgctttca agggtcccat 780

gtgccgagaa cctttggggg aggaacgaaa gttgagatta aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 170

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 170

atggatttcc aagtgcagat ttttctctttt ctcctcataa gcgcctccgt aattatgtct 60

agagctagtc aagtccaatt ggtgcaatcc ggtgccgagg ttaaaaagcc cggcgcaagt 120

gtaaaagtct cctgtaaggc cagtggctac actttcacca gctaccatat acagtgggtg 180

cggcaggcgc ctggtcaggg tctggagtgg atgggtattt ggatttatcc cggagatgga 240

agtactcaat acaatgagaa attcaagggt gtcactatga caagggatac gagcacttct 300

accgtatata tggagttgtc atctttgcga tcagaggata ccgctgtata ttattgcgca 360

cgggaaggta catattatgc catggactac tggggccaag gaaccaccgt gacggtaagc 420

tctgtcgatt ctagcggtgg cggggggctct ggcggtgggg gtagcggggg tggcggatct 480

acatcagata ttgtaatgac acagacccct ctttcacttt ccgtaacgcc aggacagccg 540

gcatcaataa gttgccgatc aagccagtct atcgtacact ccaatggtaa cacatacttg 600

gaatggtatc ttcaaaagcc cggccagagc ccgcagcttt tgatatataa agtgtccaac 660

agattcagtg gggtgccgga ccgctttagt ggatctggtt caggaacgga cttcacattg 720

aaaattagta gagttgaagc ggaagacgtg ggagtctact actgtttcca gggttcacat 780

gtgcctcgga cctttggggg aggcaccaag gttgagataa aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 171

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 171

atggacttcc aagtccaaat cttttctttt ttgttgataa gcgcatcagt tattatgtct 60

cgcgccagtc aagtacaact ggtgcagtcc ggagctgaag tgaaaaaacc aggagcaagc 120

gtgaaagtaa gttgtaaggc aagtggttac actttcacaa gctaccatat tcaatgggtc 180

cgacaggctc ctggacaggg cttggagtgg atgggcatat ggatttaccc tggtgacggg 240

tccacccagt ataatgaaaa gttcaaggga gtcacgatga ccagggacac ctctacatct 300

accgtgtata tggagctctc tagtttgcga tccgaagaca ctgccgttta ttactgtgca 360

agagaaggaa cttattacgc gatggactac tggggtcagg ggacaacagt caccgttagc 420

tccgtcgatt ccagcggggg aggtggctca ggcggggggtg gttctggggg gggcgggggc 480

acttcagata ttgtaatgac ccaaacccca ctgagtagtc cagtcacgct tggtcaaccg 540

gcaagcattt cttgcaggag ctctcagagt attgtccact ctaacgggaa tacatatttg 600

660

aggttttcag gcgtgccaga taggttctca ggctccggag cgggaaccga cttcaccctc 720

aagataagtc gggtggaagc cgaagacgta ggagtttact actgctttca aggatctcat 780

gttccacgaa cgtttgggagg aggaaccaag gtggaaataa aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 172

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 172

atggactttc aggtccaaat ttttccttc ttgctcatat ccgcgagtgt catcatgtca 60

agagcaagtc aagttcaact cgttcaatca ggagctgagg tgaaaaaacc aggggcgtct 120

gtcaaagtaa gctgcaaagc atcagggtat acgttcacga gttatcatat ccagtgggtt 180

aggcaggcgc cagggcaggg attggaatgg atgggtatct ggatttaccc gggtgacggc 240

agcactcaat acaatgagaa attcaaaggc gtaacaatga caagggacac gagcacaagc 300

acagtgtaca tggagcttag ctctttgagg tcagaggata ccgctgttta ctattgtgct 360

cgggagggta cttactatgc aatggactac tgggggcaag gcacgaccgt tacagtgagt 420

agcgtagatt cctccggggg tggcggttca ggcggcggag gctcaggcgg aggagggtca 480

acatccgatg tcgtaatgac tcagtcccct ctgtcattgc cggtgacttt gggacagcca 540

gcgtctatat cttgtaggtc ctctcaatca atagtgcatt ccaacggtaa cacctatctg 600

gaatggtatc agcaaaggcc aggacaaagt ccacgcctgc ttatatataa ggtgtctaat 660

cgattcagtg gggttcccga taggttttcc ggctctggta gcgggactga tttcacgttg 720

aaaatatcac gcgtggaagc ggaagatgtt ggggtctatt actgctttca gggtagtcat 780

gtccctcgaa cttttggcgg tggtacaaag gtagaaatca aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 173

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 173

atggattttc aggtacagat attctcattt ctccttatct cagctagtgt cataatgtcc 60

agggcgagtc aagtacaact tgtccagtca ggcgcagagg tcaagaagcc gggcgcaagc 120

gttaaggttt cctgcaaagc atccggctat acattcacgt cctatcacat ccaatgggtc 180

aggcaagcac ccggtcaagg acttgagtgg atgggcatct ggatttaccc tggagatggc 240

agtactcagt acaacgaaaa attcaaaggt gtaaccatga cccgcgacac atctacttcc 300

acagtttata tggaactcag cagtttgcgg agcgaagata ccgctgttta ctactgtgcc 360

cgagagggaa cttactacgc catggactat tggggtcaag gaacgacagt aacagttagt 420

tctgtagatt ccagtggcgg cggtgggagc gggggtgggg gatctggcgg aggcggaagt 480

acaagtgaca tcgttatgac tcagacaccc cttagtagtc ccgttacgtt gggccaaccc 540

gcgagcattt cctttcgatc ctctcagtct atagttcact caaatgggaa tacttatttg 600

gagtggtatc aacagcgccc cggacaacca ccaaggctcc tgatatacaa ggtgtccaat 660

cgattctctg gggtgcctga tagatttagc ggaagtggag ccggtacaga ttttaccctg 720

aaaatatcac gggtagaagc cgaagatgtc ggcgtctact actgtttcca gggttcccat 780

gtaccgcgaa cgttcggggg cggaacaaaa gttgagatca agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 174

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 174

atggatttcc aggttcagat atttagtttc ctcttgattt ctgccagtgt catcatgagc 60

agggcttccc aagttcagtt ggtgcaaagt ggcgctgaag tcaaaaaacc tggggcttcc 120

gttaaagtat cttgcaaggc gtccggctac actttcacat cctaccacat tcaatgggtc 180

cggcaagcgc ccggtcaggg gctcgaatgg atggggtgga tatacccagg agatggatct 240

actcagtaca acgagaaatt taaaggacgg gtgacgatga cgcgcgacac ttcaataagc 300

actgcataca tggaactgtc ccggcttagg tcagatgaca ccgcggtcta ctattgtgcg 360

agagagggta cttactatgc tatggactac tgggggcaag gcacgacggt tacagtttcc 420

tcagtcgata gttcaggcgg aggcggctcc gggggcggtg gtagtggagg gggtggatct 480

acttccgaca ttgtcatgac ccagaccccg ttgagccttt cagtgacgcc cggtcaaccc 540

gccagcataa gttgtcgatc aagccagtct attgtacact ccaatggaaa cacatatttg 600

gagtggtatc tccaaaaacc cggccaaagc cctcaactgc tcatctacaa ggtctcaaac 660

aggtttagcg gggtcccggga tcgcttctca gggtcaggat ctggtacgga ctttacactg 720

aaaatttccc gagtcgaagc ggaagacgtg ggtgtatatt actgcttcca ggggagtcat 780

gttccaagaa cctttggggg aggtacaaag gtcgaaataa aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 175

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 175

atggattttc aggtccaaat ttttccttc ttgcttatca gcgcaagtgt aatcatgtcc 60

cgcgcgtccc aagtacaact tgtgcaatct ggcgcggagg tgaaaaaacc tggagcttcc 120

gtcaaggttt cttgtaaggc ctctggctac accttcacgt cctaccacat tcagtgggtt 180

cgacaggcgc cgggccaagg actggagtgg atgggatgga tatatccagg agatggttct 240

actcagtata atgagaaatt caagggtcgc gtaacaatga cgagggatac atcaatctcc 300

accgcgtaca tggaactttc aagactccgg tcagatgaca cggcggttta ctactgtgct 360

cgggaggca cttactatgc tatggactac tgggggcaag ggacaacggt aacggtatct 420

agtgtggatt ctagtggcgg cggcggttca ggaggaggtg gttcaggcgg ggggggtagt 480

acaagtgata ttgtgatgac ccaaacaccc ctttctagcc ctgttactct gggtcaaccc 540

gcgtccataa gttgtcgaag tagtcaatcc atcgtgcata gcaacggcaa cacttacctt 600

gaatggtatc aacaacgacc cggacagccc ccgcgactgc ttatctataa agtatcaaac 660

aggttcagtg gcgtgccaga tcgattctcc ggctctgggg caggcacaga tttcacgttg 720

aaaatttctc gggtcgaggc cgaggacgtg ggcgtttatt actgtttcca ggggagtcac 780

gtccccagga cgttcgggagg aggaactaaa gtcgaaataa agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 176

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 176

atggacttcc aggtccaaat attcagcttc ctcctcattt ccgccagtgt aataatgtcc 60

agagcctcac aagtacagtt ggttcagagc ggggctgagg ttaagaaacc aggcgcgagc 120

gtcaaggtat cctgcaaggc gagtggttat actttcacta gttatcacat tcagtgggtc 180

cgacaggccc ccggtcaagg cctggagtgg atggggtgga tatatccggg agatggttct 240

acccaatata atgagaagtt taaggggaga gtcacaatga caagggacac cagtattagc 300

accgcgtata tggagctttc ccgcctgcga tcagatgaca cggccgtgta ctactgtgct 360

agggagggaa cctattatgc gatggattac tggggacagg gtactacagt cacggtctct 420

agcgtggaca gttccggggg cggtggaagc ggtggtggcg gttcaggtgg aggaggctct 480

acgagtgatg ttgtgatgac tcagtccccg ctttcacttc ccgtcaccct tgggcaaccc 540

gcaagcatct catgccgatc ctcccagtct atagtacata gtaatggcaa cacatatctt 600

gaatggtatc agcagaggcc gggtcagtct ccccgactcc ttatatataa agtgagcaac 660

agattctccg gagtaccggga tagattttcc ggctctggga gcggcaccga ctttacactg 720

aaaatttcac gggttgaagc tgaagatgtt ggggtatact attgtttcca gggttctcac 780

gtcccgagga cattcggggg aggaacgaaa gtcgaaataa agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 177

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 177

atggattttc aggtacaaat cttcagcttc ctgctcatct ccgcgagcgt aatcatgtct 60

agggcgtccc aggtgcagtt ggtgcaatca ggtgcagagg tgaagaagcc tggtgcatcc 120

gttaaagtaa gttgtaaggc aagcggatat acttttacat cctatcatat tcaatgggtc 180

agacaagcac ctggacaggg tcttgagtgg atgggctgga tctatccagg cgatggctca 240

actcaatata acgagaagtt caaggggagg gttactatga ccagggatac gtctatttcc 300

actgcgtaca tggaactctc caggttgaga agtgatgata ccgcggttta ctactgcgct 360

agagaaggaa cgtactacgc tatggattac tgggggcagg gtacaactgt caccgtctca 420

agtgtggatt cttctggggg tgggggatca ggagggggag gctccggtgg gggcgggtca 480

accagcgaca ttgtcatgac tcaaaccccc ctgagcagcc ctgtcaccct gggtcagcct 540

gcctcaatat cctttagaag ctcccaaagc atcgtccatt caaatggtaa tacctatctg 600

gagtggtatc agcaaaggcc tggtcaaccc ccgcgccttc tcatttacaa ggtgtcaaac660

aggttctccg gcgtaccggga taggttttcc ggaagcggtg ctggaaccga ctttactctc 720

aaaatctcta gggtggaagc tgaggacgtc ggtgtatact attgttttca aggctcccat 780

gttccgga catttggtgg gggaacgaag gtagaaatca agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 178

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 178

atggactttc aggttcagat tttctctttc ttgttgatct ccgctagtgt cataatgtca 60

cgggcaagtc aggtacaact cgttcagagt ggtgccgaag tgaagaaacc gggtgcctcc 120

gtaaaggtgt catgtaaagc tagtggctat acattcacaa gttatcatat ccaatgggta 180

cgacaagcac cgggacagcg actggaatgg atgggatgga tctatcctgg ggacggatct 240

acacagtaca atgagaaatt taagggacgg gtcacgataa ccagggacac atctgcttcc 300

acggcttaca tggagctttc ctccctgcgg agcgaggaca tggctgttta ctattgcgct 360

cgcgaaggga catactacgc aatggattat tggggccaag gcactaccgt gacggtctct 420

tctgtcgata gttccgggagg aggtggttca gggggaggcg gttcaggtgg gggtggatct 480

acctcagata ttgtcatgac acagacacct ttgtccttga gtgtgacacc gggtcaaccg 540

gcgagtataa gctgtcgcag ctcacaatct attgtgcata gcaacgggaa tacatatctc 600

gaatggtatc tccaaaagcc gggccaatcc ccccaacttc tcatttacaa agtttctaat 660

cgattttcag gtgtaccaga tcggttttcc gggtctggct caggtactga cttcaccttg 720

aaaatatcaa gggttgaagc tgaggatgta ggtgtgtact attgcttcca ggggtctcac 780

gttcctcgga cttttggggg gggcacaaaa gtagagatta aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 179

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 179

atggatttcc aggtgcaaat cttctcattt cttttgataa gtgcgtcagt gataatgtct 60

cgggccagtc aagtacagct tgtccaaagt ggcgctgaag tcaagaagcc gggagcctca 120

gttaaggtta gctgcaaggc ctcagggtat acttttacct cctatcatat acagtgggta 180

cgacaagcac cgggacagcg actggagtgg atgggttgga tatatccggg agatggttca 240

acccagtata atgagaagtt caaggggcga gttacgataa cccgcgatac gagtgcatca 300

acagcgtaca tggagttgag ttccctccgc agcgaggaca tggcggtata ctattgtgcc 360

agggagggga cttattatgc catggactac tgggggcagg gcacaaccgt aacagtctct 420

tctgtagaca gttcaggagg gggcggaagt ggaggtggcg gatctggtgg aggtggatct 480

acttccgaca tcgttatgac ccaaacacca ctttcatctc ccgttactct cgggcaacct 540

gctagtattt cctgtagatc ctcacaatct atagtacata gcaatggcaa tacctacctg 600

gagtggtatc aacaacgccc aggccaacca cctcgcctgc ttatctataa agtaagcaat 660

agattcagtg gtgtaccgga taggttctct ggttccggag caggaactga ctttacactc 720

aaaatcagta gggtggaggc ggaagacgtg ggagtatatt attgctttca aggttcacat 780

gtacctcgaa catttggcgg aggaactaag gttgagatta aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 180

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 180

atggatttcc aagtccagat attcagtttt cttttgataa gcgcttctgt aatcatgtct 60

cgggcgtccc aagtacaact ggtgcaatca ggggcagaag tgaaaaaacc aggtgcatcc 120

gttaaggtga gttgcaaggc ttccggctat acctttacat catatcatat tcaatgggtc 180

aggcaagcac ctggtcagcg attggaatgg atgggttgga tatatcctgg tgatgggtct 240

acacaatata acgaaaaatt caaggggcga gtgaccatca caagagatac atcagcgtca 300

acagcgtata tggaactgtc atcccttaga tcagaggaca tggcggtcta ttactgtgcc 360

agagaaggca cttattatgc aatggattat tggggacaag gaaccactgt cactgtttcc 420

agcgtagact cctccggtgg tggtggaagt ggcggcggtg ggtcaggagg gggtgggtca 480

acttctgatg tagtgatgac acagagccct ctgagcttgc ctgtgacctt gggtcagccg 540

gcctcaataa gttgtcgatc tagtcagtca atcgtccata gtaatgggaa cacatacctt 600

660

cgatttagcg gagtgccaga ccgcttttct ggttccgggt ctggcacaga ttttaccctt 720

aagatctccc gcgtggaggc ggaagacgtt ggtgtttact attgcttcca ggggtcacac 780

gttccacgca cctttgggagg aggtacgaag gtcgagatta agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 181

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 181

atggattttc aagtacagat cttctctttc ttgcttattt cagcgagcgt aatcatgagt 60

agggcatctc aagttcaact cgttcagtca ggtgctgagg taaaaaaacc aggggcttcc 120

180

cgacaagccc cggggcagcg cttggaatgg atgggctgga tttacccagg tgacggctcc 240

acgcaatata atgagaaatt taagggtaga gttactatta ccagggacac aagtgcttca 300

actgcctata tggaactgag cagccttcgg agtgaagata tggccgtata ttactgcgca 360

agggagggga cttactatgc aatggactac tggggtcagg gaacgactgt gaccgtgtcc 420

tcagttgact ccagcggtgg tggcggctct ggaggtgggg gttccggcgg aggcggaagc 480

acatctgata tagtgatgac gcaaacgcct ctttcttccc cggtaactct gggacagcca 540

gcgtcaattt catttaggtc ctcccagtca atcgtacata gtaatggaaa tacttacctg 600

gaatggtatc aacaacgacc agggcaaccg ccccgattgt tgatctataa agtgagcaat 660

cgcttttctg gcgtgcccga tcggttctca gggtctggag ctgggactga cttcacattg 720

aaaatttcca gggttgaggc cgaggatgtg ggggtttatt actgcttcca aggctcccac 780

gtcccccgca ccttcgggagg gggaaccaaa gtcgaaataa agaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 182

<211> 1599

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 182

atggattttc aagttcagat attctcattt ttgcttatat cagcctccgt aattatgtca 60

cgggcaagtc aagttcagtt ggtgcagtcc ggagcagaag ttaagaagcc cggtgcttct 120

gtgaaagtct cctgcaaagc gtctgggtac accttcacga gctaccatat acagtgggtc 180

240

acaaagtata gtcagaagtt tcaagggcga gtgacgataa ccagagatac gagtgcaagt 300

actgcataca tggaactgag ctccttgagg tccgaggata cagcggtgta ctattgcgct 360

cgggaaggga catattatgc tatggactat tggggacaag ggacaacggt aacggtgagt 420

tccgtcgatt cctcaggtgg cggaggcagt gggggcgggg gttccggcgg tggcgggtcc 480

acgagtgata tagttatgac acagaccccc ctcagccttt ctgtgacccc aggacaaccc 540

gctagtatct cttgccgcag ttctcagtcc atagtacaca gtaacggaaa tacctatctt 600

gagtggtatc ttcaaaagcc cggccagagc cctcaactct tgatatataa agtgtcaaat 660

cgattttcag gtgtgcctga tcgattctca gggtctggtt cagggacaga tttcacgctt 720

aagataagca gagtagaggc tgaagacgtg ggagtctact attgttttca ggggtcacac 780

gttccccgca cttttggtgg gggaaccaag gtggaaatca aaaaccggat ccgtggggtc 840

accgtctctt cagcgctgag caactccatc atgtacttca gccacttcgt gccggtcttc 900

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 960

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 1020

1080

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1140

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1200

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cctcgagaga 1260

1320

1380

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1440

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1500

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1560

acctacgacg cccttcacat gcaggccctg ccccctcgc 1599

<210> 183

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 183

Ser Tyr His Ile Gln

fifteen

<210> 184

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 184

Gly Tyr Thr Phe Thr Ser Tyr

fifteen

<210> 185

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 185

Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 186

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 186

Tyr Pro Gly Asp Gly Ser

fifteen

<210> 187

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 187

Glu Gly Thr Tyr Tyr Ala Met Asp Tyr

fifteen

<210> 188

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 188

Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 189

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 189

Lys Val Ser Asn Arg Phe Ser

fifteen

<210> 190

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 190

Phe Gln Gly Ser His Val Pro Arg Thr

fifteen

<210> 191

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 191

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 192

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 192

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 193

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 193

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Leu Met Thr

130 135 140

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

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

245 250

<210> 194

<211> 521

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 194

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Val Asp Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ser Asp Val Leu Met Thr

130 135 140

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

145 150 155 160

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

165 170 175

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

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Leu Glu Gln Lys Leu

245 250 255

Ile Ser Glu Glu Asp Leu Asn Arg Ile Arg Gly Val Thr Val Ser Ser

260 265 270

Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe

275 280 285

Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

290 295 300

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

305 310 315 320

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Pro Phe

325 330 335

Gly Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

340 345 350

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg

355 360 365

Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

370 375 380

Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe

385 390 395 400

Ala Ala Tyr Arg Ser Leu Glu Arg Val Arg Val Lys Phe Ser Arg Ser

405 410 415

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

420 425 430

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

435 440 445

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

450 455 460

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

465 470 475 480

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

485 490 495

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

500 505 510

Leu His Met Gln Ala Leu Pro Pro Arg

515 520

<210> 195

<211> 544

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 195

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

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

20 25 30

Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Lys Gln Arg Pro

50 55 60

Gly Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser

65 70 75 80

Thr Gln Tyr Asn Glu Lys Phe Lys Gly Lys Thr Thr Leu Thr Ala Asp

85 90 95

Lys Ser Ser Ser Thr Ala Tyr Met Leu Leu Ser Ser Leu Thr Ser Glu

100 105 110

Asp Ser Ala Ile Tyr Phe Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Val Asp Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Thr Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser

165 170 175

Leu Gly Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val

180 185 190

His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly

195 200 205

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

210 215 220

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

225 230 235 240

Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe

245 250 255

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

260 265 270

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

275 280 285

Arg Gly Val Thr Val Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe

290 295 300

Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro

305 310 315 320

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

325 330 335

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

340 345 350

Thr Arg Gly Leu Asp Pro Phe Gly Phe Trp Val Leu Val Val Val Gly

355 360 365

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

370 375 380

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

385 390 395 400

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

405 410 415

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Leu Glu Arg Val

420 425 430

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

435 440 445

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

450 455 460

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

465 470 475 480

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

485 490 495

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

500 505 510

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

515 520 525

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

530 535 540

<210> 196

<211> 1635

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 196

atggacttcc aggtgcagat cttcagcttc ctgctgatca gcgccagcgt gatcatgagc 60

cgcgctagcc aggtgcagct gcagcagagc ggccccgagc tggtgaagcc cggcgccagc 120

gtgaagatga gctgcaaggc cagcggctac accttcacca gctaccacat ccagtgggtg 180

aagcagcgcc ccggccaggg cctggagtgg atcggctgga tctaccccgg cgacggcagc 240

acccagtaca acgagaagtt caagggcaag accaccctga ccgccgacaa gagcagcagc 300

accgcctaca tgctgctgag cagcctgacc agcgaggaca gcgccatcta cttctgcgcc 360

cgcgagggca cctactacgc catggactac tggggccagg gcaccagcgt gaccgtgagc 420

agcgtcgaca gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 480

actagtgacg tgctgatgac ccagaccccc ctgagcctgc ccgtgagcct gggcgaccag 540

gtgagcatca gctgccgcag cagccagagc atcgtgcaca gcaacggcaa cacctacctg 600

gagtggtacc tgcagaagcc cggccagagc cccaagctgc tgatctacaa ggtgagcaac 660

cgcttcagcg gcgtgcccga ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 720

aagatcagcc gcgtggaggc cgaggacctg ggcgtgtact actgcttcca gggcagccac 780

gtgccccgca ccttcggcgg cggcaccaag ctggagatca agctcgagca gaagctgatc 840

agcgaggagg acctgaaccg gatccgtggg gtcaccgtct cttcagcgct gagcaactcc 900

atcatgtact tcagccactt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 960

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 1020

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctggga cccctttgggg 1080

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 1140

gccttttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 1200

aacatgactc cccgccgcc cgggcccacc cgcaagcatt accagcccta tgccccacca 1260

1320

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1380

agagaggagt acgatgtttt ggacaagaga cgtggccgggg accctgagat ggggggaaag 1440

ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1500

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1560

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1620

ctgccccctc gctaa 1635

<210> 197

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 197

ttttctagac gcgtgccacc atggccgtca tggcgcc 37

<210> 198

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 198

aagtcgacgc tagctcacac tttacaagct gtgagagaca 40

<210> 199

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 199

tcaacccgat tgtccaccat 20

<210> 200

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 200

gagtttagtc cgaaatgagg ctg 23

<210> 201

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 201

tccaaagatg tagccgcccc a 21

<210> 202

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 202

ccagtgcctc tttgctgctt tca 23

<210> 203

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 203

ctgagctcgc cagtgaaatg atg 23

<210> 204

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 204

tgctgtagtg gtggtcggag a 21

<210> 205

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 205

acctgcctta aggtggagc ca 22

<210> 206

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 206

acatgtcgca cgtctctgat ga 22

<210> 207

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 207

tgccgagc atccaaaaga 20

<210> 208

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 208

tgtattgctt tgcgttggac 20

<210> 209

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 209

aggcgctccc caagaagaca 20

<210> 210

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 210

gggctgatta gagagaggtc cct 23

<210> 211

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 211

caagacggac cagagcgaaa 20

<210> 212

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 212

ggcgggtcat gggaataac 19

<210> 213

<211> 468

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 213

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu

130 135 140

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

145 150 155 160

Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln

165 170 175

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg

180 185 190

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

195 200 205

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

210 215 220

Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr

225 230 235 240

Lys Val Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

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

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser

305 310 315 320

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

325 330 335

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

340 345 350

Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

355 360 365

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

370 375 380

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

385 390 395 400

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

405 410 415

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

420 425 430

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

435 440 445

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

450 455 460

Leu Pro Pro Arg

465

<210> 214

<211> 1470

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 214

atggctctgc ctgtgacagc tctgctgctg cctctggctc tgcttcttca tgccgccaga 60

ccatctcagg tccagctagt acaaagcggc gccgaagtaa agaaacctgg tgcctctgtg 120

aaggtgagct gcaaggccag cggctacacc ttcaccagct accacatcca gtgggttcga 180

caggcccctg gacagagact agagtggatg ggctggatct atcctggcga cggcagcacc 240

cagtacaacg agaagttcaa gggcagagtt accatcaccc gagacaccag cgccagcaca 300

360

gagggcacct actacgcaat ggattattgg ggccagggga ccaccgtgac cgtttcttct 420

ggaggcggag gttctggcgg cggaggaagt ggtggcggag gctcagatat tgtaatgacc 480

cagacacctc tgtccctgtc tgtgacacct ggacagcctg caagcatcag ctgtcggagc 540

agccagagca tcgttcacag caacggcaac acctacctgg aatggtatct gcagaagccc 600

ggacagtccc cccagctgct gatctacaag gtgtccaacc gcttcagtgg agtacccgat 660

agattttctg gcagcggctc tggcaccgac ttcaccctga agatctccag agtagaagca 720

gaggacgttg gagtgtacta ctgcttccaa ggcagccatg tgccaagaac ctttggtgga 780

ggcacaaagg tggaaatcaa gcggacaaca acacctgctc ctcggcctcc tacaccagct 840

cctacaattg ccagccagcc actgtctctg aggcccgaag cttgcaggcc tgctgctggc 900

ggagccgtgc atacaagagg actggatttc gcctgcgaca tctacatctg ggcacctctg 960

gctggaacct gtggcgtgct gctgctgagc ctggtcatca ccctgtattg ccggagcaag 1020

agaagcagac tgctgcacag cgactacatg aacatgaccc ctagacggcc cggacctacc 1080

agaaagcact accagcctta cgctcctcct agagacttcg ccgcctacag atccagagtg 1140

aagttcagca gatccgccga cgctcctgcc tatcagcagg gccaaaacca gctctacaac 1200

1260

cctgaaatgg gcggcaagcc cagacggaag aatcctcaag agggcctgta taatgagcta 1320

cagaaagaca agatggcaga ggcctacagc gagatcggaa tgaagggcga gcgcagaaga 1380

ggcaagggac acgatggact gtaccagggc ctgagcaccg ccaccaagga tacctatgat 1440

gccctgcaca tgcaggccct gcctccaaga 1470

<210> 215

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 215

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 216

<211> 42

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 216

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 217

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 217

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 218

<211> 47

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 218

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

35 40 45

<210> 219

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 219

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 220

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 220

acaacaacac ctgctcctcg gcctcctaca ccagctccta caattgccag ccagccactg 60

tctctgaggc ccgaagcttg caggcctgct gctggcggag ccgtgcatac aagaggactg 120

gatttcgcct gcgac 135

<210> 221

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 221

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

1 5 10 15

Val Ile Thr Leu Tyr Cys

twenty

<210> 222

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 222

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 223

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 223

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

twenty

<210> 224

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence

<400> 224

atctacatct gggcacctct ggctggaacc tgtggcgtgc tgctgctgag cctggtcatc 60

accctgtatt gc 72

<---

Claims (29)

1. A humanized anti-HLA-A2 antibody or antigen-binding fragment thereof, wherein said humanized anti-HLA-A2 antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 61 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 68; b) VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 68; c) VH containing the amino acid sequence of SEQ ID NO: 65 and VL containing the amino acid sequence of SEQ ID NO: 68; d) VH containing the amino acid sequence of SEQ ID NO: 66 and VL containing the amino acid sequence of SEQ ID NO: 68; e) VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 69; f) VH containing the amino acid sequence of SEQ ID NO: 65 and VL containing the amino acid sequence of SEQ ID NO: 69; g) VH containing the amino acid sequence of SEQ ID NO: 63 and VL containing the amino acid sequence of SEQ ID NO: 70. 2. A humanized anti-HLA-A2 antibody according to claim 1, wherein said heavy chain variable domain contains the amino acid sequence, SEQ ID NO: 61, and/or where said light chain variable domain contains the amino acid sequence of SEQ ID NO: 68. 3. The humanized anti-HLA-A2 antibody of claim 1, wherein said antibody is an scFv, preferably wherein said scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 81, 83, 84, 87, 88, and 91. 4. The humanized anti-HLA-A2 antibody or antigen-binding fragment thereof according to claim 3, wherein said scFv contains the amino acid sequence of SEQ ID NO: 73. 5. Chimeric antigen receptor (CAR) containing: (i) an extracellular domain containing a humanized anti-HLA-A2 antibody or antigen-binding fragment according to claim 1; (ii) a transmembrane domain; and (iii) a cytoplasmic domain including an intracellular signaling domain; wherein said CAR is capable of being expressed in an immune cell such that said CAR specifically binds to HLA-A2. 6. A CAR according to claim 5, further comprising a hinge region, preferably wherein said hinge region includes a CD8α stem region. 7. A CAR according to claim 5, wherein said transmembrane domain includes a transmembrane domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, T cell receptor alpha chain, T cell beta chain receptor, T cell receptor gamma chain, T cell receptor delta chain, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154 and any combination thereof, preferably wherein said transmembrane domain comprises a CD8 transmembrane domain. 8. CAR according to claim 5, wherein said intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of: CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, FcR gamma, FcR alpha, FcR epsilon, CD5, CD22 , CD79a, CD79b and CD66d and any combination thereof, preferably wherein said intracellular signaling domain comprises a functional signaling domain from CD3 zeta. 9. The CAR of claim 5, wherein said intracellular signaling domain further comprises a costimulatory domain, preferably wherein said costimulatory domain includes a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, antigen-1 associated with lymphocyte function ( (LFA-1) (CD11a/CD18), TNFR1 (CD120a/TNFRSF1A), TNFR2 (CD120b/TNFRSF1B), CTLA-4 (CD152), CD95, ICOS (CD278), 4-1BB (CD137), CD2, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, ICAM-1, ligand that specifically binds to CD83, IL2ra (CD25), IL6Ra (CD126), IL-7Ra (CD127), IL-13RA1, IL -13RA2, IL-33R(IL1RL1), IL-10RA, IL-10RB, IL-4R, IL-5R (CSF2RB), ARHR, BAFF receptor, IL-21R, TGFbR1, TGFbR2, TGFbR3, common gamma chain, and any combinations thereof, more preferably wherein said costimulatory domain comprises a functional signaling domain of a protein selected from CD28 and 4-1BB, and even more preferably wherein said costimulatory domain comprises a functional CD28 signaling domain. 10. The CAR of claim 5, wherein said intracellular signaling domain comprises a CD28 co-stimulatory domain and a CD3 zeta primary signaling domain. 11. A modified immune cell for promoting immune tolerance in a subject, comprising a CAR according to claim 5, preferably wherein said modified immune cell is a regulatory T cell (Treg). 12. Nucleic acid encoding a CAR according to claim 5. 13. An expression vector comprising the nucleic acid of claim 12. 14. An immune cell for promoting immune tolerance in a subject, comprising the expression vector of claim 13, preferably wherein said immune cell is a regulatory T cell (Treg). 15. A pharmaceutical composition comprising a plurality of modified immune cells according to claim 11. 16. Use of a modified immune cell according to claim 11 or a pharmaceutical composition according to claim 15 to promote immune tolerance in a subject, preferably wherein said immune tolerance is tolerance to a transplanted organ or tissue, and/or to prevent or treat graft versus host disease (GVHD) in a subject, where preferably said subject is undergoing or has undergone hematopoietic stem cell transplantation and/or to prevent or treat organ or tissue transplant rejection in the subject. 17. Use according to claim 16, wherein the subject additionally receives an immunosuppressive agent. 18. A kit for stimulating immune tolerance in a subject, the first part of which contains the modified immune cell according to claim 11 and the second part of which contains another therapeutic agent, preferably an immunosuppressive agent.

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