RU2805674C2 - New anti-hla-a2 antibodies and their application - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention discloses a novel anti-human leukocyte antigen A2 (HLA-A2) antibody and a chimeric antigen receptor (CAR) containing said anti-HLA-A2 antibody.

EFFECT: regulatory immune cells expressing the CARs of the present invention may be useful for treating or preventing transplant rejection or graft-versus-host disease (GVHD).

22 cl, 1 ex, 1 dwg

Description

Field of technology to which the invention relates

The present invention relates to molecules that bind HLA-A2, in particular to humanized antibodies against HLA-A2. The present invention also relates to recombinant molecules containing these HLA-A2 binding molecules, such as, for example, chimeric antigen receptors (CARs). Another object of the present invention is an engineered immune cell, for example a regulatory T cell, containing said CAR, and their use in therapeutic methods.

Prior Art

HLA class I antigens are polymorphic proteins expressed on all nucleated cells and are critical targets for immune recognition in the context of transplantation. In fact, the development of T cells and/or HLA class I-specific antibodies 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. Thus, finding ways to control the immune response to HLA class I proteins would be a major breakthrough in transplantation.

Classical HLA class I molecules are polymorphic and are encoded by many different alleles that have evolved in response to evolutionary pressures from infections. There are three loci that encode the classical HLA class I proteins, called loci A, B, and C. Within the HLA-A* locus, the HLA-A2 allele family is the largest and most diverse, with at least 31 different HLA- alleles. A2, which are 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. Many HLA-A2 alleles differ by only 1–9 amino acids, with most of the polymorphism concentrated around the peptide-binding groove. HLA-A2 alleles are divided into two main branches: those derived through interallelic gene conversion events from A*0201 or A*0205.

Adoptive immunotherapy with T regulatory (Treg) cells as a way to control unwanted immunity to HLA proteins and other antigens that drive graft 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. The use of Treg cell transfer to maintain c-peptide levels in type 1 diabetes has also been reported. Notably, however, it has been reported that there may be a possible transient risk of generalized immunosuppression associated with the use of polyclonal Treg cells for such cell therapy.

Data from animal studies indicate 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 disease: Treg cells isolated from pancreatic lymph nodes or activated by islet antigen are significantly better at preventing or reversing type 1 diabetes than polyclonal Treg cells. 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 a model of experimental autoimmune encephalomyelitis (EAE). Likewise, alloantigen-specific Treg cells, enriched by alloantigen-stimulated expansion in vitro or engineered to express a TCR transgene, are more effective than polyclonal Treg cells in preventing organ and tissue transplant rejection. There is some evidence that Treg cells expanded with alloantigens effectively prevent GVHD and that induction of antigen-specific Treg cells in vivo promotes acceptance of hematopoietic allografts without GVHD. Humanized mouse models have shown similar results: alloantigen-expanded human Treg cells are more potent suppressors of skin graft rejection than polyclonal Treg cells.

An alternative approach to overexpressing transgenic TCRs or antigen-stimulated expansion to enrich for 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. Once antigen is turned on, the intracellular signaling portion of the CAR can initiate an activation-related response in the immune cell.

The present invention relates to immune cells, in particular to immunoregulatory cells expressing CAR that specifically binds HLA-A2, and their therapeutic use.

The essence of the invention

The present invention relates to a humanized anti-HLA-A2 antibody having less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof, preferably from the group consisting of A25, A29, A30 and any combination thereof, compared to the BB7.2 antibody or an antibody containing the VH and VL antibodies of BB7.2, where

- the heavy chain variable region includes at least one of the following CDRs:

VH-CDR1: SYHIQ (SEQ ID NO: 1) or GYTFTSY (SEQ ID NO: 4)

VH-CDR2: WIYPGDGSTQYNEKFKG (SEQ ID NO: 2) or YPGDGS (SEQ ID NO: 5)

VH-CDR3: EGTYYAMDY (SEQ ID NO: 3)

or any CDR having an amino acid sequence that shares at least 60% identity with SEQ ID NO: 1-5, and/or

- the light chain variable region includes at least one of the following CDRs:

VL-CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 6)

VL-CDR2: KVSNRFS (SEQ ID NO: 7)

VL-CDR3: FQGSHVPRT (SEQ ID NO: 8)

or any CDR having an amino acid sequence that shares at least 60% identity with SEQ ID NO: 6-8.

In one embodiment, the heavy chain variable region includes at least one of the CDRs defined in claim 1, and the light chain variable region includes at least one of the CDRs defined in claim 1.

In one incarnation

- the heavy chain variable region includes the following CDRs: SYHIQ (SEQ ID NO: 1), WIYPGDGSTQYNEKFKG (SEQ ID NO: 2) and EGTYYAMDY (SEQ ID NO: 3); or GYTFTSY (SEQ ID NO: 4), YPGDGS (SEQ ID NO: 5) and EGTYYAMDY (SEQ ID NO: 3) or any CDRs having an amino acid sequence that shares at least 60% identity with the indicated SEQ ID NOs : 1- 5, and

- the light chain variable region includes the following CDRs: RSSQSIVHSNGNTYLE (SEQ ID NO: 6), KVSNRFS (SEQ ID NO: 7) and FQGSHVPRT (SEQ ID NO: 8) or any CDRs having an amino acid sequence that shares at least 60% identity with the indicated SEQ ID NO: 6-8.

In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9 or any amino acid sequence that has at least 60% identity with SEQ ID NO: 9.

In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 10 or any amino acid sequence that shares at least 60% identity with the specified SEQ ID NO: 10, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 is G or A.

In one embodiment, the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 or any amino acid sequence that shares at least 60% identity with the specified SEQ IDs NO: 11-13.

In one embodiment, the heavy chain variable region amino acid sequence is SEQ ID NO: 9 or any amino acid sequence that shares at least 60% identity with SEQ ID NO: 9, and the light chain variable region amino acid sequence is SEQ ID NO: 10 where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 represents L or P, X8 represents E or D, X9 represents P or R, X10 represents A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, preferably the light chain variable region amino acid sequence is selected from the group comprising SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 or any amino acid sequence that shares at least 60% identity with the specified SEQ ID NO: 10-13.

In one embodiment, said antibody is a full length antibody, single chain antibody, dimeric single chain antibody, Fv, scFv, Fab, F(ab)'2, defucosylated antibody, bispecific antibody, diabody, tribody, tetrabody, antibody fragment selected from the group consisting of a unitel, a domain antibody, and a nanobody or antibody mimetic selected from the group consisting of affitel, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, finomer, versatel, or duocalin.

In another embodiment, said antibody is a full length antibody, a single chain antibody, a dimeric single chain antibody, an Fv, a scFv, a Fab, an F(ab)'2, a defucosylated antibody, a bispecific antibody, a diabody, a tribody, a tetrabody, an antibody fragment selected from the group consisting of unitel, domain antibody, and a nanobody or antibody mimetic selected from the group consisting of affibody, alpha body, armadillo repeat protein scaffold, knottin, Kunitz domain peptide, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin , Avimer, Finomer, Versatela or Duocalin.

In one embodiment, said antibody is a scFv, preferably a scFv having the sequence SEQ ID NO: 70-72 or 74-76.

The present invention also relates to a chimeric antigen receptor (CAR) comprising:

- an extracellular domain containing the humanized anti-HLA-A2 antibody described above,

- transmembrane domain and

- cytoplasmic domain, including an intracellular signaling domain.

Another object of the present invention is the nucleic acid sequence encoding the CAR described above.

Another aspect of the invention is an immune cell comprising a CAR as described above, or a nucleic acid sequence as described above, wherein said immune cell is preferably a regulatory T cell.

The present invention further relates to a composition containing immune cells as described above, where preferably the composition is a pharmaceutical composition and further includes a pharmaceutically acceptable excipient.

The present invention also relates to an immune cell as described above for use in inducing immune tolerance in a subject in need thereof, where preferably said tolerance is tolerance to a transplanted organ or tissue.

The present invention also provides an immune cell as described above for use in the treatment of organ or tissue transplant rejection or graft-versus-host disease (GVHD).

In one embodiment, the subject additionally receives an immunosuppressive agent.

The present invention also relates to the combination of an immune cell as described herein with at least one immunosuppressive agent for the induction of immune tolerance in a subject in need thereof, or for the treatment of organ or tissue transplant rejection or graft-versus-host disease ( GVHD) at the facility that needs it.

The present invention also provides a component kit comprising primarily an immune cell as described herein and/or reagents (e.g., a nucleic acid or vector encoding an anti-HLA-A antibody or CAR of the present disclosure) for producing such immune cells and in the second part, at least one immunosuppressive agent.

The present invention also relates to a method of inducing immune tolerance in a subject in need thereof, the method comprising administering an immune cell of the present invention to the subject, wherein preferably said tolerance is tolerance to a transplanted organ or tissue.

The present invention also provides a method of treating organ or tissue transplant rejection, or graft-versus-host disease (GVHD) in a subject in need thereof, wherein the method comprises administering an immune cell described in the present invention to the subject.

In one embodiment, the method of the invention further comprises administering to the subject at least one immunosuppressive agent.

Definitions

In the present invention, the following terms have the following meanings:

In the context of the present invention, the following abbreviations for the most commonly 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.

iTerms in the singular also include the plural. For example, "element" means one element or more than one element.

The term "about", when referring to a measurable value such as magnitude, duration, etc., implies that it covers variations of ±20%, 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, since such variations are suitable for performing the disclosed methods.

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 may also be associated with detectable effector function(s), such as, for example, cytokine production or suppressive activity. The term “activated” regulatory T cells refers, among other things, to regulatory T cells that are capable of suppressing an immune response.

“Adnectins,” also known as monobodies, are well known in the art and refer to proteins designed to bind with high affinity and specificity to antigens. They belong to a class of molecules collectively called “antibody mimetics.”

As used herein, the term “alphabodies,” which may also be referred to as cell-penetrating alphabodies, refers to a type of antibody mimetics consisting of small 10-kDa proteins engineered to bind to various antigens. Alphatels are able to reach and bind to intracellular protein targets.

“Affibody” 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.

As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (Kd). The affinity can be at least 1 times greater, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, according to 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, 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 greater, or at least 1000 times greater, or greater 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 picomolar (pM), or from about 100 nM to about 1 femtomole (fM) or greater.

"Affilins" are well known in the art and refer to artificial proteins designed to selectively bind antigens. They resemble antibodies in their affinity and specificity for antigens, but not in structure, which classifies them as antibody mimetics.

The term "allogeneic" refers to any material obtained from another individual of the same species as the individual to whom the material is administered. Two or more individuals are said to be allogenic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically dissimilar enough to interact antigenically.

The terms "antibody" and "immunoglobulin" (Ig) are used interchangeably and refer to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. 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 term "antibody" also includes multispecific antibodies (eg, bispecific antibodies) and antibody fragments, provided they exhibit the desired biological activity. Antibodies can also be multimers of immunoglobulin molecules, for example, tetramers of immunoglobulin molecules.

The basic four-chain antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. L-chain from any vertebrate species can be classified into one of two clearly distinguishable types, called kappa ([kappa]) and lambda ([lambda]), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (µ), respectively. The γ and α classes are further subclassified based on relatively minor differences in SN sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each L chain is linked to an H chain by a single 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). Specific amino acid residues are believed to form the interface between the light chain and heavy chain variable domains. The pairing of VH and VL together forms one antigen-binding site. The IgM antibody is composed of five major heterotetrameric units together with an additional polypeptide called the J chain and therefore includes ten antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent communities comprising 2-5 major 4-chain units together with the J-chain. In the case of IgG, the 4-chain unit typically has a mass of about 150,000 daltons. On 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, Conn., 1994, page 71, and Chapter 6.

The term “antibody fragment” refers to at least one portion of an intact antibody, preferably an antigen binding region or variable region of an intact antibody, that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen . Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single chain antibody molecules, in particular scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragment consisting of VH domains and CHI, linear antibodies, single domain antibodies, such as sdAb (VL or VH), camel VHH domains, multispecific antibodies formed from antibody fragments, such as, for example, a divalent fragment containing two Fab fragments linked by a disulfide bridge in hinge region, and an isolated CDR or other epitope-binding fragments of the antibody. The antigen binding moiety may also be included in single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005 ). Antigen binding moieties can also be grafted into scaffolds based on polypeptides such as fibronectin type III (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). When papain cleaves antibodies, it produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to readily crystallize. The Fab fragment consists of the entire L chain together with the H chain variable domain (VH) and the first single heavy chain constant domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen-binding site. Treatment of the antibody with pepsin produces one large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments that have 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 produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical combinations of antibody fragments are also known.

As used herein, the term “antibody functional fragment or analogue” is a compound that has qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analogue of an anti-IgE antibody is a fragment that can bind to an IgE immunoglobulin in a manner that prevents or substantially reduces the ability of such a molecule to bind to the high affinity receptor, FcεRI.

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

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

“Anticalins” are well known in the art and refer to antibody mimetic technology in which the binding specificity is determined by lipocalins. Anticalins can also be formatted as dual-targeting proteins called duocalins.

The term "antigen" or "Ag" refers to a molecule that causes an immune response. This immune response may involve either the production of antibodies, the activation of specific immunologically competent cells, or both. One of ordinary skill 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 understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence encoding a protein that causes an immune response, therefore, encodes an "antigen" as that term is used herein. In addition, one skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be appreciated that 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. Moreover, one skilled in the art will appreciate that an antigen does not necessarily have to be encoded by a “gene.” It will be appreciated that the antigen may be synthesized or may be derived 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, cell, or fluid with other biological components.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (eg, a B cell, dendritic cell, and the like), that presents a foreign antigen complexed with major histocompatibility complexes (MHC) on its surface . T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

As used herein, the term “armadillo repeat protein scaffold” refers to a type of antibody mimetics corresponding to artificial peptide-binding scaffolds based on armadillo repeat proteins. Armadillo repeat proteins are characterized by an armadillo domain, consisting of tandem armadillo repeats of about 42 amino acids, which mediates interaction with peptides or proteins.

"Atrimers" are well known in the art and refer to target protein binding molecules that trimerize as a prerequisite for their biological activity. They are relatively large compared to other antibody mimetic scaffolds.

The term "autologous" refers to any material obtained from the same person to whom it is later reintroduced.

"Avimers" are well known in the art and belong to antibody mimetic technology.

“DARPins” (designed ankyrin repeat proteins) are well known in the art and refer to DRP (designed repeat protein) mimetic antibody technology designed to exploit the binding ability of non-antibody polypeptides.

As used herein, the term “BB7.2” refers to the murine hybridoma identified as ATCC deposit HB-82. BB7.2 hybridoma cells secrete a mouse monoclonal antibody of the kappa IgG2b isotype (eg, BB7.2 antibody), which was characterized by Parham P. et al. and Hilton et al. (Parham, P. et al., 1981; Hilton et al., 2013). Antibody BB7.2 is commercially available, for example, from Abcam (catalog number: ab74674), Thermo Fisher Scientific (catalog number: 17-9876-42) or Santa Cruz Biotechnology (catalog number: sc-32236). The amino acid sequences of the six complementarity determining regions (CDRs) of the BB7.2 antibody are as follows:

- Heavy chain CDR1 (HCDR1): SYHIQ (SEQ ID NO: 62);

- Heavy chain CDR2 (HCDR2): WIYPGDGSTQYNEKFKG (SEQ ID NO: 63);

- Heavy chain CDR3 (HCDR3): EGTYYAMDY (SEQ ID NO: 64);

- Light chain CDR1 (LCDR1): RSSQSIVHSNGNTYLE (SEQ ID NO: 65)

- Light chain CDR2 (LCDR2): KVSNRFS (SEQ ID NO: 66);

- Light chain CDR3 (LCDR3): FQGSHVPRT (SEQ ID NO: 67).

As used herein, the term “BB7.2 antibody” is an antibody having the VH (SEQ ID NO: 9) and VL (SEQ ID NO: 10) of the monoclonal antibody secreted by BB7.2. The BB7.2 antibody may be a full-length antibody or a fragment thereof having the VH and VL of a BB7.2-secreted monoclonal antibody, such as a scFv having a BB7.2-secreted monoclonal antibody VH and VL.

The term "binding" refers to 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, for example, binding with an affinity of 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, etc.

As used herein, the term "5' cap" (also called RNA cap, 7-methylguanosine RNA cap, or m7G RNA cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal group that is linked to the first transcribed nucleotide. Its presence is critical for ribosome recognition and protection against RNases. Cap addition is associated with transcription and occurs in concert with transcription, so that each of these processes influences the other. Shortly after the start of transcription, the 5' end of the synthesized mRNA binds to the cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions necessary for mRNA capping. Synthesis proceeds as a multi-stage biochemical reaction. The capping moiety can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

A "chimeric antibody" is an antibody molecule in which (a) the constant region (i.e., heavy and/or light chain) or part thereof is altered, substituted, or substituted such that the antigen binding site (variable region) is linked to the constant a region of a different or altered class, effector function and/or species, or with an entirely different molecule that imparts new properties to the chimeric antibody, e.g., enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or part thereof is altered, replaced, or replaced by a variable region having a different or altered antigenic specificity. Chimeric antibodies also include primatized and, in particular, humanized antibodies. In addition, chimeric antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibodies. For more information, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). (See US Pat. No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

The terms "chimeric receptor" or "chimeric antigen receptor" or "CAR" refer to a single polypeptide or set of polypeptides, usually two in the simplest embodiments, which, when found in an immune cell, confer target ligand specificity to the cell and generate an intracellular signal . In some embodiments, the set of polypeptides are adjacent to each other. In some embodiments, the chimeric receptor is a chimeric fusion protein comprising a set of polypeptides. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to each other, for example, can link 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 “conserved sequence modifications” refers to amino acid modifications that do not significantly affect or alter the biological function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the protein by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A "conservative substitution" is the replacement of an amino acid with another amino acid that has similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Therefore, amino acid substitutions are generally based on the relative similarity of the side chain amino acid substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Typical substitutions that take into account various of the above characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions can further be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been identified in the field. 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), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in the antibody or chimeric receptor of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody or chimeric receptor can be tested for binding to HLA-A2.

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

The term “costimulatory molecule” refers to a cognate 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 signaling domain may be an intracellular portion of a costimulatory molecule. The co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors.

A “cytotoxic cell” includes cytotoxic CD8+ T cells, natural killer (NK) cells, and neutrophils that are capable of mediating cytotoxic responses.

“Derived from,” as the term is used herein, indicates a bond between the first and second molecule. Generally, it refers to the structural similarity between the first molecule and the second molecule and does not imply or include a process or source limitation for the first molecule that is derived from the second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that it performs the desired function, namely the ability to generate a signal under appropriate conditions. This does not mean or limit the specific process of creating an intracellular signaling domain, for example, it does not mean that to provide an intracellular signaling domain one has to start with the CD3zeta sequence and remove the unwanted sequence or introduce mutations to achieve the intracellular signaling domain.

The term "diabodies" refers to small antibody fragments produced by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains, so that interchain but not intrachain pairing of the V domains is achieved, resulting in a divalent fragment, then there is a fragment that has two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” 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).

"Domain antibody" is well known in the art and refers to the smallest functional antibody binding units corresponding to the variable regions of the heavy or light chains of antibodies.

The term "coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as templates for the synthesis of other polymers and macromolecules in biological processes that have either a specific nucleotide sequence (e.g., rRNA, tRNA and mRNA), or a certain sequence of amino acids and the biological properties resulting from it. Thus, a gene, cDNA, or RNA encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the sequence of the mRNA and usually indicated in sequence listings, and the 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 specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "protein or RNA encoding nucleotide sequence" may also include introns to the extent that the protein encoding nucleotide sequence may, in some versions, contain intron(s).

The term "endogenous" refers to any material from or produced within an organism, cell, tissue or system.

The terms "engineered" or "modified" are used interchangeably and refer to a cell that has been transfected, transformed, or transduced.

"Evasins" are well known in the art and belong to the class of chemokine-binding proteins.

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

The term "expression" refers to the transcription and/or translation of a specific 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 contains a sufficient number of cis-acting elements for expression; other expression elements may be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, including, but not limited to, cosmids, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that include the recombinant polynucleotide.

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

"Framework" or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

"Fv" is the minimal antibody fragment that includes the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. The folding of these two domains results in the formation of six hypervariable loops (three loops each of the H and L chains) that contribute amino acid residues for antigen binding and provide the antibody with antigen-binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

“Phenomers” are well known in the art and refer to proteins that belong to the class of antibody mimetics. They are attractive binding molecules due to their high thermostability and reduced immunogenicity.

The term "graft-versus-host disease" or "GVHD" as used herein refers to a medical complication following receipt of 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 resulting from other forms of tissue graft. GVHD can also occur after a blood transfusion.

As used herein, the term “HLA-A subtype” refers to the protein encoded by an allele of the HLA-A gene. As used herein, the term “HLA-A*25:01” refers to the HLA protein designated “HLA-A*25:01” according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors. The terms "HLA-A*29:02" and "HLA-A*30:01" refer to the HLA proteins designated "HLA-A*29:02" and "HLA-A*30:01", respectively.

As used herein, the term “HLA-A2” refers to human leukocyte antigen (HLA) proteins, including cell surface proteins encoded by the HLA-A*02 family of alleles at the HLA-A* locus of the HLA complex. HLA proteins covered by the term "HLA-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 type antigen include "HLA-A2", HLA-A02" and "HLA-A*2". Various naming systems have been developed that identify the HLA proteins encoded by this family of alleles, including the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors. The term "HLA-A2" refers to HLA proteins encoded by alleles designated under 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 : eleven". Allele designations may be in italics. Allele designations beginning with "HLA-A*02:" followed by 2, 3, or 4 additional digits may constitute the entire designation or the initial part of the designation. The term "HLA-A2" also refers to HLA proteins identified with designations that begin with "HLA-A*02" according to 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 “anti-HLA-A2 antibody” refers to an antibody that preferentially or specifically binds to HLA-A2.

As used herein, the term “HLA-A*03” refers to HLA proteins, including cell surface proteins encoded by the HLA-A*03 family of alleles at the HLA-A* locus of the HLA complex. HLA proteins covered 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 the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with “HLA-A*03:”.

The terms "HLA-A*11", "HLA-A11" and "A11" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*11 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered by the terms "HLA-A*11", "HLA-A11" and "A11" include HLA proteins identified as belonging to the HLA-A*11 antigen type by serological testing or genotyping. Additional names for the HLA-A*11 antigen type include "HLA-A11". The terms "HLA-A*11", "HLA-A11" and "A11" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with "HLA-A*11:".

The terms "HLA-A*23", "HLA-A23" and "A23" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*23 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered by the terms "HLA-A*23", "HLA-A23" and "A23" include HLA proteins identified as belonging to the HLA-A*23 antigen type by serological testing or genotyping. Additional names for the HLA-A*23 antigen type include "HLA-A23". The terms "HLA-A*23", "HLA-A23" and "A23" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*23:".

The terms "HLA-A*25", "HLA-A25" and "A25" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*25 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered 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 HLA-A*25 antigen type include "HLA-A25". The terms "HLA-A*25", "HLA-A25" and "A25" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*25:".

The terms "HLA-A*26", "HLA-A26" and "A26" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*26 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered by the terms "HLA-A*26", "HLA-A26" and "A26" include HLA proteins identified as belonging to the HLA-A*26 antigen type by serological testing or genotyping. Additional names for the HLA-A*26 antigen type include "HLA-A26". The terms "HLA-A*26", "HLA-A26" and "A26" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*26:".

The terms "HLA-A*29", "HLA-A29" and "A29" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*29 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered 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 terms "HLA-A*29", "HLA-A29" and "A29" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*29:".

The terms "HLA-A*30", "HLA-A30" and "A30" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*30 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered 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 HLA-A*30 antigen type include "HLA-A30". The terms "HLA-A*30", "HLA-A30" and "A30" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*30:".

The terms "HLA-A*31", "HLA-A31" and "A31" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*31 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered 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 terms "HLA-A*31", "HLA-A31" and "A31" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*31:".

The terms "HLA-A*33", "HLA-A33" and "A33" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*33 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered 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 HLA-A*33 antigen type include "HLA-A33". The terms "HLA-A*33", "HLA-A33" and "A33" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*33:".

The terms "HLA-A*34", "HLA-A34" and "A34" as used herein refer to the HLA proteins, including the cell surface proteins encoded by the HLA-A*34 family of alleles in the HLA-A locus of the HLA gene complex . The HLA proteins covered by the terms "HLA-A*34", "HLA-A34" and "A34" include HLA proteins identified as belonging to the HLA-A*34 antigen type by serological testing or genotyping. Additional names for the HLA-A*34 antigen type include "HLA-A34". The terms "HLA-A*34", "HLA-A34" and "A34" refer to HLA proteins encoded by alleles designated according to the HLA naming system developed in 2010 by the WHO Committee on HLA System Factors, which begin with " HLA-A*34:".

"Humanized" forms of non-human (eg, 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. In general, humanized antibodies and antibody fragments thereof 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 the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and potential. In some cases, human immunoglobulin Fv framework region (FR) residues are replaced by corresponding non-human residues. In addition, the humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Such antibodies are designed to retain the binding specificity of the non-human antibody from which the binding regions are derived and to prevent an immune reaction against the non-human antibody. These modifications can further improve and optimize the characteristics of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are human immunoglobulin sequences. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. 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.

“Fully human” refers to an immunoglobulin, such as, for example, an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin. Such antibodies can be obtained from transgenic mice or other animals that have been engineered to produce specific human antibodies in response to antigen (see, for example, Green et al., (1994) Nature Genet 7:13; Lonberg et al. , (1994) Nature 368: 856; Taylor et al., (1994) Int Immun 6: 579, all descriptions of which are incorporated herein by reference). A fully human antibody can also be constructed by genetic or chromosomal transfection techniques, as well as phage display technology, all of which are known in the art (see, for example, McCafferty et al., (1990) Nature 348: 552-553). Human antibodies can also be generated by in vitro activated B cells (see, for example, US Pat. Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entirety).

The term “hypervariable region” as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically includes amino acid residues from the “complementarity determining region” or “CDR” (e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and about about 31- 35 (H1), 50-65 (H2) and 95-102 (H3) in VH when numbered according to the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or residues from the “hypervariable loop” (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in VL and 26-32 (H1), 52-56) (H2) and 95-15 101 (H3) in VH when numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); and/or residues from the hypervariable loop/CDR (eg, residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in VL and 27-38 (H1), 56). -65 (H2) and 105-120 (H3) in VH when numbered according to the IMGT numbering system; Lefranc, M. P. et al., Nucl. Acids Res. 27: 209-212 (1999), Ruiz, M. et al., Nucl. Acids Res. 28: 219-221 (2000)). Optionally, the antibody has symmetrical insertions at one or more of the following sites 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in VL and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in VH when numbered according to AHo (Honneger, A. and Plunkthun, AJ Mol. Biol. 309: 657-670 (2001)).

The term "identity" or "homology", when used in a relationship between the sequences of two or more polypeptides or nucleic acid sequences, refers to the degree of sequence relatedness between polypeptides or nucleic acid sequences, determined by the number of matches between strings of two or more amino acid residues or nucleotide residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with the alignment of gaps (if any) resolved by a particular mathematical model or computer program (i.e., "algorithms"). When the subunit position in both of two molecules is occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by an 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 ten-subunit-long polymer) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g. 9 out of 10) are the same or homologous, the two sequences are 90% homologous. The identity of related polypeptides or nucleic acid sequences can be easily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New 10 Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to provide the best match between the tested sequences. Methods for determining identity are described in publicly available computer programs. Preferred computer software methods for determining identity between two sequences include the GCG software package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.) , BLASTP, BLASTN and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith-Waterman algorithm can also be used to determine identity.

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. Examples of immune cells include, but are not limited to, lymphocytes (T cells, B cells, and natural killer (NK) cells) and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

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 T cell-mediated and/or B cell immune responses. 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 influenced by T cell activation, such as the production of antibodies (humoral responses) and the activation of cytokine-sensitive 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.

As used herein, the term “immune accommodation” refers to a condition in a transplant recipient in which the organ or tissue graft functions normally despite the recipient having antibodies specific to the organ or tissue graft.

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 a specific immunological response (which is considered to be mediated at least in part by an antigen -specific effector T-lymphocytes, B-lymphocytes, antibodies or their equivalents); b) delay in the onset or progression of a specific immunological response; or c) reduced risk of the onset or progression of a specific immunological response. "Specific" immunological or immune tolerance occurs when immunological or immune tolerance is induced preferentially against certain antigens over others.

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

As used herein, “instructions” include publication, writing, diagram, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructions of a kit of the invention may, for example, be attached to a container that includes the antibody, cell, and/or composition of the invention, or supplied with a container that includes the antibody, cell, and/or composition of the invention. Alternatively, the instructions may be delivered separately from the container for the purpose of sharing the teaching material and the antibody, cell and/or composition by the recipient.

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 may be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof.

"Intracellular signaling domain", as used herein, refers to the intracellular portion of the molecule. The intracellular signaling domain generates a signal that promotes the immune function of the cell containing the chimeric receptor. Examples of immune function in a chimeric receptor T cell may include cytolytic activity, suppressive activity, and helper activity, including cytokine secretion.

As used herein, the term “in vitro transcribed RNA” refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, in vitro transcribed RNA is generated from an in vitro transcription vector. An in vitro transcription vector includes a template that is used to generate transcribed RNA in vitro.

The term "isolated" means altered or removed from its 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 its natural state, is isolated. The isolated nucleic acid or peptide may exist in substantially purified form or may exist in a non-native environment, such as, for example, a host cell. Typically, an isolated nucleic acid or peptide preparation includes a nucleic acid or peptide with a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, more 95% pure, greater than 96% pure, greater than 97% pure, greater than 98% pure, or greater than 99% pure.

An "isolated antibody" is an antibody that has been separated and/or extracted from a component of its natural environment. Contaminants in its natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein components. In preferred embodiments, the antibody is purified: (1) to greater than 95 wt.% antibody, as determined by the Lowry method, and most preferably greater than 99 wt.%; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating beaker sequencer; or (3) to homogeneity, as shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue staining or, preferably, silver staining. The isolated antibody includes the antibody in situ in the recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody will be obtained by at least one purification step.

An "isolated nucleic acid" or "isolated nucleotide sequence" is a nucleic acid that is substantially separated from other genomic DNA sequences and from proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. The term covers a nucleic acid sequence that has been removed from its natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. Substantially pure nucleic acid includes isolated forms of nucleic acid. Of course, this refers to the nucleic acid that was originally isolated and does not exclude genes or sequences that are subsequently added to the isolated nucleic acid by human hands.

An "isolated polypeptide" is one that has been identified and separated and/or extracted from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to contain greater than 95 wt% polypeptide as determined by the Lowry method, and most preferably greater than 99 wt% (2) to an extent sufficient to obtain at least 15 N- residues terminal or internal amino acid sequence using a spin-beaker sequencer or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. The isolated polypeptide includes the polypeptide in situ in recombinant cells, since at least one component of the polypeptide's natural environment will not be present. Typically, however, the isolated polypeptide will be obtained through at least one purification step.

As used herein, 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.

As used herein, the term “Kunitz domain peptide” refers to a type of antibody mimetics and is based on the active domains of proteins that inhibit protease function.

The term "lentivirus" refers to a genus in the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect nondividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most efficient methods for a gene delivery vector. Examples of lentiviruses include, but are not limited to, HIV, SIV and FIV.

The term “lentiviral vector” refers to a vector derived from at least a portion of a lentiviral genome, including in particular the self-inactivating lentiviral vector provided in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, 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 one of ordinary skill in the art.

The term "ligand" refers to a member of a ligand/receptor pair and binds to another member of the pair.

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

The term "nucleic acid" or "polynucleotide" refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the 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 conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the explicitly stated 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)).

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

A "native sequence" polynucleotide is one that has the same nucleotide sequence as a naturally occurring polynucleotide. A "native sequence" polypeptide is a polypeptide with the same amino acid sequence as a polypeptide (eg, antibody) obtained from nature (eg, any species). Such native sequence polynucleotides and polypeptides may be isolated from nature or may be produced by recombinant or synthetic methods.

The terms "operably linked" or "transcriptional control" refer to the functional relationship between a regulatory sequence and a heterologous nucleic acid sequence leading to 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 related DNA sequences can be adjacent to each other and, for example, optionally connect two protein-coding regions that are in the same reading frame.

As used herein, the term “functional tolerance” refers to the clinical situation in which there is stable graft function in which there is no 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.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. The polypeptide is not limited to a specific length: it must contain at least two amino acids, and no restrictions are placed on the maximum number of amino acids that the polypeptide sequence may contain. Peptides, oligopeptides and proteins are included in the definition of polypeptide, and such terms may be used interchangeably herein unless specifically stated otherwise. 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 also commonly referred to in the art as proteins, of which there are many types. In one embodiment, as used herein, the term “peptides” refers to a linear polymer of amino acids linked to each other by peptide bonds, preferably with a chain length of less than about 50 amino acid residues; "polypeptide" refers to a linear polymer of at least 50 amino acids linked together by peptide bonds; and protein specifically refers to a functional entity formed from one or more peptides or polypeptides, optionally glycosylated and, optionally, non-polypeptide cofactors. The term also does not refer to or exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The polypeptide can be a whole protein or a subsequence thereof. "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 naturally occurring peptide, a recombinant peptide, or a combination thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences containing CDRs and capable of binding antigen.

The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that does not cause adverse, allergic or other adverse reactions when administered to an animal, preferably a human. The term includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents, and the like. For administration to humans, drugs must meet standards of sterility, pyrogenicity, general safety and purity as required by regulatory authorities such as the FDA or EMA.

As used herein, the term “poly(A)” is a series of adenosine monophosphate attached to mRNA. In a preferred embodiment of the transient expression construct, the polyA is from 50 to 5000 adenosine monophosphates, preferably greater than or equal to 64, more preferably greater than or equal to 100, most preferably greater than or equal to 300 or 400 adenosine monophosphates. Poly(A) sequences can be modified chemically or enzymatically to modulate the functionality of the mRNA, such as localization, stability, or translation efficiency.

As used herein, the term “polyadenylation” refers to the covalent bonding of a polyadenylyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3'-poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to pre-mRNA by the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and its associated protein help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has ceased, the mRNA strand is cleaved by an endonuclease complex associated with RNA polymerase. A cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence (such as, for example, a DNA sequence) recognized by a cell's synthetic machinery, or introduced by a synthetic machinery, necessary to initiate specific transcription of a polynucleotide sequence, thereby allowing expression of a gene product operably linked to promoter/regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. A promoter/regulatory sequence may, for example, be a sequence that expresses a gene product in a tissue-specific manner.

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). A first antibody is "less reactive" to a molecule (eg, an HLA molecule) than a second antibody when the first antibody exhibits reduced binding to the molecule compared to the second antibody. Approaches are known to quickly compare the reactivity of first and second antibodies to 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 the ability to react (or bind) to specific HLA molecules. Such flow cytometric approaches are amenable to high-throughput analysis of antibody reactivity.

The term “recombinant protein or peptide” refers to a protein or peptide (eg, antibody or CAR) that is produced using recombinant DNA technology, such as, for example, a protein or peptide (eg, antibody or CAR) expressed by a bacteriophage or yeast expression system. The term should also be understood to mean a protein or peptide (e.g., antibody or CAR) that has been produced by the synthesis of a DNA molecule encoding a protein or peptide (e.g., antibody or CAR), and where the DNA molecule expresses the protein or peptide (e.g., antibody or CAR) or amino acid sequence causing a protein or peptide (eg, antibody or CAR), where the DNA sequence or amino acid sequence was obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

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 thereby maintain immune system homeostasis and tolerance to self-antigens. “Regulatory immune cells” can also influence non-immune cells, leading to clinical improvements such as tissue repair or regeneration. Regulatory immune cells may include, but are not limited to, 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.

“Regulatory T lymphocyte,” “regulatory T cell,” “T regulatory cell,” “Treg cell,” or “Treg,” as used herein, are synonymous and have the standard definition used in the art. 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 called suppressor T cells. Treg cells are often, but not always, characterized by the expression of the forkhead family transcription factor, Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They may also express CD25. As used herein, and unless otherwise noted, Tregs include “natural” Tregs that develop in the thymus, induced/adaptive/peripheral Tregs that arise 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, which were created using recombinant DNA technology. 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 Tregs is by targeting T effector cells with IL-10 or TGF-β. T cells can also be converted into Treg cells by transfecting or transducing the Foxp3 gene into a mixed population of T cells. A T cell that induces Foxp3 expression adopts a Treg phenotype, and such recombinant Tregs are also defined herein as “Tregs”.

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 recipient's immune system attacking the transplanted organ or tissue. Rejection can occur from a few days to a few weeks after transplantation (acute) or from several months to several years after transplantation (chronic).

The term "single chain Fv" or "scFv" refers to a fusion protein containing at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH ), where the light and heavy chain variable regions are continuously linked into one polypeptide chain. In one embodiment, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains that allows the scFv to form a desired structure for antigen binding, for example, a synthetic linker, such as a short flexible polypeptide linker. Thus, scFv is able to be expressed as a single chain polypeptide and retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv used herein may have the VL and VH variable regions in any order, for example, 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 .

As used herein, an antibody or CAR is said to be “immunospecific,” “specific for,” or “specifically binds” an antigen if it reacts at a detectable level with an antigen, preferably with an affinity constant Ka greater than or equal to about 10 ⁴ M ^-1 or greater than or equal to about 10 ⁵ M ^-1 , greater than or equal to about 10 ⁶ M ^-1 , greater than or equal to about 10 ⁷ M ^-1 , or greater than or equal to about 10 ⁸ M ^-1 , or greater than or equal to about 10 ⁹ M ^-1 , or greater or equal to about 10 ¹⁰ M ^-1 . The affinity of an antibody for its cognate antigen is also typically expressed as a dissociation constant, Kd, and in some embodiments, an antibody binds specifically to an antigen if it binds to a Kd of less than or equal to 10 ^-4 M, less than or equal to about 10 ^-5 M, less than or equal to about 10 ^-6 M, less than or equal to about 10 ^-7 M, or less than or equal to about 10 ^-8 M, or less than or equal to about 5.10 ^-9 M, or less than or equal to about 10 ^-9 M, or less or equal to about 5.10 ^-10 M, or less than or equal to about 10 ^-10 M. The affinity of antibodies or CARs can be easily determined using conventional techniques, such as those described by Scatchard et al., (Ann. NY Acad. Sci. USA 51:660 (1949)). The binding properties of an antibody to antigens, cells or tissues thereof can generally be determined and assessed using immunodetection methods, including, for example, immunofluorescence-based assays such as immunohistochemistry (IHC) and/or fluorescence-activated cell sorting (FACS) . In one embodiment, the term “specifically binds” refers to an antibody, CAR or ligand that recognizes and binds to a binding partner present in a sample, but where the antibody or CAR or ligand does not substantially recognize or bind other molecules in the sample.

The term "signal transduction pathway" refers to the biochemical relationships between various signal transduction molecules that play a role in transmitting a signal from one part of the cell to another part of the cell.

The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through specific signaling pathways, generating second messengers or functioning as effectors that respond to such messengers.

As used herein, the term “stem cell” generally includes pluripotent or multipotent stem cells. “Stem cells” include, but are not limited to, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow-derived cells, etc.).

The term “stimulation” refers to the primary response induced by the binding of a stimulatory molecule (eg, a TCR/CD3 complex or a chimeric receptor) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signaling through the TCR/CD3 complex or signaling through chimeric receptor signaling domains. Stimulation can mediate altered expression of certain molecules.

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

The term "subject" is intended to include living organisms in which an immune response can be elicited. In one embodiment, the subject is a warm-blooded animal, preferably a mammal (including humans, pets, farm and zoo animals, sporting or domestic animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.). etc.), and more preferably - people. In one embodiment, the subject may be a "patient", that is, a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care, or has been/is/will be the subject of a medical procedure or is being monitored for the development of a target disease or condition. In one embodiment, the subject is an adult (eg, a subject over 18 years of age). In another embodiment, the subject is a child (eg, a subject under 18 years of age). In one embodiment, the subject is male. In another embodiment the object is a woman.

The term "substantially purified cell" refers to a cell that is substantially free of other cell types. An essentially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In one embodiment, a substantially purified cell refers to a cell that is at least about 75% free, 80% free, or 85% free, and preferably about 90%, 95%, 96%, 97%, 98 % or 99.% free from other cell types 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 one embodiment, a population of substantially purified cells refers to a population of cells that is at least about 75% homogeneous, 80% homogeneous, or 85% homogeneous, and preferably about 90%, 95%, 96%, 97%, 98 %, or 99% homogeneous. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated 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 "T cells" includes all types of immune cells that express CD3, including T helper cells (CD4+ cells), CD8+ T cells (eg, cytotoxic CD8+ T cells, regulatory CD8+ T cells), T regulatory cells ( Tregs), gamma delta T cells and double negative T cells.

The terms “therapeutically effective amount” refer to the amount of immune cells or composition, as described herein, effective to achieve a particular biological result. Thus, the terms “effective amount” or “therapeutically effective amount” mean the level or number of cells or compositions that are targeted, without causing significant negative or adverse side effects, to (1) delay or prevent the onset of the target disease or condition; (2) slowing or stopping the progression, exacerbation or worsening of one or more symptoms of the target disease or condition; (3) causing improvement in symptoms of the target disease or condition; (4) reducing the severity or incidence of the target disease or condition; or (5) curing the target disease or condition. A therapeutically effective amount may be administered prior to the onset of the target disease or condition for prophylactic or prophylactic effect. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of the target disease or condition for therapeutic effect.

The term "transfected" or "transformed" or "transduced" refers to the process by which an 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 an object's primary cell and its offspring.

The term "transfer vector" refers to a composition of material that includes an isolated nucleic acid, and which can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, ionic or amphiphilic linked polynucleotides, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

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 period of time for expression is less than the period of time for expression of the gene if it is integrated into the genome or contained in a stable plasmid replicon in the host cell .

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

“Treat” or “treatment” refers to both therapeutic treatment and prophylactic or preventive measures; where the goal is to prevent or slow down (reduce) the target pathological condition or disorder. Thus, in one embodiment, these terms refer to the reduction or attenuation of the progression, severity and/or duration of the target disease or condition, or the reduction of one or more symptoms (preferably one or more noticeable symptoms) of the target disease or condition resulting from the administration of one or multiple therapies (eg, one or more therapeutic agents, such as Treg cells of the invention). In specific embodiments, the terms “treat” and “treating” refer to improving at least one measurable physical parameter of the target disease or condition. In other embodiments, the terms “treat” and “treating” refer to inhibiting the progression of a target disease or condition either physically, such as by stabilizing a discernible symptom, or physiologically, such as by stabilizing a physical parameter, or both. Those in need of treatment include those who already have the disorder, as well as those who are likely to have the disorder or those whose disorder is to be prevented. A subject is successfully "treated" if, after receiving a therapeutic amount of cells or compositions according to the present invention, the subject exhibits an observable and/or measurable reduction in the number of pathogenic cells; reducing the percentage of the total number of cells that are pathogenic; relief to some extent of one or more symptoms associated with a specific disease or condition; reduction in morbidity and mortality and/or improvement in quality of life. The above parameters for assessing successful treatment and improvement of the disease can be easily measured using routine procedures familiar to the doctor.

“Unitelo” is well known in the art and refers to an antibody fragment that lacks 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 rather than a divalent binding region of IgG4 antibodies.

The term "variable" refers to the fact that certain segments of the V domains vary widely 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 not evenly distributed over the range from 110 to 130 amino acids of the variable domains. Instead, 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 9–12 amino acids long. Each of the variable domains of the native heavy and light chains includes four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions that form loops connecting and in some cases forming part of the β-sheet structure. Hypervariable regions on each chain are held together in close proximity to the FR and, together with hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the antibody's involvement in antibody-dependent cellular cytotoxicity (ADCC).

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy chain variable domain may be referred to as "VH" or " _VH ". The light chain variable domain may be referred to as "VL" or " _VL ". These domains are typically the most variable portions of the antibody and include antigen binding sites.

A polynucleotide "variant" as used herein is a polynucleotide that generally differs from the polynucleotide specifically disclosed herein by one or more substitutions, deletions, additions and/or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more polynucleotide sequences of the invention and assessing one or more biological activities of the encoded polypeptide, as described herein, and/or using any of a number of methods, well known in the field. A "variant" polypeptide, as used herein, is a polypeptide that generally differs from the polypeptide specifically disclosed herein by one or more substitutions, deletions, additions and/or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences and assessing one or more biological activities of the polypeptide as described herein and/or using any of a number of methods well known in the art. this area. Modifications can be made to the structure of the polynucleotides and polypeptides of the present invention and still provide a functional molecule that encodes a variant or derivative of the polypeptide with the desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent or even improved variant or portion of the polypeptide of the invention, one of ordinary skill in the art will typically change one or more codons of the DNA coding sequence. For example, some amino acids can be replaced by other amino acids in the structure of a protein without noticeably losing its ability to bind other polypeptides (eg, antigens) or cells. Since it was the binding ability and nature of the protein that determined its biological functional activity, certain substitutions in the amino acid sequence and, of course, in its implied DNA coding sequence could be made and still produce a protein with similar properties. Thus, it is contemplated that various changes may be made to the peptide sequences of the present invention or to the corresponding DNA sequences that encode the peptides without appreciable loss of their biological utility or activity. In many cases, a polypeptide variant will contain one or more conservative substitutions. The variant may also contain, or alternatively contain, non-conservative changes. In a preferred embodiment, the variant polypeptides differ from the native sequence by substitution, deletion or addition of five amino acids or less. Variants can also be (or alternatively) modified, for example, by deletion or addition of amino acids that have minimal impact on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

Versatela is well known in the art and belongs to another antibody mimetic technology. They are small 3-5 kDa proteins with >15% cysteines that form a high disulfide density scaffold replacing the hydrophobic core that typical proteins have.

The term "xenogeneic" refers to any material obtained from an individual of another species. The term "xenograft" refers to a graft obtained from an individual of another species.

The term "zeta" or alternatively "zeta chain", "CD3 zeta" or "TCR zeta" is defined as a protein reported as GenBank Acc. No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and “zeta stimulating domain” or, alternatively, “CD3 zeta stimulating domain” or “ TCR zeta stimulatory domain" is defined as amino acid residues of the cytoplasmic zeta chain domain or functional derivatives thereof that are sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the cytoplasmic zeta domain includes residues 52 to 164 from GenBank Acc. No. BAG36664.1 or equivalent residues from a non-human species, eg mouse, rodent, monkey, ape, etc., which are functional orthologues thereof.

Detailed description

The first object of the present invention is an antibody directed against HLA-A2, preferably a humanized antibody directed against HLA-A2.

In one embodiment, 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: 62; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 63; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 64; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 65; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 66; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 67.

In one embodiment, an antibody of the invention binds 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: 62; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence SEQ ID NO: 63; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 64; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 65; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 66; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 67.

In one embodiment, an anti-HLA-A2 antibody of the invention competes for binding to HLA-A2 and/or binds to the same HLA-A2 epitope as the BB7.2 antibody.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof, according to compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03:01, A11:01, A23:01, A25:01, A26:01, A29:02, A30 : 01, A31: 01, A33: 01, A34: 01 and any combination thereof, compared to the BB7.2 antibody or compared to an antibody containing the VH and VL of the BB7.2 antibodies, preferably compared to the BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from three or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or over an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from three or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from four or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or over an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from four or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from five or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from five or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from six or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or over an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from six or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from seven or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from seven or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from eight or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 it compared to the BB7.2 antibody or compared to an antibody containing the VH and VL of the BB7.2 antibodies, preferably compared to the BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from eight or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from nine or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 over BB7.2 antibody or over an antibody containing VH and VL of BB7.2 antibodies, preferably over BB7.2 sc7v. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from nine or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to BB7.2 scFv.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity against each of the HLA-A subtypes selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33 and A34 compared to BB7.2 antibody or compared to an antibody containing VH and VL BB7.2 antibodies, preferably compared to BB7.2 scFv. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity for each of the HLA-A subtypes selected from the group consisting of A03:01, A11:01, A23:01, A25:01, A26:01, A29:02 , A30:01, A31:01, A33:01 and A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL of the BB7.2 antibodies, preferably compared to the BB7.2 scFv.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to the BB7.2 antibody or compared to the BB7.2 antibody . an antibody containing VH and VL antibodies BB7.2 is preferred over sc7v BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25:01, A29:02, A30:01, and any combination thereof, compared to the BB7 antibody. 2 or compared to an antibody containing VH and VL antibodies BB7.2, preferably compared to sc7v BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A25, A29 and A30 compared to the BB7.2 antibody or compared to the antibody containing VH and VL antibodies BB7.2, preferably over sc7v BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A25:01, A29:02, and A30:01, compared to the BB7.2 antibody or compared to an antibody containing VH and VL antibodies BB7.2, preferably compared to sc7v BB7.2.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A25, A29 and A30, compared to the BB7.2 antibody or compared to the antibody containing VH and VL antibodies BB7.2, preferably over sc7v BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A25:01, A29:02 and A30:01, compared to the BB7 antibody. 2. or compared to an antibody containing VH and VL antibodies BB7.2, preferably compared to sc7v BB7.2.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity for each of the HLA-A subtypes selected from the group consisting of A25, A29 and A30 compared to the BB7.2 antibody or compared to an antibody containing VH and VL BB7.2 antibodies, preferably over sc7v BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity for each of the HLA-A subtypes selected from the group consisting of A25:01, A29:02 and A30:01 compared to the BB7.2 antibody or both with an antibody containing VH and VL antibodies BB7.2, preferably over sc7v BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A25 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A29 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A30 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A3 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A11 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A26 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A31 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A32 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A33 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A34 compared to a BB7.2 antibody or compared to an antibody comprising the VH and VL antibodies of BB7.2, preferably compared to a BB7.2 scFv.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A25:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A29:02 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A30:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A03:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A11:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A26:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A31:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A33:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv . In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A34:01 compared to the BB7.2 antibody or compared to an antibody containing the VH and VL antibodies of BB7.2, preferably compared to the BB7.2 scFv .

Methods for measuring HLA-A2 antibody reactivity to HLA-A* subtypes are known to one of ordinary skill in the art and include, but are not limited to, single antigen assays such as, for example, the FlowPRA Single Antigen Antibody available from ONE LAMBDA.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof compared with the BB7.2 antibody or with an antibody containing the VH and VL antibodies of BB7.2, when measured under test conditions A.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to the BB7.2 antibody or an antibody containing VH and VL BB7.2 antibodies when measured under Test A conditions.

Test A:

0.25.10 ⁶ T cells expressing CAR containing anti-HLA-A2 antibody or BB7.2 antibody, or BB7.2 VH and VL antibodies (mA2 CAR)) were incubated with a panel of FlowPRA single antigen antibody beads (FL1HD01 , FL1HD02, FL1HD03, FL1HD04, FL1HD06 and FL1HD08, One Lambda) and fixable viability dye (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 using a fixable dye to assess viability. Single antigen beads were then selected for signal above a threshold after excluding dead cells and doublets. The number of beads per HLA was then determined from their corresponding 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 CAR-Treg binding compared to the control (cells not expressing CAR) was determined by subtracting the number of beads in the CAR-Treg from the number of beads in the control sample, then dividing by the average number of beads in the non-CAR-expressing control. and multiplying by 100.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof compared with the BB7.2 antibody or compared with an antibody containing the VH and VL of BB7.2 when measured under test B conditions.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to the BB7.2 antibody or compared to the BB7.2 antibody . antibody containing VH and VL of BB7.2 as measured under test B conditions.

Test B:

0.25.10 ⁶ T cells expressing anti-HLA-A2 antibody or BB7.2 antibody, or BB7.2 VH and VL antibodies or a truncated NGFR transduction marker (NGFR) on the cell surface (such as, for example, as part of a chimeric antigen receptor described herein) were incubated with FlowPRA single antigen antibody beads (FL1HD, One Lambda) and viability fixative dye (FVD, 65-0865-14, eBioscience), then fixed with 0.5% formaldehyde and analyzed using flow cytometry. For analysis, dead cells were first removed using a fixable dye to assess viability. Single antigen beads were then selected for signal above a threshold after excluding dead cells and doublets. The number of beads per HLA was then determined by their corresponding PE intensity. Data were normalized by dividing the number of negative beads in the sample by the number of negative beads in the Treg-NGFR sample multiplied by the number of negative beads in the Treg-NGFR sample. Relative percentage 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, and multiplied by 100.

In one embodiment, an anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, statistically inferior to the reactivity of the BB7.2 antibody or statistically inferior to the reactivity of an antibody containing the VH and VL of the BB7.2 antibody, preferably when measured under Test A or Test B 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 A25, A29, A30, statistically inferior to the reactivity of the BB7.2 antibody, or statistically inferior to the reactivity of the antibody , containing VH and VL antibodies BB7.2, preferably when measured under Test A or Test B conditions.

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

In one embodiment, an anti-HLA-A2 antibody of the invention is reactive to at least one HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, inferior to the reactivity of the BB7.2 antibody or an antibody containing VH and VL of the BB7.2 antibody, preferably the anti-HLA-A2 antibody of the invention has relative binding to at least one HLA-A* selected from the group consisting of A03, A11 , A23, A25, A26, A29, A30, A31, A33, A34, inferior to that of the BB7.2 antibody or an antibody containing the VH and VL of the BB7.2 antibody when measured under the conditions of test A or test B, more preferably the relative binding measured for the anti-HLA-A2 antibody of the invention, is at most about 90%, 80%, 70%, 60%, 50%. 40%, 30%, 20%, 10% or less relative binding measured for the BB7.2 antibody or for an antibody containing the VH and VL of the BB7.2 antibody.

In one embodiment, an anti-HLA-A2 antibody of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of A25, A29, A30, inferior to the reactivity of the BB7.2 antibody, or the reactivity of an antibody containing VH and The VL antibody BB7.2, preferably the anti-HLA-A2 antibody of the invention has relative binding to at least one HLA-A subtype selected from the group consisting of A25, A29, A30 inferior to the relative binding of the BB7.2 antibody or antibody, containing VH and VL antibodies BB7.2 when measured under test A or test B conditions, more preferably the relative binding measured for the anti-HLA-A2 antibody of the invention 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 or for an antibody containing the VH and VL of the BB7.2 antibody.

In an embodiment, said antibody is a monoclonal antibody.

In another embodiment, said antibody is a polyclonal antibody.

In one embodiment, the heavy chain variable region includes at least one of the following CDRs:

VH-CDR1: SYHIQ (SEQ ID NO: 1)

VH-CDR2: WIYPGDGSTQYNEKFKG (SEQ ID NO: 2)

VH-CDR3: EGTYYAMDY (SEQ ID NO: 3)

The numbering and definition of the CDR corresponds to the Kabat definition.

In one embodiment, the heavy chain variable region includes at least one of the following CDRs:

VH-CDR1: GYTFTSY (SEQ ID NO: 4)

VH-CDR2: YPGDGS (SEQ ID NO: 5)

VH-CDR3: EGTYYAMDY (SEQ ID NO: 3)

The numbering and definition of CDR corresponds to the definition of Chothia.

In one embodiment, the light chain variable region includes at least one of the following CDRs:

VL-CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 6)

VL-CDR2: KVSNRFS (SEQ ID NO: 7)

VL-CDR3: FQGSHVPRT (SEQ ID NO: 8)

The numbering and definition of the CDR corresponds to the Kabat or Chothia definitions.

In one embodiment of the invention, the anti-HLA-A2 antibody contains in its heavy chain one VH-CDR1 (SYHIQ (SEQ ID NO: 1)), one VH-CDR2 (WIYPGDGSTQYNEKFKG (SEQ ID NO: 2)) and/or one VH- CDR3 (EGTYYAMDY (SEQ ID NO: 3)).

In one embodiment of the invention, the anti-HLA-A2 antibody contains 3 CDRs in its heavy chain: VH-CDR1 (SYHIQ (SEQ ID NO: 1)), VH-CDR2 (WIYPGDGSTQYNEKFKG (SEQ ID NO: 2)) and VH-CDR3 ( EGTYYAMDY (SEQ ID NO: 3)).

In one embodiment of the invention, the anti-HLA-A2 antibody contains in its heavy chain one VH-CDR1 (GYTFTSY (SEQ ID NO: 4)), one VH-CDR2 (YPGDGS (SEQ ID NO: 5)) and/or one VH- CDR3 (EGTYYAMDY (SEQ ID NO: 3)).

In one embodiment of the invention, the anti-HLA-A2 antibody contains 3 CDRs in its heavy chain: VH-CDR1 (GYTFTSY (SEQ ID NO: 4)), VH-CDR2 (YPGDGS (SEQ ID NO: 5)) and VH-CDR3 ( EGTYYAMDY (SEQ ID NO: 3)).

In one embodiment of the invention, the anti-HLA-A2 antibody contains in its light chain one VL-CDR1 (RSSQSIVHSNGNTYLE (SEQ ID NO: 6)), one VL-CDR2 (KVSNRFS (SEQ ID NO: 7)) and/or one VL- CDR3 (FQGSHVPRT (SEQ ID NO: 8)).

In one embodiment of the invention, the anti-HLA-A2 antibody contains in its light chain 3 CDRs: VL-CDR1 (RSSQSIVHSNGNTYLE (SEQ ID NO: 6)), VL-CDR2 (KVSNRFS (SEQ ID NO: 7)) and VL-CDR3 ( FQGSHVPRT (SEQ ID NO: 8)).

In one embodiment of the invention, the anti-HLA-A2 antibody comprises:

- in its heavy chain 3 CDR SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; And

- in its light chain 3 CDR SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In one embodiment of the invention, the anti-HLA-A2 antibody comprises:

- in its heavy chain 3 CDR SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 3; And

- in its light chain 3 CDR SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

According to the invention, any of the CDRs 1, 2 and 3 of the heavy and light chains can be characterized as having an amino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96 identical %, 97%, 98%, 99% of certain CDRs or sets of CDRs listed in the corresponding SEQ ID NO: 1-8.

In one embodiment of the invention, the anti-HLA-A2 antibody comprises a heavy chain variable region of the sequence SEQ ID NO: 9.

(SEQ ID NO: 9)

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSS

In one embodiment of the invention, the heavy chain variable region of an anti-HLA-A2 antibody has a sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% identity with SEQ ID NO: 9.

In one embodiment of the invention, the anti-HLA-A2 antibody comprises a light chain variable region of the sequence SEQ ID NO: 10.

(SEQ ID NO: 10)

DX ₁ VMTQX ₂ PX ₃ X ₄ LX ₅ VX ₆ X ₇ GX ₈ X ₉ X ₁₀ X ₁₁ ISCRSSQSIVHSNGNTYLEWYX ₁₂ QKPGQX ₁₃ PRLLIYKVSNRFSGX ₁₄ PDRFSGSGSGTDFTLX ₁₅ ISRX ₁₆ EX ₁₇ EDX ₁₈ X ₁₉ VYYCFQGSHVP RTFGGGTKLEIKR

where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I , X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A.

In one embodiment of the invention, the anti-HLA-A2 antibody light chain variable region has a sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% identity with SEQ ID NO: 10.

In one embodiment, the light chain region has the sequence SEQ ID NO: 11 or any sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% identity with SEQ ID NO: 11.

(SEQ ID NO: 11)

DVVMTQTPLSLPVTLGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKR

In one embodiment, the light chain region has the sequence SEQ ID NO: 12 or any sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% identity with SEQ ID NO: 12.

(SEQ ID NO: 12)

DVVMTQSPSSSLSVTLGDRVSISCRSSQSIVHSNGNTYLEWYQQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKR

In one embodiment, the light chain region has the sequence SEQ ID NO: 13 or any sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% identity with SEQ ID NO: 13.

(SEQ ID NO: 13)

DIVMTQSPATLSVSPGERATISCRSSQSIVHSNGNTYLEWYQQKPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCFQGSHVPRTFGGGTKLEIKR

In one embodiment, an HLA-A2 antibody of the invention includes a heavy chain variable region having the sequence SEQ ID NO: 9 and a light chain variable region having the sequence SEQ ID NO: 10, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 is G or A, or any sequence that shares at least about 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9-10.

In one embodiment, an HLA-A2 antibody of the invention includes a heavy chain variable region having the sequence SEQ ID NO: 9 and a light chain variable region having the sequence SEQ ID NO: 11, or any sequence that has at least about 60 %, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NOs: 9 and 11.

In one embodiment, an HLA-A2 antibody of the invention includes a heavy chain variable region having the sequence SEQ ID NO: 9, and a light chain variable region having the sequence SEQ ID NO: 12, or any sequence that has at least about 60 %, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9 and 12.

In one embodiment, an HLA-A2 antibody of the invention includes a heavy chain variable region having the sequence SEQ ID NO: 9, and a light chain variable region having the sequence SEQ ID NO: 13, or any sequence that has at least about 60 %, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9 and 13.

According to the invention, one, two, three, four or more amino acids of the heavy chain or light chain variable regions may be replaced by another amino acid.

In one embodiment, in an antibody of the invention, said variable regions and CDR sequences may contain modifications of conserved sequences. Conserved sequence modifications refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Another aspect of the invention is an isolated nucleotide sequence encoding the heavy chain variable region of SEQ ID NO: 9. In one embodiment, said nucleic acid sequence is SEQ ID NO: 86.

(SEQ ID NO: 86)

CAGGTCCAGCTAGTACAAAGCGGCCCTGAAGTAAAGAAACCTGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACCACATCCAGTGGGTTCGACAGGCCCCTGGACAGGGACTAGAGTGGATCGGCTGGATCTATCCTGGCGACGGCAGCACCCAGTACAACGAGAAGTTCAAGGGCAGAGTTACCATCACCGCCGACAAGAGCACCAGCACAGCCTATATG GAGCTGAGCAGCCTGACCAGCGAGGACACAGCTGTTTACTATTGTGCCAGAGAGGGCACCTACTACGCAATGGATTATTGGGGCCAGGGGACCAGCGTGACCGTTTCTTCT

Another aspect of the invention is an isolated nucleotide sequence encoding a light chain variable region having the sequence SEQ ID NO: 11. In one embodiment, said nucleic acid sequence is SEQ ID NO: 87.

(SEQ ID NO: 87)

GATGTTGTAATGACCCAGACACCTCTGAGCCTGCCTGTGACCCTGGGAGAACCAGCATCCATCAGCTGTCGGAGCAGCCAGAGCATCGTTCACAGCAACGGCAACACCTACCTGGAATGGTATCTACAGAAGCCCGGACAGAGCCCCAGGCTGCTGATCTACAAGGTGTCCAACCGCTTCAGTGGTGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCT CCAGAGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCTTCCAAGGCAGCCATGTGCCAAGAACCTTTGGTGGAGGCACAAAGCTGGAAATCAAGCGG

Another aspect of the invention is an isolated nucleotide sequence encoding a light chain variable region having the sequence SEQ ID NO: 12. In one embodiment, said nucleic acid sequence is SEQ ID NO: 88.

(SEQ ID NO: 88)

GATGTTGTAATGACCCAGAGCCCTAGTAGCCTGTCTGTGACCCTGGGAGATCGAGTATCCATCAGCTGTCGGAGCAGCCAGAGCATCGTTCACAGCAACGGCAACACCTACCTGGAATGGTATCAACAGAAGCCCGGACAGAGCCCCAGGCTGCTGATCTACAAGGTGTCCAACCGCTTCAGTGGTGTGCCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACGATC TCCAGAGTGGAACCAGAGGACCTGGGCGTGTACTACTGCTTCCAAGGCAGCCATGTGCCAAGAACCTTTGGTGGAGGCACAAAGCTGGAAATCAAGCGG

Another aspect of the invention is an isolated nucleotide sequence encoding a light chain variable region of SEQ ID NO: 13. In one embodiment, said nucleic acid sequence is SEQ ID NO: 89

(SEQ ID NO: 89)

GATATTGTAATGACCCAGAGCCCTGCAACACTGTCTGTGTCCCCTGGAGAACGAGCAACAATCAGCTGTCGGAGCAGCCAGAGCATCGTTCACAGCAACGGCAACACCTACCTGGAATGGTATCAACAGAAGCCCGGACAGGCCCCCAGGCTGCTGATCTACAAGGTGTCCAACCGCTTCAGTGGAATACCCGATAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACGATCTCCA GATTAGAACCAGAGGACTTTGCAGTGTACTACTGCTTCCAAGGCAGCCATGTGCCAAGAACCTTTGGTGGAGGCACAAAGCTGGAAATCAAGCGG

Another object of the invention is an expression vector containing nucleic acid sequences encoding an anti-HLA-A2 antibody according to the invention.

Another object of the invention is an isolated host cell containing said vector. Said host cell can be used to recombinantly produce antibodies of the invention.

In one embodiment, said antibody is an antibody molecule selected from the group consisting of a full length antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, an Fv, a scFv, a Fab, a F(ab)'2, a defucosylated antibody, a bispecific antibody , diabodies, triatebodies, tetrabodies.

In another embodiment, the antibody is an antibody fragment selected from the group consisting of a unitbody, a domain antibody, and a nanobody.

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

The antibody fragments and derivatives of the present invention (which are encompassed by the term "antibody" as used herein unless otherwise indicated or clearly contrary to context) can be prepared by methods known in the art. "Fragments" include a portion of an intact antibody, typically an antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; dimers; any antibody fragment that is a polypeptide having a primary structure consisting of one contiguous sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single-chain polypeptide”), including, but not limited to, (1) single-chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or fragment thereof, which includes three light chain variable domain CDRs, without an associated heavy chain portion, and (3) single chain polypeptides containing only one heavy chain variable region or fragment thereof , containing three CDRs of the heavy chain variable region, without an associated light chain fragment; and multispecific antibodies formed from antibody fragments. The present antibody fragments can be prepared using standard methods.

For example, Fab or F(ab')2 fragments can be obtained by protease digestion of isolated antibodies according to conventional techniques. It is understood that immunoreactive fragments can be modified using known methods, for example, to slow clearance in vivo and obtain a more desirable pharmacokinetic profile, the fragment can be modified with polyethylene glycol (PEG). Methods for binding and site-specific conjugation of PEG to a Fab' fragment are described, for example, in Leong et al., Cytokines 16 (3): 106-119 (2001) and Delgado et al., Br. J. Cancer 5 73 (2): 175-182 (1996), the descriptions of which are incorporated herein by reference.

In one embodiment, the antibody of the invention is a scFv or scFab, preferably a scFv.

In one embodiment, the antibody of the invention is a scFv comprising the sequence SEQ ID NO: 9 and the sequence SEQ ID NO: 10, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 represents S or T, X5 represents P or S, X6 represents T or S, X7 represents L or P, X8 represents E or D, X9 represents P or R, X10 represents A or V, X11 represents is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, in any order for the N-terminus and C-terminus of scFv. Preferably, the antibody of the invention is a scFv containing the sequence of SEQ ID NO: 9 and the sequence of SEQ ID NO: 11, 12 or 13 in any order from the N-terminus to the C-terminus of the scFv.

In one embodiment, the sequence SEQ ID NO: 9 is N-terminal and the sequence SEQ ID NO: 10, 11, 12 or 13 is C-terminal.

In one embodiment, the scFv of the invention comprises, from N-terminus to C-terminus: SEQ ID NO: 9, optional linker, SEQ ID NO: 10, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A (preferably one of SEQ ID NOs: 11-13).

In one embodiment, the scFv of the invention comprises, from N-terminus to C-terminus: SEQ ID NO: 10 where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A (preferably one of SEQ ID NO: 11-13), an optional linker, SEQ ID NO: 9.

In one embodiment, the scFv includes a linker. Examples of linkers include, but are not limited to, GS linkers as described herein. In one embodiment, the linker includes or consists of the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 18).

In one embodiment, the scFv of the invention comprises or consists of the sequence SEQ ID NO: 69 or any amino acid sequence with at least about 95-99% identity to SEQ ID NO: 69

(SEQ ID NO: 69)

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDX ₁ VMTQX ₂ PX ₃ X ₄ LX ₅ VX ₆ X ₇ GX ₈ X ₉ X ₁₀ X ₁₁ ISCRSSQSIVHSNGNTYLEWYX ₁₂ QKPGQX ₁₃ PRLLIYKVSNRFSGX ₁₄ PDRFSGSGSGTDFTLX ₁₅ ISRX ₁₆ EX ₁₇ EDX ₁₈ X ₁₉ VYYCFQGSHVPRTFGGGTKLEIKR

where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I , X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 is G or A. SEQ ID NO: 69 consists of N-terminus to C-terminus SEQ ID NO: 9, SEQ ID NO: 18 and SEQ ID NO: 10.

In one embodiment, the scFv of the invention comprises or consists of the sequence SEQ ID NO: 70, 71 or 72 or any amino acid sequence with at least about 95-99% identity to SEQ ID NO: 70, 71 or 72. SEQ ID NO: 70-72 consist from N-terminus to C-terminus of SEQ ID NO: 9, SEQ ID NO: 18 and SEQ ID NO: 11-13, respectively.

(SEQ ID NO: 70)

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVTLGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPRLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKR

(SEQ ID NO: 71)

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQSPSSLSVTLGDRVSISCRSSQSIVHSNGNTYLEWYQQKPGQSPRLLIYKV SNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKR

(SEQ ID NO: 72)

QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGERATISCRSSQSIVHSNGNTYLEWYQQKPGQAPRLLIYKVSN RFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCFQGSHVPRTFGGGTKLEIKR

In one embodiment, the scFv of the invention further includes an S residue at the N-terminus to facilitate cleavage of an optional leader sequence.

Therefore, in one embodiment, the scFv of the invention has sequence 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A.

In one embodiment, the scFv of the invention comprises or consists of the sequence SEQ ID NO: 74, 75 or 76.

(SEQ ID NO: 74)

A VSNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKR

(SEQ ID NO: 75)

A VSNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKR

(SEQ ID NO: 76)

SQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGERATISCRSSQSIVHSNGNTYLEWYQQKPGQAPRLLIYKV SNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCFQGSHVPRTFGGGTKLEIKR

Another object of the invention is a composition comprising essentially consisting of or consisting of at least one anti-HLA-A2 antibody according to 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 hereinabove, is the only therapeutic agent or agent with biological activity in the specified composition.

Another object of the invention is a pharmaceutical composition containing at least one anti-HLA-A2 antibody according to the invention and a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers include, but are not limited to, media, solvents, coatings, isotonic and absorption retarding 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 vehicles include, but are not limited to, water, neutral saline, phosphate-buffered saline, saline or other physiologically buffered saline or other solvent such as glycol, glycerol, 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, but are not limited to, sugars, sodium chloride, and the like.

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

Examples of additives 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 human serum albumin) suitable for administration in vivo.

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, buffers 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 carboxymethylcellulose , polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat.

Another object of the invention is a medicament containing, consisting of or consisting essentially of at least one anti-HLA-A2 antibody according to the invention, as described above.

Another object of the invention is the use of at least one anti-HLA-A2 antibody according to the invention for the detection of HLA-A2 in a sample, preferably a biological sample, in vitro or in vivo.

Examples of assays in which the anti-HLA-A2 antibody of the invention may be used include, but are not limited to, ELISA, sandwich ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, and immunoprecipitation.

Another aspect of the invention is a method for detecting HLA-A2 in a sample, comprising contacting the sample with an anti-HLA-A2 antibody of the invention and detecting an anti-HLA-A2 antibody bound to HLA-A2, indicating the presence of HLA-A2 in the sample.

In one embodiment of the invention, the sample is a biological sample. Examples of biological samples include, but are not limited to, bodily fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluid, urine, amniotic fluid, abdominal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid and alveolar macrophages, tissue lysates and extracts prepared from diseased tissue or graft.

In one embodiment of the invention, the term “sample” is intended to refer to a sample taken from an individual prior to any analysis.

In one embodiment of the invention, the anti-HLA-A2 antibody of the invention is directly labeled with a detectable label and can be detected directly. In another embodiment, the protein of the invention is unlabeled (and referred to as the first/primary antibody), and the secondary antibody or other molecule that can bind the anti-HLA-A2 antibody is labeled. As is well known in the art, the secondary antibody is selected so that it can specifically bind a particular species and class of primary antibody.

The presence of an anti-HLA-A2 antibody/HLA-A2 complex in a sample can be detected and measured by detecting the presence of a labeled secondary antibody. For example, after leaching unbound secondary antibody from a well containing the primary antibody/antigen complex or from a membrane (eg, nitrocellulose or nylon membrane) containing the complex, the bound secondary antibody can be developed and detected based on chemiluminescence for an example label.

Labels for an anti-HLA-A2 antibody or secondary antibody include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Examples of such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; examples of prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin; examples of fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansine chloride, or phycoerythrin; examples of luminescent material include, but are not limited to, luminal; examples of magnetic agents include gadolinium; and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

Another object of the invention is a kit containing at least one anti-HLA-A2 antibody according to the invention, preferably a monoclonal anti-HLA-A2 antibody.

By "kit" is meant any product of manufacture (eg, packaging or container) containing at least one reagent, preferably an antibody, for specifically detecting HLA-A2 expression.

The kit may be advertised, distributed or sold as a unit to implement the methods of the present invention. In addition, any or all of the kit reagents may be provided in containers that protect them from the external environment, such as sealed containers.

The kits may also contain a package insert describing the kit and how to use it.

Sandwich ELISA kits typically include a capture antibody, optionally immobilized on a solid support (eg, a microtiter plate), and a detection antibody bound to a detectable substance, such as, for example, HRP, a fluorescent tag, a radioisotope, beta-galactosidase, and alkaline phosphatase.

In another embodiment, the detectable substance is immobilized on a solid support (eg, a microtiter plate).

The present invention also relates to a chimeric antigen receptor (CAR) specific for HLA-A2. CARs are chimeric protein molecules that combine antibody specificity for a target antigen with a receptor-activated intracellular domain of an immune cell.

According to the present invention, a CAR comprises in one or more polypeptides (i) an extracellular HLA-A2 binding domain, wherein said extracellular domain includes or consists of an anti-HLA-A2 antibody (preferably an anti-HLA-A2 scFv) of the present invention, (ii) optionally an extracellular a hinge domain, (iii) an optional transmembrane domain, and (iv) an intracellular signaling domain. In one embodiment, the chimeric receptor further includes a tag and/or leader sequence.

In one embodiment, the CAR of the present invention competes for binding to an HLA-A2 antibody comprising: heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 62; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 63; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 64; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 65; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 66; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 67.

In one embodiment, the CAR of the present invention binds 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: 62; heavy chain complementarity determining region 2 (HCDR2) having the amino acid sequence of SEQ ID NO: 63; heavy chain complementarity determining region 3 (HCDR3) having the amino acid sequence of SEQ ID NO: 64; light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 65; light chain complementarity determining region 2 (LCDR2) having the amino acid sequence of SEQ ID NO: 66; and light chain complementarity determining region 3 (LCDR3) having the amino acid sequence of SEQ ID NO: 67.

In one embodiment, the CAR of the invention competes for binding to HLA-A2 and/or binds to the same HLA-A2 epitope as the BB7.2 antibody.

In one embodiment, the CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof, compared to a CAR containing BB7.2, or CAR containing VH and VL antibodies BB7.2. In one embodiment, the CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03:01, A11:01, A23:01, A25:01, A26:01, A29:02, A30:01, A31: 01, A33:01, A34:01 and any combination thereof compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from three or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from three or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from four or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from four or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from five or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from five or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from six or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from six or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from seven or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from seven or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from eight or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from eight or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from nine or more of A03, A11, A23, A25, A26, A29, A30, A31, A33, and A34 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from nine or more of A03:01, A11:01, A23:01, A25:01, A26: 01, A29:02, A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity against each of the HLA-A subtypes selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33 and A34 compared to CAR containing BB7.2, or to CAR containing VH and VL antibodies BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity for each of the HLA-A subtypes selected from the group consisting of A03:01, A11:01, A23:01, A25:01, A26:01, A29:02 , A30:01, A31:01, A33:01, and A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

In one embodiment, the CAR of the invention is less reactive to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to antibody BB7.2, preferably compared to scFv BB7.2, or compared with an antibody containing VH and VL antibodies BB7.2. In one embodiment, the CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25:01, A29:02, A30:01, and any combination thereof, compared to antibody BB7.2, preferably compared to sc7v BB7.2 or compared to an antibody containing the VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A25, A29 and A30, compared to a CAR containing BB7.2, or CAR containing VH and VL antibodies BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from one or more of A25:01, A29:02, and A30:01, compared to a CAR containing BB7 .2 or to CAR containing VH and VL antibodies BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A25, A29 and A30, compared to a CAR containing BB7.2, or CAR containing VH and VL antibodies BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to at least one HLA-A subtype selected from two or more of A25:01, A29:02 and A30:01, compared to a CAR containing BB7. 2 or to CAR containing VH and VL antibodies BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity against each of the HLA-A subtypes selected from the group consisting of A25, A29 and A30, compared to a CAR containing BB7.2 or a CAR containing VH and VL antibodies BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity against each of the HLA-A subtypes selected from the group consisting of A25:01, A29:02 and A30:01, compared to a CAR containing BB7.2 or CAR containing VH and VL antibodies BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A25 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A29 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A30 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A3 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A11 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A26 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A31 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A32 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A33 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A34 compared to a CAR containing BB7.2 or a CAR containing VH and VL antibodies of BB7.2.

In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A25:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A29:02 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, the anti-HLA-A2 antibody of the invention has less reactivity to HLA-A30: compared to a CAR containing BB7.2, or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A03:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A11:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A26:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A31:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A33:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2. In one embodiment, an anti-HLA-A2 antibody of the invention has less reactivity to HLA-A34:01 compared to a CAR containing BB7.2 or a CAR containing the VH and VL antibodies of BB7.2.

Methods for measuring CAR reactivity to HLA-A subtypes are known to one of ordinary skill in the art and include, but are not limited to, single antigen assays such as, for example, the FlowPRA Single Antigen Antibody provided by ONE LAMBDA.

In one embodiment, a CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof, compared to a CAR containing BB7.2 antibody or compared to CAR containing VH and VL BB7.2 antibodies when measured under Test A conditions.

In one embodiment, the CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to the BB7.2 antibody or compared to an antibody comprising the VH and VL of the BB7 antibodies. 2 when measured under test conditions A.

Test A:

0.25.10 ⁶ T cells expressing CAR containing anti-HLA-A2 antibody or BB7.2 antibody or BB7.2 VH and VL antibodies (mA2 CAR) were incubated with a panel of FlowPRA anti-single antigen antibody beads (FL1HD01, FL1HD02, FL1HD03). FL1HD04, FL1HD06 and FL1HD08, One Lambda) and fixable viability dye (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 using a fixable dye to assess viability. Single antigen beads were then selected for signal above a threshold after excluding dead cells and doublets. The number of beads per HLA was then determined by their corresponding peak PE intensity. 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 Tregs compared to the control (non-CAR-expressing cells) was determined by subtracting the number of beads in the CAR-Treg from the number of beads in the control sample, then dividing the average number of beads in the non-CAR-expressing control multiplied by by 100.

In one embodiment, a CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, and any combination thereof, compared to a CAR containing BB7.2 antibody or compared to CAR containing VH and VL BB7.2 antibodies when measured under test B conditions.

In one embodiment, the CAR of the invention has less reactivity to an HLA-A subtype selected from the group consisting of A25, A29, A30, and any combination thereof, compared to the BB7.2 antibody or compared to an antibody comprising the VH and VL of the BB7 antibodies. 2 when measured under test conditions B.

Test B:

0.25.10 ⁶ T cells expressing anti-HLA-A2 antibody or BB7.2 antibody, or BB7.2 VH and VL antibodies or a truncated NGFR transduction marker (NGFR) on the cell surface (such as, for example, as part of a chimeric antigen receptor described herein) were incubated with FlowPRA single antigen antibody beads (FL1HD, One Lambda) and viability fixative dye (FVD, 65-0865-14, eBioscience), then fixed with 0.5% formaldehyde and analyzed using flow cytometry. For analysis, dead cells were first removed using a fixable dye to assess viability. Single antigen beads were then selected for signal above a threshold after excluding dead cells and doublets. The number of beads per HLA was then determined by their corresponding PE intensity. Data were normalized by dividing the number of negative beads in the sample by the number of negative beads in the Treg-NGFR sample multiplied by the number of negative beads in the Treg-NGFR sample. Relative percentage 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.

In one embodiment, the CAR of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, statistically inferior to the reactivity of the CAR containing the BB7.2 antibody or a CAR containing the VH and VL of the BB7.2 antibody, preferably when measured under Test A or Test B conditions.

In one embodiment, the CAR of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of A25, A29, A30, statistically inferior to the reactivity of the BB7.2 antibody or the reactivity of an antibody containing the VH and VL of the BB7.2 antibody , preferably when measured under Test A or Test B conditions.

In one embodiment, the term “statistically inferior” means that the reactivity (ie, for example, relative binding under test A or test B conditions) measured for the CAR of the invention is lower than the reactivity measured for BB7. 2 antibodies or for an antibody containing VH and VL antibodies BB7.2, with a p-value of at least about 0.05, preferably at least about 0.01, more preferably at least about 0.005, and more preferably at least about 0.001 specifically when analyzed using two-way ANOVA, Dunnett's post hoc test.

In one embodiment, the CAR of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26, A29, A30, A31, A33, A34, inferior to the reactivity of the CAR containing a BB7.2 antibody, or a CAR comprising the VH and VL of a BB7.2 antibody, preferably the CAR of the invention has relative binding to at least one HLA-A subtype selected from the group consisting of A03, A11, A23, A25, A26 , A29, A30, A31, A33, A34 inferior to that of the CAR containing the BB7.2 antibody, or one of the CARs containing the VH and VL antibodies of BB7.2, when measured under the conditions of test A or test B, more preferably the relative binding measured for an anti-HLA-A2 antibody of the invention is at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the relative binding measured for the BB7 antibody .2 or for an antibody containing VH and VL antibodies BB7.2.

In one embodiment, the CAR of the invention has reactivity to at least one HLA-A subtype selected from the group consisting of A25, A29, A30, inferior to the reactivity of the CAR containing the BB7.2 antibody, or the reactivity of the CAR containing the VH and VL antibodies BB7.2, preferably the CAR of the invention has a relative binding to at least one HLA-A subtype selected from the group consisting of A25, A29, A30, inferior to the relative binding of a CAR containing antibody BB7.2 or the relative binding of a CAR containing The VH and VL of the BB7.2 antibody when measured under test A or test B conditions, more preferably the relative binding measured for the anti-HLA-A2 antibody of the invention is at most about 90%, 80%, 70%, 60%, 50 %, 40%, 30%, 20%, 10% or less relative binding measured for the BB7.2 antibody or for an antibody containing the VH and VL of the BB7.2 antibody.

The present invention also provides a nucleic acid sequence encoding a CAR described above, wherein said nucleic acid sequence comprises i) an HLA-A2 binding domain nucleic acid sequence (preferably, wherein said HLA-A2 binding domain is an anti-HLA-A2 antibody, preferably an anti-HLA-A2 scFv as described above), (ii) an optional extracellular hinge domain nucleic acid sequence, (iii) an optional transmembrane domain nucleic acid sequence, (iv) one or more intracellular signaling domain nucleic acid sequences. In one embodiment, the nucleic acid sequence further includes a sequence encoding a tag and/or a leader sequence.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention is a scFv containing the sequence SEQ ID NO: 9 and the sequence SEQ ID NO: 10, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 is G or A, in any order from N-terminus to C-terminus of scFv. Preferably, the HLA-A2 binding domain contained in the CAR of the invention is a scFv comprising the sequence of SEQ ID NO: 9 and the sequence of SEQ ID NO: 11, 12 or 13 in any order from the N-terminus to the C-terminus of the scFv.

In one embodiment, the sequence SEQ ID NO: 9 is N-terminal and the sequence SEQ ID NO: 10, 11, 12 or 13 is C-terminal.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention includes, from N-terminus to C-terminus: SEQ ID NO: 9, optional linker, SEQ ID NO: 10, where X1 is V or I, X2 is is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 represents P or R, X10 represents A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents is V or L, X17 is A or P, X18 is L or F, and X19 is G or A (preferably one of SEQ ID NOs: 11-13).

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention comprises, from N-terminus to C-terminus: SEQ ID NO: 10 wherein X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A (preferably one of SEQ ID NO: 11-13), optional linker, SEQ ID NO: 9.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention includes a linker. Examples of linkers include, but are not limited to, GS linkers as described herein. In one embodiment, the linker includes or consists of the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 18).

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention includes or consists of the sequence SEQ ID NO: 69, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents V or L, X17 represents A or P, X18 represents is L or F and X19 is G or A. SEQ ID NO: 69 consists of, from N-terminus to C-terminus, SEQ ID NO: 9, SEQ ID NO: 18 and SEQ ID NO: 10.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention includes or consists of the sequence SEQ ID NO: 70, 71 or 72. SEQ ID NO: 70-72 consist of N-terminus to C-terminus of SEQ ID NO: : 9, SEQ ID NO: 18 and SEQ ID NO: 11-13, respectively.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention further includes an S residue at the N-terminus to facilitate cleavage of an optional leader sequence.

Therefore, in one embodiment, the HLA-A2 binding domain contained in the CAR of the invention has the sequence 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T , X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 represents G or A.

In one embodiment, the HLA-A2 binding domain contained in the CAR of the invention includes or consists of the sequence SEQ ID NO: 74, 75 or 76.

In one embodiment, the HLA-A2 binding domain may be linked to the transmembrane domain via a hinge domain.

In one embodiment, a short oligo- or polypeptide linker, preferably having a length in the range of 2 to 10 amino acids, can form a hinge domain. In one embodiment, the term “linker” refers to a flexible polypeptide linker.

For example, the glycine-serine doublet provides a particularly suitable hinge domain (GS linker). In one embodiment, the hinge domain is a Gly/Ser linker. Examples of Gly/Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers.

Examples of G2S linkers include, but are not limited to, GGS.

G3S linkers contain the amino acid sequence (Gly-Gly-Gly-Ser)n, also referred to as (GGGS)n or (SEQ ID NO: 14)n, where n is a positive integer equal to or greater than 1 (such as, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10). Examples of G3S linkers include, but are not limited to, GGGSGGGSGGGSGGGS (SEQ ID NO: 15).

Examples of G4S linkers include, but are not limited to, (Gly4 Ser) corresponding to GGGGS (SEQ ID NO: 16); (Gly4 Ser)2 corresponding to GGGGSGGGGS (SEQ ID NO: 17); (Gly4Ser)3 corresponding to GGGGSGGGGSGGGGS (SEQ ID NO: 18); and (Gly4 Ser)4 corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19).

Also included within the scope of the invention are the hinge domains described in WO2012/138475, which is incorporated herein by reference.

In one embodiment, the hinge domain includes the amino acid sequence AGSSSSGGSTTGGSTT (SEQ ID NO: 20), the amino acid sequence GTTAASGSSGGSSSGA (SEQ ID NO: 21), the amino acid sequence SSATATAGTGSSTGST (SEQ ID NO: 22) and/or the amino acid sequence TSGSTGTAASSTSSTST (SEQ ID NO: 23 ).

In one embodiment, the hinge domain is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 24).

In another embodiment, the hinge domain is a KIR2DS2 hinge corresponding to KIRRDSS (SEQ ID NO: 25).

In one embodiment, the hinge domain includes or consists of a CD8 hinge amino acid sequence (SEQ ID NO: 26) or an amino acid sequence with 95-99% identity to SEQ ID NO: 26. In one embodiment, the hinge domain is a CD8 hinge encoded by a nucleic acid sequence SEQ ID NO: 27 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 27.

(SEQ ID NO: 26)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

(SEQ ID NO: 27)

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

In another embodiment of the invention, the hinge domain includes or consists of an IgG4 hinge amino acid sequence (SEQ ID NO: 28) or an amino acid sequence with 95-99% identity to SEQ ID NO: 28. In one embodiment, the hinge domain is an IgG4 hinge encoded by a nucleic acid sequence acid SEQ ID NO: 29 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 29.

(SEQ ID NO: 28)

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

(SEQ ID NO: 29)

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCA ATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCG TGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

In another embodiment, the hinge domain includes or consists of an IgD hinge amino acid sequence (SEQ ID NO: 30) or an amino acid sequence with 95-99% identity to SEQ ID NO: 30. In one embodiment, the hinge domain is an IgD hinge encoded by a nucleic acid sequence SEQ ID NO: 31 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 31.

(SEQ ID NO: 30)

RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVS GFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH

(SEQ ID NO: 31)

AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTG GCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCACAGCTCTGAT GGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAG CACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT

In another embodiment, the hinge region includes or consists of a CD28 hinge amino acid sequence (SEQ ID NO: 32) or an amino acid sequence with 95-99% identity to SEQ ID NO: 32. In one embodiment, the hinge domain is a CD28 hinge encoded by the nucleic acid of SEQ ID NO: 33 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 33.

(SEQ ID NO: 32)

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

(SEQ ID NO: 33)

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC

Examples of transmembrane domains that can be used in a chimeric receptor of the invention include, but are not limited to, T cell receptor alpha, beta or zeta chain transmembrane domains or CD28, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, 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), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D and/or NKG2C.

In one embodiment, the transmembrane domain includes or consists of an amino acid sequence of a CD8 transmembrane domain (SEQ ID NO: 34) or an amino acid sequence with 95-99% identity to SEQ ID NO: 34. In another embodiment, the transmembrane domain includes or consists of an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence with 95-99% identity to SEQ ID NO: 34.

(SEQ ID NO: 34)

IYIWAPLAGTCGVLLLSLVITLYC

In another embodiment, the transmembrane domain is encoded by the nucleotide sequence of the CD8 transmembrane domain (SEQ ID NO: 35) or a nucleotide sequence with 95-99% identity to SEQ ID NO: 35.

(SEQ ID NO: 35)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

In another embodiment, the transmembrane domain includes or consists of the amino acid sequence of the CD28 transmembrane domain (SEQ ID NO: 36) or an amino acid sequence with 95-99% identity to SEQ ID NO: 36. In one embodiment, the transmembrane domain is the CD28 transmembrane domain encoded by the sequence nucleic acid SEQ ID NO: 37 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 37.

(SEQ ID NO: 36)

FWVLVVVGGVLACYSLLVTVAFIIFWV

(SEQ ID NO: 37)

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG

In one embodiment, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic amino acids such as valine or leucine.

The cytoplasmic domain, which includes the intracellular signaling domain of the CAR, is responsible for activating at least one of the effector functions of the immune cell (eg, a 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 inhibiting or suppressing the induction and/or proliferation of other immune cells. In addition, Treg effector function may involve effects on non-immune cells that result in improved clinical conditions, such as promoting tissue repair or regeneration. Thus, the term "intracellular signaling domain" refers to the part of the protein that transduces the effector function signal and directs the immune cell to perform its specialized function. Although the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the intact circuit if it conveys a signal to an effector function. The term "intracellular signaling domain" is thus intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the signal to effector function.

In one embodiment, the intracellular signaling domain may include all or all of the native intracellular signaling domain of the molecule from which it is derived, or a functional fragment or derivative thereof.

In accordance with one embodiment, the intracellular signaling domain comprises at least one (such as, for example, one) primary T cell signaling domain (or a sequence derived therefrom) and optionally one or more intracellular domains of a T cell co-stimulatory molecule (or sequence(s) derived from them).

In one embodiment, the intracellular signaling domain of a chimeric receptor of the invention consists of at least one (eg, one) primary signaling domain.

In one embodiment, the intracellular signaling domain includes one or more intracellular domains of a T cell co-stimulatory molecule. In one embodiment, the intracellular signaling domain consists of one or more intracellular domains of a T cell co-stimulatory molecule.

In another embodiment, the intracellular signaling domain of a chimeric receptor of the invention includes at least one co-stimulatory domain and at least one (eg, one) primary signaling domain.

In another embodiment, the intracellular signaling domain of a chimeric receptor of the invention includes at least two costimulatory domains and at least one (eg, one) primary signaling domain.

In one embodiment of the invention, the primary T cell signaling domain comprises a protein signaling domain selected from the group CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, conventional FcR gamma (FCER1G), FcR beta (Fc epsilon Rib) , CD79a, CD79b, Fc gamma RIIa, DAP10 and DAP 12 and sequences derived from them.

In one embodiment, the primary T cell signaling domain includes or consists of a functional CD3 zeta signaling domain.

In one embodiment, the primary T cell signaling domain comprises or consists of the amino acid sequence of the CD3 zeta domain of SEQ ID NO: 38, 39, 40 or 41 or an amino acid sequence with 95-99% identity to SEQ ID NO: 38-41.

(SEQ ID NO: 38)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 39)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 40)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 41)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In another embodiment, the CD3 zeta primary signaling domain comprises or consists of an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence SEQ ID NO: 38-41 or amino acid sequence with 95-99% identity to SEQ ID NO: 38-41.

Thus, in one embodiment, the nucleic acid sequence encoding the primary T cell signaling domain includes or consists of a CD3 zeta domain nucleic acid sequence of SEQ ID NO: 42 or SEQ ID NO: 43 or a nucleotide sequence with 95-99% identity to SEQ ID NO: 42 or SEQ ID NO: 43.

(SEQ ID NO: 42)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

(SEQ ID NO: 43)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

Primary T cell signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS).

Examples of ITAMs containing primary intracellular T cell signaling domains that are particularly useful in the invention include, but are not limited to, CD3-zeta domains, conventional FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD66b, CD79a, CD79b, DAP10 and DAP12.

In one embodiment, the primary T cell signaling domain includes a modified ITAM domain, for example, a mutated ITAM domain that has had altered (eg, increased or decreased) activity compared to 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 shortened 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 of a chimeric receptor of the invention comprises a T cell primary signaling domain (such as, for example, a CD3 zeta signaling domain) in combination with one or more co-stimulatory signaling domains. Costimulatory signaling domain(s) refers to a portion of a chimeric receptor containing at least one intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is required for an effective lymphocyte response to an antigen.

Examples of intracellular domains of a T cell co-stimulatory molecule include, but are not limited to, the signaling domains of proteins selected from the group CD27, CD28, 4-1BB (CD137), MHC class I molecules, BTLA, toll receptor ligand, OX40, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR , ARHR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160 (BY55), CD19, CD19a, CD4, CD8 alpha, CD8 beta, IL2ra, ILRR beta, IL2R gamma, IL7R alpha , IL-13RA1/RA2, IL-33R (IL1RL1), IL-10RA/RB, IL-4R, IL-5R (CSF2RB), IL-21R, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a/CD18, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, CT 4 (CD152), CD95, TNFR1 (CD120a/TNFRSF1A), TNFR2 (CD120b/TNFRSF1B), TGFbR1/2/3, 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, common gamma chain, a ligand that specifically binds to CD83, NKp44, NKp30, NKp46 or NKG2D and any combination thereof.

In one embodiment of the invention, the chimeric receptor includes at least one intracellular domain of a T cell co-stimulatory molecule selected from the group consisting of 4-1BB, ICOS, CD27, OX40, CD28, CTLA4 and PD-1, and any combination thereof.

In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence of the 4-1BB intracellular domain (SEQ ID NO: 44) or an amino acid sequence with 95-99% identity to SEQ ID NO: 44. In another embodiment, the T cell costimulatory signaling domain -cells includes or consists of an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 44.

(SEQ ID NO: 44)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence of the CD27 intracellular domain (SEQ ID NO: 45) or an amino acid sequence with 95-99% identity to SEQ ID NO: 45. In another embodiment, the T cell costimulatory signaling domain includes or consists of an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 45.

(SEQ ID NO: 45)

QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP

In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence of the CD28 intracellular domain (SEQ ID NO: 46) or an amino acid sequence with 95-99% identity to SEQ ID NO: 46. In another embodiment, the T cell costimulatory signaling domain includes or consists of an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 46.

(SEQ ID NO: 46)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

In one embodiment of the invention, the chimeric receptor includes a combination of at least two intracellular domains of a T cell co-stimulatory molecule, preferably selected from a CD28 intracellular domain, a CD27 intracellular domain, and a 4-1BB intracellular domain.

In one embodiment, the chimeric receptor comprises a 4-1BB intracellular domain amino acid sequence (SEQ ID NO: 44) or an amino acid sequence with 95-99% identity to SEQ ID NO: 44 and a CD27 intracellular domain amino acid sequence (SEQ ID NO: 45) or amino acid sequence sequence with 95-99% identity to SEQ ID NO: 45.

In another embodiment, the chimeric receptor comprises a 4-1BB intracellular domain amino acid sequence (SEQ ID NO: 44) or an amino acid sequence with 95-99% identity to SEQ ID NO: 44 and a CD28 intracellular domain amino acid sequence (SEQ ID NO: 46) or amino acid sequence sequence with 95-99% identity to SEQ ID NO: 46.

In yet another embodiment, the chimeric receptor comprises a CD27 intracellular domain amino acid sequence (SEQ ID NO: 45) or an amino acid sequence with 95-99% identity to SEQ ID NO: 45 and a CD28 intracellular domain amino acid sequence (SEQ ID NO: 46) or an amino acid sequence with 95-99% identity to SEQ ID NO: 46.

In one embodiment, the chimeric receptor comprises a 4-1BB intracellular domain amino acid sequence (SEQ ID NO: 44) or an amino acid sequence with 95-99% identity to SEQ ID NO: 44 and a CD27 intracellular domain amino acid sequence (SEQ ID NO: 45) or amino acid sequence a sequence with 95-99% identity to SEQ ID NO: 45 and an amino acid sequence of the intracellular domain of CD28 (SEQ ID NO: 46) or an amino acid sequence with 95-99% identity to SEQ ID NO: 46.

Thus, in one embodiment, the nucleic acid sequence encoding the T cell co-stimulatory signaling domain includes a 4-1BB intracellular domain nucleic acid sequence (SEQ ID NO: 47) or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 47 and/or a CD27 intracellular domain nucleic acid sequence (SEQ ID NO: 48) or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 48, and/or a CD28 intracellular domain nucleic acid sequence (SEQ ID NO: 49) or nucleic acid sequence with 95-99% identity to SEQ ID NO: 49.

(SEQ ID NO: 47)

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

(SEQ ID NO: 48)

CAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCC

(SEQ ID NO: 49)

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

In one embodiment, the intracellular signaling domain of the chimeric receptor of the invention comprises:

- amino acid sequence of the intracellular domain 4-1BB SEQ ID NO: 44 or amino acid sequence with 95-99% identity to SEQ ID NO: 44 and/or amino acid sequence of the intracellular domain CD27 SEQ ID NO: 45 or amino acid sequence with 95-99% identity with SEQ ID NO: 45, and/or the amino acid sequence of the intracellular domain of CD28 SEQ ID NO: 46 and/or the amino acid sequence with 95-99% identity with SEQ ID NO: 46; And

- amino acid sequence of the CD3-zeta intracellular domain SEQ ID NO: 38-41 or amino acid sequence with 95-99% identity with SEQ ID NO: 38-41;

where the sequences contained in the intracellular domain are expressed in the same framework and as a single polypeptide chain.

Thus, in one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of the chimeric receptor of the invention comprises:

- a nucleic acid sequence of the 4-1BB intracellular domain SEQ ID NO: 47 or a nucleic acid sequence with 95-99% identity to SEQ ID NO: 47 and/or a nucleic acid sequence of the CD27 intracellular domain SEQ ID NO: 48 or a nucleic acid sequence with 95 -99% identity to SEQ ID NO: 48, and/or CD28 intracellular domain nucleic acid sequence SEQ ID NO: 49 or nucleic acid sequence with 95-99% identity to SEQ ID NO: 49; And

- nucleic acid sequence of the CD3-zeta intracellular domain SEQ ID NO: 42 or SEQ ID NO: 43, or a sequence with 95-99% identity to SEQ ID NO: 42 or SEQ ID NO: 43.

In one embodiment, the intracellular signaling domain of a chimeric receptor of the invention includes at least two different domains (e.g., a primary signaling domain and at least one intracellular T cell co-stimulatory molecule domain) that may be randomly associated with each other or in that order.

Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can form a link between the individual signaling domains. In one embodiment, a glycine-serine doublet (GS) is used as a suitable linker. In one embodiment, a single amino acid is used as a suitable linker, eg alanine (A), glycine (G).

In another embodiment, the intracellular signaling domain of a chimeric receptor of the invention includes two or more, for example 2, 3, 4, 5 or more, co-stimulatory signaling domains.

In another embodiment, two or more, eg 2, 3, 4, 5 or more, costimulatory signaling domains are separated by a linker molecule, eg a linker molecule as described above.

In one embodiment, the intracellular signaling domain of a chimeric receptor of the invention comprises a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41) and a CD28 co-stimulatory signaling domain (preferably SEQ ID NO: 46).

In another embodiment, the intracellular signaling domain of a chimeric receptor of the invention comprises a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41) and a 4-1BB co-stimulatory signaling domain (preferably SEQ ID NO: 44).

In another embodiment, the intracellular signaling domain of a chimeric receptor of the invention comprises a CD3 zeta signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41) and a CD27 signaling domain (preferably SEQ ID NO: 45).

In one embodiment, the chimeric receptor of the invention further includes a leader sequence located N-terminal to the HLA-A2 binding domain. A non-limiting example of a leader sequence is a CD8 leader sequence, which may contain or consist of the sequence SEQ ID NO: 50.

(SEQ ID NO: 50)

MALPVTALLLPLALLLHAARP

In one embodiment, the chimeric receptor further includes a label, such as, for example, a label for quality control, enrichment, in vivo tracking, or the like. The specified tag can be localized at the N-terminus, C-terminus and/or inside. Examples of tags that can be used in the chimeric receptor of the invention are well known to one skilled in the art. For example, but without limitation, a tag used in the invention may be a tag selected from the group consisting of a hemagglutinin tag, a polyarginine tag, a polyhistidine tag, a Myc tag, a Strep tag, an S-tag, a HAT tag, a 3xTag Flag, calmodullin binding peptide tags, SBP tags, chitin binding domain tags, GST tags, maltose binding protein tags, fluorescent protein tags (eg, eGFP having the sequence SEQ ID NO: 68), T7 tags, V5 tags, and Xpress tags. -tags. Other examples of the label include, but are not limited to, NWSHPQFEK (SEQ ID NO: 51) or SAWSHPQFEK (SEQ ID NO: 52).

(SEQ ID NO: 51)

NWSHPQFEK

(SEQ ID NO: 52)

SAWSHPQFEK

(SEQ ID NO: 68)

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG PVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

In one embodiment, the chimeric receptor of the invention further includes a ribosomal cleavage site located C-terminal to the intracellular signaling domain. A non-limiting example of a ribosomal cleavage site is the P2A ribosomal cleavage site sequence ASGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 61).

According to a first embodiment, the chimeric receptor of the invention comprises an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents V or L, X17 represents A or P, X18 represents is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), optionally an extracellular hinge domain, a transmembrane domain and a primary T cell signaling domain.

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 marker-binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 marker-binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention comprises an HLA-A2 marker-binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

According to a second embodiment, the chimeric receptor of the invention comprises an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents V or L, X17 represents A or P, X18 represents is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), optionally an extracellular hinge domain, a transmembrane domain, one intracellular T cell co-stimulatory molecule domain, and a primary signaling domain T cells.

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SEQ ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SEQ ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

According to a third embodiment, the chimeric receptor of the invention comprises an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents V or L, X17 represents A or P, X18 represents is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), optionally an extracellular hinge domain, a transmembrane domain, two intracellular T cell co-stimulatory molecule domains, and a primary signaling domain T cells.

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD8 hinge domain (preferably SEQ ID NO: 26); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SED ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; IgG4 hinge domain (preferably SEQ ID NO: 28); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain, preferably containing the amino acid sequence of SEQ ID NO: 30; CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; an IgD hinge domain (preferably SEQ ID NO: 30); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In another embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 34); CD8 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SED ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); intracellular domain 4-1BB (preferably SED ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD8 transmembrane domain (preferably SEQ ID NO: 34); CD27 intracellular domain (preferably SEQ ID NO: 45); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD27 intracellular domain (preferably SEQ ID NO: 45); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); intracellular domain 4-1BB (preferably SEQ ID NO: 44); CD28 intracellular domain (preferably SEQ ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41). In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain; CD28 hinge domain (preferably SEQ ID NO: 32); CD28 transmembrane domain (preferably SEQ ID NO: 36); CD27 intracellular domain (preferably SED ID NO: 45); CD28 intracellular domain (preferably SED ID NO: 46); and a CD3-zeta primary signaling domain (preferably SEQ ID NO: 38, 39, 40 or 41).

In one embodiment, the chimeric receptor of the invention includes (i) an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 represents S or T, X12 represents L or Q, X13 represents S or A, X14 represents V or I, X15 represents K or T, X16 represents V or L, X17 represents A or P, X18 represents is L or F and X19 is G or A, more preferably one of SEQ ID NOs: 70-72 or 74-76), (ii) human CD28 hinge region, (iii) human CD28 transmembrane domain, (iv) intracellular domain Human CD28 and (v) the intracellular domain of the human CD3ζ chain.

In one embodiment, a portion of the chimeric receptor comprising the human CD28 hinge region, the human CD28 transmembrane domain, the human CD28 intracellular domain, and the human CD3ζ chain intracellular domain corresponds to the amino acid sequence of SEQ ID NO: 53 or 54 or an amino acid sequence with 95-99% identity to SEQ ID NO: 53 or 54.

(SEQ ID NO: 53)

AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 54)

AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In another embodiment, the chimeric receptor of the invention includes (i) an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A , X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), (ii) human CD8 hinge region, (iii) human CD8 transmembrane domain, (iv) intracellular human 4-1BB domain and (v) human CD3ζ intracellular domain.

In another embodiment, the chimeric receptor of the invention includes (i) an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A , X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), (ii) human CD8 hinge region, (iii) human CD8 transmembrane domain, (iv) intracellular human 4-1BB domain and (v) human CD3ζ intracellular domain. In one embodiment, a portion of the chimeric receptor comprising a human CD8 hinge region, a human CD8 transmembrane domain, a human 4-1BB intracellular domain, and a human CD3ζ intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 55, 56, 57 or 58, or any amino acid sequence with 95-99% identity to SEQ ID NO: 55-58.

(SEQ ID NO: 55)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 56)

NWSHPQFEKMHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 57)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 58)

NWSHPQFEKMHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76) associated with the amino acid sequence of SEQ ID NO: 55-58 or a sequence or amino acid sequence with 95-99% identity to SEQ ID NO: 55-58.

In one embodiment, the chimeric receptor of the invention includes or consists of the sequence set forth in SEQ ID NO: 77, or any amino acid sequence with 95-99% identity to SEQ ID NO: 77.

(SEQ ID NO: 77)

SQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDX₁VMTQX₂PX₃X₄LX₅VX₆X₇GX₈X₉X₁₀X_elevenISCRSSQSIVHSNGNTYLEWYX₁₂QKPGQX₁₃PRLLIYKVSNRFSGX₁₄PDRFSGSGSGTDFTLX₁₅ISRX₁₆EX₁₇EDX₁₈X₁₉VYYCFQGSHVPRTFGGGTKLEIKRRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I , X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A.

In one embodiment, the chimeric receptor of the invention further includes a leader sequence at the N-terminus, preferably having the sequence SEQ ID NO: 50, and includes or consists of the sequence SEQ ID NO: 81, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F, and X19 is G or A, or any amino acid sequence with 95-99% identity to SEQ ID NO: 81.

(SEQ ID NO: 81)

MALPVTALLLPLALLLHAARPSQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDX₁VMTQX₂PX₃X₄LX₅VX₆X₇GX₈X₉X₁₀X_elevenISCRSSQSIVHSNGNTYLEWYX₁₂QKPGQX₁₃PRLLIYKVSNRFSGX14PDRFSGSGSGTDFTLX15ISRX16EX17EDX18X₁₉VYYCFQGSHVPRTFGGGTKLEIKRRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention includes or consists of the sequence set forth in SEQ ID NO: 78, or any amino acid sequence with 95-99% identity to SEQ ID NO: 78.

(SEQ ID NO: 78)

SQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVTLGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPRLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKRRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention further includes a leader sequence at the N-terminus, preferably having the sequence of SEQ ID NO: 50, and includes or consists of the sequence of SEQ ID NO: 82 or any amino acid sequence with 95-99% identity to SEQ ID NO: : 82.

(SEQ ID NO: 82)

MALPVTALLLPLALLLHAARPSQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVTLGEPASISCRSSQSIVHSNGNTYLEWYL QKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIKRRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention includes or consists of the sequence set forth in SEQ ID NO: 79, or any amino acid sequence with 95-99% identity to SEQ ID NO: 79.

(SEQ ID NO: 79)

A VSNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKRRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention further includes a leader sequence at the N-terminus, preferably having the sequence SEQ ID NO: 50, and includes or consists of the sequence SEQ ID NO: 83 or any amino acid sequence with 95-99% identity to SEQ ID NO : 83.

(SEQ ID NO: 83)

MALPVTALLLPLALLLHAARPSQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQSPSSLSVTLGDRVSISCRSSQSIVHSNGNTYLEWY QQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEPEDLGVYYCFQGSHVPRTFGGGTKLEIKRRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention includes or consists of the sequence set forth in SEQ ID NO: 80, or any amino acid sequence with 95-99% identity to SEQ ID NO: 80.

(SEQ ID NO: 80)

SQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGERATISCRSSQSIVHSNGNTYLEWYQQKPGQAPRLLIYKV SNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCFQGSHVPRTFGGGTKLEIKRRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention further includes a leader sequence at the N-terminus, preferably having the sequence SEQ ID NO: 50, and includes or consists of the sequence SEQ ID NO: 84 or any amino acid sequence with 95-99% identity to SEQ ID NO : 84.

(SEQ ID NO: 84)

MALPVTALLLPLALLLHAARPSQVQLVQSGPEVKKPGASVKVSCKASGYTFTSYHIQWVRQAPGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLTSEDTAVYYCAREGTYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGERATISCRSSQSIVHSNGNTYLEWYQ QKPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCFQGSHVPRTFGGGTKLEIKRRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In another embodiment, the chimeric receptor of the invention includes (i) an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A , X4 is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76), (ii) human CD8 hinge region, (iii) human CD8 transmembrane domain, (iv) intracellular human CD28 domain and (v) human CD3ζ intracellular domain. In one embodiment, a portion of the chimeric receptor comprising a human CD8 hinge region, a human CD8 transmembrane domain, a human CD28 intracellular domain, and a human CD3ζ intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 59 or 60, or any amino acid sequence from 95-99 % identity with SEQ ID NO: 59 or 60.

(SEQ ID NO: 59)

MHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR

(SEQ ID NO: 60)

MHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment, the chimeric receptor of the invention includes an HLA-A2 binding domain (preferably SEQ ID NO: 69 or 73, where X1 is V or I, X2 is T or S, X3 is L or S or A, X4 is is S or T, X5 is P or S, X6 is T or S, X7 is L or P, X8 is E or D, X9 is P or R, X10 is A or V, X11 is S or T, X12 is L or Q, X13 is S or A, X14 is V or I, X15 is K or T, X16 is V or L, X17 is A or P, X18 is L or F and X19 is G or A, more preferably one of SEQ ID NO: 70-72 or 74-76) related to the amino acid sequence of SEQ ID NO: 59-60 or a sequence or amino acid sequence with 95-99% identity to SEQ ID NO: 59-60.

The present invention also relates to an immune cell expressing a CAR as described above, and to a population of such immune cells.

In one embodiment, a nucleic acid encoding a CAR of the present invention is introduced into an immune cell, thereby producing an engineered cell expressing the CAR of the present invention on the surface of the cell.

According to the invention, the immune cell of the invention is genetically modified to express a chimeric receptor that recognizes HLA-A2. Thus, the immune cell of the invention can be defined as a redirected immune cell.

As used herein, the term “redirected” refers to an immune cell bearing a chimeric receptor, as described herein, that confers on the immune cell the ability to bind and be activated by a ligand (i.e., HLA-A2) that is different from the ligand to which the immune cell is or would be specific or by which it would be activated. In one embodiment, the chimeric receptor 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 of the invention is a mammalian immune cell, preferably a human immune cell.

In one embodiment, the immune cell of the invention has been frozen and thawed.

In one embodiment, the immune cell is selected from the group consisting of lymphocytes, myeloid cells, and any combination thereof.

In one embodiment, the immune cell is a lymphocyte 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 CD4+ T cells, CD8+ T cells, γδ T cells, double negative (DN) T cells, and any combination thereof.

In one embodiment, the immune cell is a CD4+ T cell, such as, for example, a T helper cell, a regulatory T cell, and any combination thereof.

In one embodiment, the immune cell is a CD4+ regulatory T cell (Treg). 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), preferably a CD4+ FOXP3+ regulatory T cell.

In one embodiment, the immune cell is a CD8+ T cell, such as, for example, a cytotoxic CD8+ T cell or a CD8+ regulatory T cell.

Preferably, the immune cell is a CD8+ regulatory T cell (Treg). 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 immune cell is an INFγ + IL10 + IL34 + CD8+ CD45RClow regulatory T cell.

In one embodiment, the immune cell is a γδ T cell, preferably a regulatory γδ T cell.

In one embodiment, the immune cell is a DN T cell, preferably a regulatory DN T cell.

In one embodiment, the immune cell is a B cell, preferably a regulatory B cell. In one embodiment, the regulatory B cell is a CD19+CD24hiCD38hi B cell.

In one embodiment, the immune cell is an NK cell, preferably a regulatory NK cell.

In one embodiment, the immune cell is a myeloid-derived cell, preferably selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, or any combination thereof.

In one embodiment, the immune cell is a macrophage, preferably a regulatory macrophage.

In one embodiment, the immune cell is a dendritic cell, preferably a regulatory dendritic cell.

Preferably, the immune cell is a regulatory immune cell, such as, for example, any regulatory immune cell suitable for use in cell therapy.

In one embodiment, the regulatory immune cell is selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory γδ T cell, a regulatory DN T cell, a regulatory B cell, a 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. Examples of CD8+ regulatory T cells include, but are not limited to, 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 regulatory immune cell is a regulatory γδ T cell.

In one embodiment, the regulatory immune cell is a regulatory DN T cell.

In one embodiment, the regulatory immune cell is a regulatory B cell. Examples of regulatory B cells include, but are not limited to, CD19+CD24hiCD38hi B cells.

In one embodiment, the regulatory immune cell is a regulatory NK cell.

In one embodiment, the regulatory immune cell is a regulatory macrophage.

In one embodiment, the regulatory immune cell is a regulatory dendritic cell.

In one embodiment, the regulatory immune cell is a regulatory T cell, particularly a thymus-derived Treg or an adaptive or induced Treg. Examples of Tregs include, but are not limited to, a CD4+ FOXP3+ regulatory T cell or a CD4+ 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+. Preferably, 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 permeabilized prior to flow cytometry analysis, allowing intracellular proteins to be detected.

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 using FACS.

The terms “expressing (or +)” and “non-expressing (or -)” are well known in the art and refer to the level of expression of the cell marker of interest, in which the level of expression of the cell marker corresponding to “+” is high or medium, also called “+/-”, and the expression level of the cell marker corresponding to “-” is 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 the population of cells being analyzed 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 different populations of cells.

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 staining intensity are designated as “hi,” and cells falling in the top half of the population are classified as “+.” These cells falling below 50% fluorescence intensity are designated as "lo", and cells below 5% are designated as "-" cells.

The level of expression of a cellular 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 population of cells stained with a fluorescently labeled antibody with irrelevant specificity, but with the same isotype, the same fluorescent probe and originating from the same species (referred to as isotype control). Cells from a population stained with fluorescently labeled antibodies specific for that marker and showing equivalent MFI or lower MFI than cells stained with isotype controls do not express that marker and are therefore designated as (-) or negative. Cells from a population stained with a fluorescently labeled antibody specific for that marker and exhibiting an MFI value superior to that of cells stained with isotype controls expressing that marker and are therefore designated (+) 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, an 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 on 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, the CAR of the invention includes a primary T cell signaling domain (such as, for example, CD3zeta), and the second receptor includes a costimulatory signaling domain (such as, for example, 4-1BB, CD28, or a combination of a 4-1BB costimulatory signaling domain 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 a costimulatory signaling domain of 4-1BB and CD28), and the second receptor includes a primary T cell signaling domain (such as, for example, CD3zeta ).

Therefore, in accordance with these embodiments, full activation of the immune cell of the invention requires both binding of the CAR of the invention to HLA-A2 and 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 the autoimmune response.

Examples of ligands that may be recognized by the second receptor include, but are not limited to, food antigens from the normal human diet, self-antigens, inhaled allergens, ingested allergens, or contact allergens.

The term "food antigen from the 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. Food antigens may 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 autoantigen 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 graft 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 protein, oligodendrocyte-specific protein (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, human cartilage glycoprotein 39 (HCgp39) peptides, HSPs, heterogeneous nuclear ribonucleoprotein (hnRNP) A2 peptides, hnRNP B1, hnRNP D, Ro60/52, HSP60, HSP65 , HSP70 and HSP90, BiP, keratin, vimentin, fibrinogen, collagen peptide types I, III, IV and V, 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, 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, plectin, 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 graft rejection or GVHD. Examples of such antigens include, but are not limited to, graft-specific or host-specific MHC, β2-microglobulin, ABO system antigens, Rh system antigens (particularly C, c, E et e and D system antigens), 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 vaccination), HLA-B (particularly during the first two years after vaccination ), 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, I antigen, desmoglein 1 or 3 (Dsg1/3), BP180, BP230, nicotinic postsynaptic acetylcholine receptors, platelet integrin, GpIIb:IIIa, collagen (e.g. alpha-3 chain (IV) 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, antineutrophil 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), anti-liver and kidney microsome-1 antibodies (ALKM-1), anti-liver cytosol antibodies -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 IA2 or ICA512) PLA2R1 and THSD7A1.

In one embodiment, said ligand is selected from the group consisting of ovalbumin, MOG, type II collagen moieties, 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 includes 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 includes an extracellular ligand binding domain comprising an HLA-A2 binding domain and another ligand binding domain recognizing a ligand other than HLA-A2. In one embodiment, said ligand binding domain is a bifunctional antibody that recognizes both HLA-A2 and a separate ligand.

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

The invention also relates to an isolated and/or substantially purified population of immune cells as defined above.

Thus, one aspect of the invention is an isolated and/or substantially purified population of immune cells, wherein the cells of the population contain a CAR, wherein said CAR recognizes HLA-A2.

As used herein, the term "isolated population" refers to a cell population that is removed from its natural environment (such as peripheral blood) and that is isolated, purified, or separated and is at least about 75% free, 80% free , 85% free and preferably about 90%, 95%, 96%, 97%, 98%, 99% free from other cells with which it is naturally present but which lack the cell surface markers on which it was isolated cells.

The present invention also relates to an enriched population of immune cells as defined above.

In one embodiment of the invention, the isolated, purified and/or enriched population of immune cells according to the invention was frozen and thawed.

Another object of the invention is a vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) described above.

Examples of vectors that can be used in the present invention include, but are not limited to, DNA vector, RNA vector, plasmid, phagemid, phage derivative, animal virus and cosmid.

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 manuals on virology and molecular biology. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses.

Typically, a suitable vector includes an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease convenience sites, and one or more selectable markers (for example, as described in WO 01/96584; WO 01/29058; and US6 326,193, incorporated herein by reference).

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 methods known in the art. The recombinant virus can then be isolated and delivered to the target 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. Typically, as regards the main promoter, they are located in the region of 30-110 bp. upstream of the start site, although it has recently been shown that a number of promoters also contain functional elements downstream of the start site, and enhancer elements are typically located -2000 bp. up the sequence from the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the distance between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that 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 growth factor-la (EF-la). 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 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Moreover, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothioneine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. In addition, bidirectional promoters that provide efficient and coordinated expression of two or more genes may also be of interest in the present invention. Examples of bidirectional promoters include, but are not limited to, the promoters described in US2006200869, incorporated herein by reference, disclosing a bidirectional promoter comprising i) a first minimal promoter sequence derived from cytomegalovirus (CMV) or mouse mammary tumor virus (MMTV) genomes and ii) a fully effective promoter sequence derived from an animal gene.

To assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into an immune cell may also contain a selectable marker gene, such as, for example, CD34 or a reporter gene, or both, to facilitate identification and selection of expressers. cells from a population of cells that were attempted to be transfected or infected through viral vectors. In other aspects, a selectable marker can be applied to a single DNA fragment and used in a cotransfection 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.

In some embodiments of the invention, suicide gene technology may be used. Various suicide gene technologies have been described in the field depending on their mechanism of action (Jones et al. Frontiers in Pharmacology, 2014 (5): 254). Examples of gene-directed enzyme prodrug therapy (GDEPT) that convert a non-toxic drug into a toxic drug include herpes simplex virus thymidine kinase (HSV-TK) and cytosine deaminase (CD). Other examples are chimeric proteins consisting of a drug binding domain associated with apoptotic components, such as, for example, inducible Fas systems (iFas) or inducible caspase 9 (iCasp9). Other examples include therapeutic antibody mediated systems, such as inducing overexpression of c-myc on the surface of an engineered cell to induce their deletion by administering an anti-c-myc antibody. A similar system describes the use of EGFR compared to the c-myc system.

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 expressed by the recipient organism or tissue and that encodes a polypeptide whose expression results in some easily detectable property, such as enzymatic activity. Reporter gene expression was analyzed at appropriate times after DNA injection into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (eg, Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known methods or obtained commercially. In general, the construct with the 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 readily introduced into a host cell, for example 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, membrane disruption, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanoparticles (nanospheres, 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).

When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. It is envisaged that lipid formulations will be used to introduce 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 lipid-bound nucleic acid may be encapsulated in the aqueous interior of the liposome, embedded in the lipid bilayer of the liposome, attached to the liposome through a linking 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, mixed with a lipid, combined with a lipid, suspended in a lipid, contained 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 can be naturally occurring or synthetic lipids. For example, lipids include fat droplets that occur naturally 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). Lipid stock solutions in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a variety of single- and multilayer lipid carriers formed by 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 multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-organization to form closed structures and trap water and solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also covered. For example, lipids can 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 a host cell, various assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, “molecular biology” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example, by immunological means (ELISA and Western blotting) or assays described herein, to identify agents within the scope of the invention.

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 as a form of transient transfection. RNA can be produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). 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 source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, 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 to be PCR includes an open reading frame. The DNA may come from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene or portion 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, including 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not typically 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 portions of DNA that are ligated together may be from the same organism or from more than one organism.

PCR can be used to create a template for in vitro transcription of mRNA that is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to the regions of DNA that will 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 mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the putative target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion 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' UTR. Primers can also be designed to amplify a portion of a gene that encodes a specific domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are prepared by synthetic methods that 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. "Upstream" is used herein to refer to the location 5', the DNA sequence that is 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 lies downstream of the DNA sequence to be amplified. The term "downstream" is used herein to refer to the location 3' of the DNA sequence to be amplified relative to the coding strand.

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

Chemical structures capable of providing translation stability and/or efficiency may also be used. RNA preferably has 5' and 3' UTRs. In one embodiment, the 5'-UTR has a length of zero to 3000 nucleotides. The length of the 5' and 3' UTR sequences to be added to the coding region can be modified 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 required to achieve optimal translation efficiency after transfection of the transcribed RNA. The 5' and 3' UTRs may be 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 through any other template modifications. The use of UTR sequences that are not endogenous to the gene of interest may be useful for altering the stability and/or translational efficiency of RNA. For example, it is known that AU-rich elements in 3' UTR sequences can reduce mRNA stability. Therefore, 3'UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of the UTRs that are well known in the art.

In one embodiment, the 5'-UTR may contain a 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 modified by adding the 5' UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but do not appear to be required for all RNAs to ensure efficient translation. The requirement of Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5'-UTR may be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent degradation of the mRNA by an 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 transcribed sequence. 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 before the open reading frame to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as 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 determine 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 is polyadenylated after transcription.

On a linear DNA template, phage 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).

The generally accepted 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 obtained from bacterial cells are often heavily contaminated with deletions and other aberrations. This makes cloning procedures not only labor-intensive and time-consuming, but often unreliable. That is why a method that allows the construction of DNA templates with a polyA/T 3' region without cloning is highly desirable.

A polyA/T segment of transcriptional DNA template can be generated 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 RNA degradation. In general, 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 following 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 RNA translation efficiency. In addition, the addition of various 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 analogues can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogues can further enhance RNA stability.

5' caps on RNA also provide stability to RNA molecules. In a preferred embodiment, the RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is provided 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)).

RNAs 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 artificially engineered sequence that initiates cap-independent ribosome binding to the 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 which 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 into cells using a retroviral or lentiviral vector. CAR-expressing retroviral and lentiviral vectors can be delivered into 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, bound or naked vectors. The method used can be used for any purpose where stable expression is required or sufficient.

In another embodiment, CAR sequences are delivered into cells using in vitro transcribed mRNA. In vitro transcribed CAR mRNA can be delivered into various types of eukaryotic cells, as well as into 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 expansion 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, thymic tissue, tissue from the site of infection, ascites, pleural effusion, splenic 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 a subject using any number of techniques known to one of ordinary skill in the art, such as Ficoll™ separation or the Sepax separation system. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis may 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 cleaning solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca 2+ -free, Mg 2+ -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 the culture medium.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, for example, by PERCOLL™ gradient centrifugation or by countercurrent centrifugation. A specific subset of T cells, such as CD4+ CD25+, in particular CD4+ CD25+ CD127lo, such as, for example, CD4+ CD25+ CD127loCD45RA+, can be further selected by positive or negative selection methods. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient to positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period is from 30 minutes to 36 hours or more, and all integer values in between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred embodiment, the time period is from 10 to 24 hours. In one preferred embodiment, the incubation time is 24 hours. To isolate T cells from leukemia patients, using a longer incubation time, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or in immunocompromised individuals. Thus, by simply shortening or lengthening the time that T cells are allowed to bind to CD3/CD28 beads and/or increasing or decreasing the ratio of beads to T cells (as described later herein), T cell subpopulations can be preferentially selected. cells for or against at the start of the culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, T cell subsets can be preferentially selected for or against at the start of culture or at other desired times. One skilled in the art will recognize that multiple rounds of selection may also be used in the context of this invention.

In another embodiment, it may be desirable to perform a selection procedure and use “unselected” cells during the activation and expansion process. "Unselected" cells may also undergo additional rounds of selection. Enrichment of the T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to negatively selected cells. One method is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry, which uses a panel of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the 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 regulatory T cells typically having the following phenotype CD4+, CD25+, CD62Lhi, GITR+ and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted using anti-CD25 conjugated beads or other similar selection method.

Cell and surface concentrations (eg, particles such as beads) can be varied to isolate the desired cell population using positive or negative selection. 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) to ensure maximum cell-bead contact. 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 is used. In a further embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml are 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. Use of high concentrations may result in increased cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations allows for more efficient capture of cells that may poorly express target antigens of interest, such as CD28-negative T cells, or from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, and etc.)). Such cell populations may have therapeutic value and would be desirable to obtain.

T cells for stimulation can also be frozen after washing. Without wishing to be bound by theory, we assume that the freezing and thawing step provides a more uniform product by removing granulocytes and to some extent monocytes from the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be useful in this context, one method involves the use of 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% plasmalyte-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 a rate of 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 were thawed and washed as described herein and left for one hour at room temperature before activation.

Also contemplated within the scope of the invention is the collection of blood or apheresis product samples from a 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 as needed, and the desired cells, such as T cells, can be isolated and frozen for subsequent use in T cell therapy for any number of diseases or conditions. that would benefit from T cell therapy such as that described herein. In one embodiment, a blood sample or apheresis is collected from a normally healthy subject. In some embodiments, a blood sample or apheresis is collected from a normally healthy subject who is at risk of developing disease but has not yet developed disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and used at a later date.

Whether before or after genetic modification of T cells (preferably Treg cells) to express the desired CAR, T cells can be activated and expanded, typically using methods as described, for example, in US 6,352,694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and US20060121005, incorporated herein by reference.

Typically, a population of T cells (preferably Treg cells) of the invention is expanded by contacting a surface to which an agent is attached, a stimulatory signal associated with the CD3/TCR complex, and a ligand that stimulates a co-stimulatory molecule on the surface of the T cell (preferably Treg) cell population. In particular, the T (preferably Treg) cell population can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (for example, bryostatin) in combination with a calcium ionophore. To co-stimulate an accessory molecule on the surface of T cells, a ligand is used that binds the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-0CD28 antibody under conditions suitable to stimulate T cell proliferation. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the proliferation of CD4+ T cells. Examples of anti-CD28 antibodies include, but are not limited to, 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France). Other propagation methods commonly known in the art may be used (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(l-2):53-63, 1999).

In some embodiments, the primary stimulatory signal and the co-stimulatory signal for a population of T cells (preferably Treg) cells can be provided by different protocols. For example, the agents providing each signal may be in solution or bound to the surface. When bound to a surface, agents can be bound to the same surface (i.e., in a “cis” formation) or to other surfaces (i.e., in a “trans” formation). Alternatively, one agent may be bound to 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 cross-linked 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, US 20040101519 and 20060034810, incorporated herein by reference, for artificial antigen presenting cells (aAPC), which are contemplated for use in the activation and expansion of T cells in the present invention.

In one embodiment, the two agents are immobilized on the beads, either on the same bead, ie "cis", or on separate beads, ie "trans". For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen binding fragment thereof, and the agent providing the costimulation signal is an anti-CD28 antibody or an 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 antibody bound to the beads is used for CD4+ T cell expansion and T cell growth. In some aspects of the present invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to the increase observed using a 1:1 ratio. 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 associated with particles than anti-CD3 antibodies, that is, the CD3:CD28 ratio is less than one. In some embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a ratio of 1:100 CD3:CD28 antibodies bound to beads is used. In another embodiment, a 1:75 ratio of CD3:CD28 antibodies bound to beads is used. In a further embodiment, a 1:50 ratio of CD3:CD28 antibodies bound to the beads is used. In another embodiment, a 1:30 ratio of CD3:CD28 antibodies bound to beads is used. In one preferred embodiment, a 1:10 ratio of CD3:CD28 antibodies bound to beads is used. In another embodiment, a 1:3 ratio of CD3:CD28 antibodies bound to beads is used. In another embodiment, a 3:1 ratio of CD3:CD28 antibodies bound to beads 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 will be readily appreciated by those skilled in the art, the particle to cell ratio 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 cell to particle ratio is in the range of 1:100 to 100:1, and any integer values in between, and in other embodiments, the ratio is in the range of 1:9 to 9:1, and any integer values in between may 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, however some preferred values 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 and 15:1, with one preferred ratio being at least 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 preferred particle:cell ratio is 1:5. In other embodiments, the particle to cell ratio may vary depending on the day of stimulation. For example, in one embodiment, the particle-to-cell ratio is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for up to 10 days in final ratios. 1:1 to 1:10 (based on the number of cells on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment of the invention, 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 particle to cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment of the invention, 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 depending on the particle size and the size and type of cells.

In other embodiments of the present invention, T cells (preferably 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, the agent-coated beads and cells are not separated but cultured together before culturing. 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 cell surface markers, thereby causing stimulation of the cells.

For example, cell surface proteins can be ligated, allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 × 28 beads) to contact cells in the Treg cell population. In one embodiment, cells (eg, 104-109 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 calcium and magnesium). Again, those skilled in the art will readily understand that any concentration of cells can be used. For example, the target cell may be very rare in the sample and constitute only 0.01% of the sample, or the entire sample (i.e., 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 particles and cells are mixed (ie, increase the concentration of cells) to ensure maximum 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 is used. In a further embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml are 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. Use of high concentrations may result in increased cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations allows for more efficient capture of cells that may poorly express target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and would be desirable in certain embodiments.

In one embodiment of the present invention, the mixture may be cultured for anywhere from a few hours (about 3 hours) to about 14 days, or any hourly integer value 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, such that the T cell culture time may be 60 days or more. Conditions suitable for T cell culture include suitable media (eg, minimal essential medium or RPMI Media 1640 or X-vivo 15 (Lonza)), which 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 additives known to one 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. The media may include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, either without serum or supplemented with appropriate amounts serum (or plasma) or a certain set of hormones and/or the amount of cytokines sufficient for the growth and reproduction of T cells. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the cell cultures that are to be injected into the subject. The target cells are maintained under conditions necessary to support growth, such as suitable temperature (eg, 37°C) and atmosphere (eg, air plus 5% CO2).

T cells that have been stimulated for different times may have different characteristics. For example, typical blood mononuclear cell or peripheral blood cell products have a population of helper T cells (Th, CD4+) that is larger than a population of cytotoxic or suppressor T cells (Tc, CD8+). Expression of T cells ex vivo by stimulation of the CD3 and CD28 receptors results in the formation of a T cell population that, until about 8-9 days, consists predominantly of Th cells, while after about 8-9 days the T cell population includes all a large population of Tc cells. Accordingly, depending on the purpose of treatment, it may be advantageous to administer to the subject a population of T cells consisting predominantly of Th cells. Likewise, if an antigen-specific subset of Tc cells has been isolated, it may be advantageous to expand that subset to a greater extent.

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

In one embodiment of the invention, T cells can be cultured in the presence of rapamycin to produce 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, where the T cells are cultured with a T cell receptor (TCR)/CD3 activator such as, for example, TCR/CD3 antibodies, a TCR costimulator 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 expression of a CAR may 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 a very small functional TCR on its surface. Alternatively, the T cell may express a substantially disrupted 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 cause an adverse immune response in the host.

In another embodiment, the genetically modified cells described herein can be engineered so that they do not express functional HLA on their surface. For example, an immune cell described herein can be engineered to reduce cell surface expression of HLA, such as 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 a 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 expression of a functional TCR and/or HLA can be generated by any suitable method, including knockout or knockdown of one or more TCR and/or HLA subunits. For example, an immune cell may involve TCR and/or HLA knockdown using siRNA, shRNA, clustered regularly interspaced 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 (combining the TAL effector with the 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 expression and/or HLA expression can be inhibited using siRNA or shRNA that targets the nucleic acid encoding the TCR and/or HLA in the T cell. Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system, such as, for example, a lentiviral expression system. Exemplary siRNAs and shRNAs that suppress the expression of HLA class I and/or HLA class II genes are described, for example, in US 2007/0036773. Typical shRNAs that suppress the expression of TCR components are described, for example, in US2012/0321667.

As used herein, the term “CRISPR” or “TCR and/or HLA CRISPR” or “TCR and/or HLA inhibition CRISPR” refers to a set of regularly interspaced clustered short palindromic repeats, or a system containing such a set of repeats. "Cas", as used herein, refers to a CRISPR-associated protein. The "CRISPR/Cas" system refers to a system derived from CRISPR and Cas, which 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 eubacterial genomes and 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, enhancing, or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This is achieved by introducing a plasmid containing a specially engineered CRISPR and one or more suitable Cas into a eukaryotic cell. A CRISPR sequence, sometimes called a CRISPR locus, contains alternating repeats and spacers. In naturally occurring CRISPR spacers, they typically contain sequences foreign to the bacterium, 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. The 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 siRNA. Pennisi (2013) Science 341: 833–836. Thus, the CRISPR/Cas system can be used to edit the TCR and/or HLA gene (adding or deleting a base pair) or to introduce a premature stop codon, which thereby reduces TCR and/or HLA expression. Alternatively, a CRISPR/Cas system such as RNA interference can be used to reversibly disable 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 that inhibit TCR and/or HLA can be created using technology known in the art, for example, 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 produced, 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 “TCR and/or HLA TALEN” or “TCR and/or HLA inhibition TALEN” 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 fusing the TAL effector DNA-binding domain with a DNA cleavage domain. Transcription activator-like activators (TALEs) can be designed to bind any desired DNA sequence, including part of the TCR and/or HLA gene. By combining an engineered TALE with a DNA cleavage domain, it is possible to obtain a restriction enzyme that is specific for any desired DNA sequence, including the TCR and/or HLA sequence. They can then be introduced into a 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; Moscou et al. (2009) Science 326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA-binding domain includes a highly conserved 33-34 amino acid repeat sequence, with the exception of the 12th and 13th amino acids. These two positions vary greatly, showing a strong correlation with the recognition of specific nucleotides. Thus, they can be designed to bind to the DNA sequence of interest. To produce TALEN, the TALE protein is fused to a nuclease (N), which is wild-type or mutated Fokl endonuclease. Several mutations of Fokl have been generated for use in TALEN; for example, they improve the specificity or activity of 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 the 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 distinct 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 within the cell to create a double-strand break (DSB). A mutation can be introduced at the break site if repair mechanisms do not properly repair the break through a non-homologous end junction. For example, improper repair can lead to a frameshift mutation. Alternatively, foreign DNA may 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 into the wt TCR and/or HLA gene, thereby reducing TCR and/or HLA expression. TALENs specific for sequences in the TCR and/or HLA can be created using any method known in the art, including various designs 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 comprises a Fokl nuclease domain (or its derivative) 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. The zinc finger is a small protein structural motif stabilized by one or more zinc ions. The zinc finger may contain, for example, Cys2His2 and can 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 create zinc fingers (and combinations thereof) that recognize specific sequences, which include phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must dimerize to cleave DNA. Thus, a ZFN pair is required for targeting to 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. Just like TALEN, ZFN can create a double-strand break in DNA, which can create a frameshift mutation if repaired incorrectly, resulting in decreased TCR and/or HLA expression and quantity in the cell. ZFN can also be used with homologous recombination to mutate the TCR and/or HLA gene. ZFNs specific for sequences in the 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 "TCR and/or HLA meganuclease" or "TCR and/or HLA inhibition meganuclease" refers to a monomeric endonuclease with large (>14 base pairs) recognition sites that can be used to edit the TCR and/or HLA gene . Meganucleases (mn) are monomeric proteins with natural nuclease activity that are derived from bacterial homing endonucleases and engineered to a unique target site. Homing endonucleases are DNA-cleaving enzymes that can generate double-strand breaks at discrete 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 (usually 20 to 30 base pairs). Homing endonucleases are extremely common and are found in microbes, as well as phages and viruses. The LAGLIDADG and His-Cys box enzymes (which are the most sequence specific of these enzymes) depend on antiparallel β-sheets that are anchored in the major grooves of the DNA of their target sites (Flick et al., 1998; Jurica et al., 1998 ). There they establish an array of sequence-specific and non-specific contacts that are distributed unevenly across several successive base pairs (Chevalier et al., 2003; Scalley-Kim et al., 2007).

The LAGLIDADG (LHE) family of homing endonucleases is a major 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 possess one conserved LAGLIDADG motif (SEQ ID NO: 85) per protein chain form homodimeric proteins that cleave palindromic and near-palindromic DNA target sequences, whereas meganucleases that contain two such motifs per protein chain form larger pseudosymmetric 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 the DNA, which can create a frameshift mutation if repaired incorrectly, 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 the TCR and/or HLA gene. MegaTALs are hybrid monomeric nucleases produced by fusing minimal TAL (transcription activator-like) effector domains with 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. MegaTALs can be engineered to target the TCR and/or HLA and thereby create a double-strand break in the DNA, which can create a frameshift mutation if repaired incorrectly, resulting in decreased expression and amount of HLA in the cell. The meganuclease I-OnuI (mn) variant was used to construct TCRα-megaTAL to knock out the T cell receptor alpha (TCRα) gene. TCRα mn was fused to a 10.5-repeat TALE array designed to bind the DNA sequence upstream of the TCRα mn binding site. MegaTAL targeting TCRα was found to achieve extremely high gene disruption without detectable 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 short-term persistence in the patient due to shortened telomeres in the T cell; Accordingly, transfection with the telomerase gene can lengthen T cell telomeres and improve T cell persistence in the 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, a genetically modified immune cell of the invention ectopically expresses a telomerase subunit, for example a telomerase catalytic subunit, for example TERT, for example hTERT. In some aspects, this disclosure relates to a method of producing a CAR-expressing immune cell, comprising contacting the immune cell with a nucleic acid encoding a telomerase subunit, e.g., a telomerase catalytic subunit, e.g., TERT, e.g., hTERT. The cell may be contacted with the nucleic acid before, simultaneously with, or after contact with the construct encoding the CAR.

Another object of the invention is a composition comprising, consisting of or consisting essentially of at least one immune cell or population according to the invention.

In one embodiment, said composition contains, consists of, or consists essentially of at least one isolated and/or substantially purified population of immune cells according to the invention.

In one embodiment, said composition was frozen and thawed.

Another object of the invention is a pharmaceutical composition containing, consisting of or consisting essentially of at least one immune cell or population, as described above, and at least one pharmaceutically acceptable excipient.

Another object of the invention is a medicinal product containing, consisting of or consisting essentially of at least one population of immune cells, as described above.

In one embodiment, the pharmaceutical composition or drug includes an isolated and/or substantially purified population of immune cells according to the invention.

As used herein, the term “consisting essentially of” in relation to a pharmaceutical composition or drug means that at least one immune cell or population of the invention is the only therapeutic agent or agent having biological activity in the pharmaceutical composition or drug.

Such compositions and drugs may contain buffers such as neutral-buffered saline, phosphate-buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives.

Administration of the compositions may be by any convenient route, including aerosol inhalation, injection, oral administration, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (IV), or intraperitoneal. In one aspect, the immune cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.

In one embodiment, at least one immune cell or population of the present invention is administered by intravenous injection.

The compositions of the present invention are thus, in one embodiment, formulated for intravenous administration.

In one embodiment, an engineered immune cell of the invention may be used as a means to recruit said immune cell to sites of a specific immune or inflammatory response and to activate the immune cell to suppress the immunological activity of immune effector cells at those sites. Thus, CAR-mediated recruitment and activation of immune cells may thus provide a method for preventing or treating 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 as described above, preferably selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory γδ T cell, a regulatory DN T cell , a regulatory B cell, a regulatory NK cell, a regulatory macrophage, a regulatory dendritic cell, and any combination thereof.

In one embodiment, the immune cell is a regulatory T cell. Although the activation of regulatory T cells is antigen-dependent, the suppressive effects of these cells are 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 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 HLA-A2 target antigen may be present or expressed at the site or target tissue of an unwanted immune or inflammatory response mediated by immune effector cells.

In one embodiment, immune cells engineered with the CARs 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 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 given to the recipient through transplantation. Accordingly, CAR-mediated redirection and activation of immune cells provides a method of inhibiting the rejection of inappropriate cells and/or tissues by immune effector cells in transplant recipients or inhibiting the pathogenic effects of transplanted immunocompetent cells in the case of GVHD. In one embodiment, the mismatched cells and/or tissues comprise cells and/or tissues that are HLA-A2 mismatched.

Thus, another aspect of the present invention is a method of treating one or more diseases, disorders, symptoms or conditions associated with organ or tissue transplantation (eg, organ or tissue rejection/dysfunction, GVHD and/or conditions associated therewith) to a subject, wherein the method comprises administering to the subject an engineered immune cell with a CAR or a 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 object of the present invention is a CAR-engineered immune cell, or a composition described herein, for use in the treatment of one or more diseases, disorders, symptoms or conditions associated with organ or tissue transplantation (eg, rejection/ organ or tissue dysfunction, GVHD and/or related conditions) in the subject.

The CAR-modified immune cells of the present invention can further be used to stimulate immune tolerance, functional tolerance and/or immune accommodation in a subject, particularly after organ or tissue transplantation.

Thus, another aspect of the present invention is a method of promoting immune tolerance, functional tolerance and/or immune accommodation in a subject, a method comprising administering to the subject a CAR engineered immune cell or a pharmaceutical composition as described herein. In one embodiment, the method may be intended to promote immune tolerance, functional tolerance, and/or immune accommodation to a transplanted organ or tissue in a subject.

In one embodiment of the invention, a CAR-engineered immune cell (preferably a CAR-engineered Treg cell) of the invention is administered at the same time as, before, or after organ or tissue transplantation.

In one embodiment, the CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention can be used to prevent or treat organ or tissue transplant rejection. Examples of organ or tissue transplant rejection include, but are not limited to, hyperacute organ or tissue transplant rejection and antibody-mediated organ or tissue transplant rejection.

In one embodiment, the method of the invention comprises administering the CAR-engineered Immune cells (CAR-engineered Tregs) of the present invention to a subject receiving 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.

In one embodiment, the CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention can be used as a therapy for inhibiting graft rejection after transplantation, including, but not limited to, allograft rejection or xenograft rejection.

Another aspect of the invention is a method of preventing or treating organ or tissue transplant rejection in a subject, comprising administering to the subject CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the invention or a pharmaceutical composition containing said immune cells.

Thus, another object of the present invention is a CAR-engineered immune cell (preferably a 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 object.

Another aspect of the invention is a method of increasing graft survival in a subject, comprising administering to the subject CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention, or a pharmaceutical composition comprising them.

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 receiving any therapy with immunosuppressive agents.

In one embodiment, administration of an immune cell or composition of the invention allows the amount of immunosuppressant therapy received by the subject to be reduced.

In one embodiment, the graft is an allograft. In one embodiment, the graft may be exposed to CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention concurrently, before or after transplantation of the graft into the 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, bone, or islet cells. However, the invention is not limited to a particular type of transplantation.

In one embodiment, the donor graft may be "preconditioned" or "preconditioned" by treating the organ or tissue graft prior to transplantation into the recipient with immune cells engineered with the CARs 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 an HLA-A subtype selected from the group consisting of HLA-A03, HLA-A11, HLA-A23, HLA-A25, HLA-A26, HLA - A29, HLA-A30, HLA-A31, HLA-A33 and HLA-A34. In one embodiment, the graft host or recipient is HLA-A2 negative and is positive for an HLA-A subtype selected from the group consisting of HLA-A25, HLA-A29 and HLA-A30.

In one embodiment, the graft is HLA-A2 positive.

In one embodiment, the CAR-engineered immune cells (preferably 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 includes administering CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention to a subject receiving 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 can occur after 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 particular type of transplantation.

In one embodiment, GVHD may occur following a hematopoietic stem cell transplant.

Thus, another aspect of the invention is a method of preventing or treating graft-versus-host disease (GVHD) in a subject, comprising administering to the subject CAR-engineered immune cells (preferably CAR-engineered Treg cells) or a pharmaceutical composition in the form described in this document.

In one embodiment, the invention relates to a method of contacting a donor graft, such as a biocompatible mesh or donor tissue, organ or cell, with CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention, simultaneously, up to or after transplantation of a transplant to the recipient.

In one embodiment, CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention can be used to attenuate, inhibit, or reduce the adverse graft response of a donor against a recipient, thereby preventing or treating GVHD.

Thus, another object of the present invention is a method of preventing or delaying the onset of GVHD in a subject, the method comprising administering to the subject CAR-engineered immune cells (preferably CAR-engineered Treg cells) or a pharmaceutical composition as described 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 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 for the entire life of the object.

In one embodiment, the subject is not receiving any therapy with immunosuppressive agents.

In one embodiment, the subject is subjected to immunosuppressive therapy. In one embodiment, immune cells of the invention are administered to a subject for the purpose of reducing a therapeutically effective amount of said immunosuppressive therapy.

In one embodiment, GVHD may be acute GVHD or chronic GVHD.

In one embodiment, the donor graft may be "preconditioned" or "pretreated" by treating the graft prior to transplantation into the recipient with CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the invention to reduce the immunogenicity of the graft against the recipient, thereby 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 can be associated with the graft. After the graft has been treated with the recipient's cells or tissue, the cells or tissue may be removed from the graft. The treated graft may then be further contacted with CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention to reduce, inhibit or eliminate the activity of immune effector cells that were activated by the cell treatment or tissue from the recipient. After this treatment of the graft with the CAR-engineered immune cells (preferably CAR-engineered Treg cells) of the present invention, the CAR-engineered immune cells can be removed from the graft prior to transplantation into the recipient. However, some CAR-engineered immune cells (eg, CAR-engineered Treg cells) can adhere to the graft and can therefore be administered to the recipient via the graft. In this situation, CAR-engineered immune cells (eg, CAR-engineered Treg 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 an HLA-A subtype selected from the group consisting of HLA-A03, HLA-A11, HLA-A23, HLA-A25, HLA-A26, HLA - A29, HLA-A30, HLA-A31, HLA-A33 and HLA-A34. In one embodiment, the graft host or recipient is HLA-A2 negative and is positive for an HLA-A subtype selected from the group consisting of HLA-A25, HLA-A29 and HLA-A30.

In one embodiment, the graft is HLA-A2 positive.

Another aspect of the invention is a method of increasing the population of immune cells in a subject, where the immune cells are modified to express a chimeric antigen receptor (CAR) of the invention, the method comprising administering to the subject an engineered immune cell described elsewhere herein, wherein the administered engineered immune cells produce the progeny population of immune cells in an object.

In one embodiment, the immune cell is a regulatory immune cell, preferably selected from the group consisting of 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, regulatory dendritic cells, and any combination thereof, and more preferably regulatory T cells.

In one embodiment, the CAR engineered 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.

Thus, another aspect of the present invention is a method of 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) of the invention, the method comprising administering the regulatory T cells to the subject, as described herein document where a persistent population of modified regulatory T cells is maintained 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 of the invention may be capable of self-renewal and reactivation in vivo to suppress the immune response of a target cell. In one embodiment, the CAR Treg cells may be memory CAR Treg cells that can be reactivated to suppress the immune response of a 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 individual or species as a whole) to produce 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 to the transplant recipient. In one embodiment, the CAR Treg cells of the present invention may be autologous to the transplant recipient.

In one embodiment, the object may be a mammal. In one embodiment, the subject may be a person.

In one embodiment, it may be desirable to administer activated T cells to a subject, and then subsequently re-draw blood (or perform apheresis), activate T cells from it according to the present invention, and reintroduce these activated and expanded T cells to the subject. This process may be repeated several times every few weeks. In some embodiments, T cells can be activated by drawing 10 to 400 cc of blood ^. In some embodiments, T cells are activated by drawing blood volumes of 20 cc ^, 30 ^cc , 40 ^cc , 50 ^cc , 60 ^cc , 70 ^cc , 80 ^cc , 90 cc ^, or 100 ^cc . 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-engineered immune cells of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components, including, but not limited to, IL-2 or other cytokines or cell populations.

In one embodiment, the pharmaceutical compositions of the present invention may contain a CAR engineered 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, for example, 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 can be administered to a subject by any suitable route, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The pharmaceutical compositions described herein can be administered to a subject by parenteral administration. The pharmaceutical compositions described herein may be administered to a subject by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intrasternal, intravenous (IV) injection, infusion, or intraperitoneal routes. In one embodiment, the CAR-modified immune cell compositions of the present invention can be administered to a subject by intradermal or subcutaneous injection. In another embodiment, the CAR-modified immune cell compositions of the present invention can be administered by intravenous injection. In one embodiment, compositions of CAR-modified immune cells 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 can be administered in a manner suitable for preventing or treating a disease. The amount and frequency of administration will be determined by factors such as the subject's condition and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials.

When an "effective amount" or a "therapeutic amount" is indicated, the exact amount of the compositions of the present invention to be administered may be determined taking into account individual differences in age, weight, antibody titer and condition of the subject. In general, it can be stated that a pharmaceutical composition containing CAR engineered immune cells as described herein can be administered at a dosage of 1 x 10 ⁴ to 1 x 10 ⁹ cells/kg body weight or 1 x 10 ⁵ to 100 x 10 ⁵ cells/kg body weight, including all integer values in these ranges. CAR-engineered immune cell compositions can also be administered multiple times at these dosages. Immune cells engineered with CARs can be administered using infusion techniques that are commonly known in immunotherapy. The optimal dosage and treatment regimen for a particular subject can be easily 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 the administration of another active agent.

In one embodiment, the CAR-engineered immune cells of the present invention may be administered to a subject in combination (e.g., before, concurrently, or after) with any number of suitable treatments, including, but not limited to, treatment with agents such as antiviral therapies, chemotherapy, radiation , immunosuppressants, antibodies, immunoablative agents, cytokines and radiation.

In one embodiment, the CAR-engineered immune cells of the present invention can be administered in combination with an immunosuppressant. Any immunosuppressant known in the art can be used.

Examples of immunosuppressants include, without limiting to the specified, calciuminein inhibitors, such as cyclosporine, tacrolimus, azathioprine, methotrexate, methoxylen, rapamycin, mycophenolate Mofenolic acid, sodium mycophenolat, 6-mercaptopurine, 6-tyguanin, rituximab, inhibitors, inhibitors. mtor, 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.

The CAR-engineered immune cells of the present invention can be administered to a subject before, after, or simultaneously with an immunosuppressant.

The CAR-engineered immune cells of the present invention and/or an immunosuppressant can be administered to the subject after transplantation. Alternatively or in addition, the CAR-engineered immune cells of the present invention and/or an immunosuppressant may be administered to the subject prior to transplantation. The CAR-engineered immune cells of the present invention and/or an immunosuppressant can be administered to the subject during transplant surgery.

In one embodiment, the method of the invention of administering CAR-engineered immune cells to a subject is carried out after initiation of immunosuppressive therapy.

In some embodiments, the method is performed more than once, for example, to monitor a transplant recipient over time and, if applicable, across different immunosuppressive therapy regimens.

In some embodiments, immunosuppressive therapy is reduced if the transplant recipient is predicted to be tolerant to the transplant. In some embodiments, no immunosuppressive therapy is administered, for example, immunosuppressive therapy is discontinued if the transplant recipient is predicted to be tolerant to the transplant.

Thus, the present invention further provides a set of parts comprising, firstly, an immune cell or population of immune cells of the present invention, and in a second part, another active agent, including, but not limited to, antiviral therapy, chemotherapy, radiation therapy, immunosuppressants, antibodies, immunoablative agents, cytokines and radiation. Preferably, a kit of part of the present invention includes in a first part an immune cell or population of immune cells according to the present invention and in a second part one or more immunosuppressants.

In one embodiment, the set of parts includes one or more reagents (eg, a nucleic acid or expression vector encoding an anti-HLA-A*CAR antibody of the present invention) for producing cells of the present invention.

Examples of immunosuppressants include, without limiting to the specified, calciuminein inhibitors, such as cyclosporine, tacrolimus, azathioprine, methotrexate, methoxylen, rapamycin, mycophenolate Mofenolic acid, sodium mycophenolat, 6-mercaptopurine, 6-tyguanin, rituximab, inhibitors, inhibitors. mtor, 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.

Another aspect of the invention is a method of preventing or treating organ or tissue transplant rejection in a subject, comprising administering to the subject (i) at least one immunosuppressive agent and (ii) CAR-engineered immune cells (preferably CAR-engineered Treg cells). CAR) according to the invention or a pharmaceutical composition containing these immune cells.

Thus, another object 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 organ or tissue transplant rejection in a subject.

Another aspect of the invention is a method of increasing the graft survival period of 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 (preferably 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 a hematopoietic stem cell transplant.

Thus, another aspect 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 (preferably CAR-engineered Treg cells ) or a pharmaceutical composition as described herein.

In one embodiment, the invention relates to a method of contacting a donor graft, for example a biocompatible mesh or donor tissue, organ or cell, with at least one immunosuppressive agent and CAR-engineered immune cells (preferably Treg cells, engineered with CAR) 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 (preferably CAR-engineered Treg cells) of the present invention can be used to attenuate, inhibit, or reduce an adverse graft response against a recipient, such thus preventing or treating GVHD.

Thus, another aspect 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 compositions as described herein.

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, concurrently, or after) the transplant.

The CAR-engineered immune cells of the present invention can be administered following diagnosis of organ or tissue transplant rejection, followed by subsequent 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 combination with (eg, before, concurrently, or after) a bone marrow transplant.

In another embodiment, the CAR engineered immune cells of the present invention can be administered following B cell ablative therapy, for example, agents that react with CD20, such as rituximab. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, following transplantation, subjects may receive an infusion of 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, a subject (eg, a human) receives an initial administration of at least one immune cell or population of the invention and one or more subsequent administrations, wherein the one or more subsequent administrations are administered in less than 15 days, e.g., 14, 13 , 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days after the previous administration.

In one embodiment, a therapeutically effective amount of immune cells of the invention is or is to be administered to a subject.

In one embodiment, the number of immune cells, at least one population of immune cells according to 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 a subject is from about 10 ² to about 10 ⁹ , from about 10 ³ to about 10 ⁸ , from about 10 ⁴ to about 10 ⁷ , or from about 10 ⁵ to about 10 ⁶ cells.

In another embodiment, the number of immune cells of the invention administered to a subject is from about 10 ⁶ to about 10 ⁹ , from about 10 ⁶ to 10 ⁷ , from about 10 ⁶ to 10 ⁸ , from about 10 ⁷ to 10 ⁹ , from about 10 ⁷ to 10 ⁸ , from about 10 ⁸ to 10 ⁹ . In another embodiment, the number of immune cells of the invention administered to a subject is about 10 ⁶ , about 10 ⁷ , about 10 ⁸ or about 10 ⁹ .

In one embodiment, the number of immune cells, at least one population of immune cells according to the invention, administered to the subject is at least 110 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 a subject is from about 10 ⁴ to 10 ⁹ cells/kg body weight or from 10 ⁵ to 10 ⁸ cells/kg body weight, including all integer values in these ranges.

In one embodiment, a subject (eg, a human) is given more than one administration of at least one immune cell or population of the invention per week, for example, 2, 3 or 4 administrations of a genetically modified immune cell or population of the invention are administered per week.

Another object of the present invention is an article containing materials useful for the prevention and/or treatment of transplant rejection or GVHD.

The article may include a container and a label or insert in the package or on 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 for preventing and/or treating an immunological condition such as graft rejection or GVHD, and may have a sterile access opening (for example, the container may be an intravenous solution bag or a vial with a stopper suitable for piercing with a needle for subcutaneous injections). At least one active agent in the composition is a CAR-engineered immune cell of the present invention, preferably a CAR-engineered 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 product may further comprise a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered 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 various purposes (eg, to prevent or treat transplant rejection or GVHD). Kits may be provided that contain CAR engineered immune cells. As with the product, the kit may include a container and a label or insert in or associated with the container. The container includes a composition containing at least one CAR-engineered immune cell, preferably a CAR-engineered Treg, of the present invention. Additional containers may be included that contain, for example, diluents and buffers. The label or package insert may contain a description of the composition as well as instructions for intended use.

Brief description of drawings

Figure 1 is a combination of graphs showing the cross-reactivity of the anti-HLA-A2 CAR construct of the invention with conventional HLA-A*HLA-B allelic variants. GFP + Treg cells expressing the CAR construct of the invention (or the mouse anti-HLA-A2 CAR construct) were incubated with Flow Panel Reactive Single Antigen beads and viability fixative dye for 30 minutes at room temperature. Samples were then washed, fixed, and analyzed by flow cytometry. Data were analyzed as described in Methods. Data in A and B show the percentage of binding relative to control Treg cells expressing only GFP, normalized by the number of HLA-negative beads collected by the cytometer. Data points above the line represent values that differed by more than two standard deviations from the mean of the pellet-only control and are thus statistically significant (p < 0.05). Data are averages of two independent experiments. Mean ± SEM.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1:

Materials and methods

Generation of humanized HLA-A2-specific scFvs. The sequence for the HLA-A2-specific scFv was derived from the sequence disclosed in patent application US2013/0078243 A1. Codon optimized gene sequences encoding the humanized scFv were synthesized by GeneArt, Life Technologies (Regensburg) as “Strings” using a codon optimization algorithm provided by the supplier. The ScFvs were flanked at the N-terminus by a Kozak sequence and a CD8 signal sequence. The constructs were further flanked by BamHI and MfeI restriction sites, which allowed in-frame cloning of the CD8 hinge region of a self-inactivating lentiviral plasmid expressing CAR. The PGK promoter-driven CAR expression plasmid contained the human CD8 hinge and CD8 transmembrane domain followed by the human intracellular costimulatory domain 41BB fused to human CD3ζ. Downstream of the CAR sequence, the EGFP reporter protein was linked in frame through a self-cleavage 2A site to detect gene transfer in cells.

Obtaining HLA-A2-CAR. Viral vectors were generated by polyethylenimine-based transient transfection in HEK 293T cells using 3rd generation packaging plasmids (Trono lab, EPFL (École polytechnique fédérale de Lausanne)).

Jurkat NFAT. Jurkat-Lucia NFAT reporter cell lines were cultured in RPMI (Gibco, Life technologies) supplemented with 10% fetal calf serum. Cells were transduced using incubation at varying concentrations of viral vectors to achieve 30–50% transduction to avoid multiple vector integrations. Expression and presentation of properly folded CARs were demonstrated by binding to an APC-conjugated HLA-A2 multimer (Dextramer, WB2666, Immudex; Copenhagen, Denmark). Activation through CARs was achieved by combining HLA-A2-specific CARs with HLA-A2-positive or HLA-A2-negative cell lines. The secretion of luciferase depending on the activation of NFAT in the medium was analyzed on a luminometer (Glowmax, Promega) using the QUANTI-Luc substrate (Invivogen).

Sorting, transduction and expansion of Tregs. CD4+ T cells were isolated from HLA-A2− donors through RosetteSep (Stemcell) and enriched for CD25+ cells (Miltenyi) before sorting live CD4+ CD127loCD25hi Tregs using FACSAria II (BD Biosciences). Sorted Tregs were stimulated with L cells and αCD3 mAb (OCT3; 200 ng/ml) in 1000 U/ml IL-2. One day later, cells were transduced with lentivirus at a multiple infection rate of 10 virus particles:1 cell. On day 7, cells were restimulated with L cells as described above and expanded for 5 days. To test the effects of HLA-A2-mediated stimulation, Tregs were cultured overnight with IL-2 restriction (100 U/ml) and then restimulated with irradiated anti-CD3/anti-CD28 loaded K562.64 cells or K562.64.HLA cells -A2 in a 1:2 ratio (K562:T cells) for 24 hours.

Flow cytometry. For phenotypic analysis, cells were stained with a fixative dye for viability (FVD, 65-0865-14, eBioscience; 423102, Biolegend) and for surface markers before fixation/permanentilization using the FOXP3/Transcription Factor Staining Buffer kit (eBioscience) and staining for intracellular proteins. Samples were read on a Cytoflex (Beckman-Coulter) and results were analyzed using FlowJo software versions 9.9.4 and 10.3 (Tree Star). Surface staining was performed for CD3 (564465, BD Biosciences), CD4 (317410, Biolegend), CD25 (130-091-024, Miltenyi), LAP (25-9829-42, eBioscience), CD69 (310946, Biolegend), CD154 ( 555702, BD Biosciences) and CD127 (48-1278-42, eBiosciences). Tetramer staining was performed with HLA-A*02:01 monomers converted to streptavidin-allophycocyanin tetramers (PJ27S, Prozyme). Intracellular staining was performed with CTLA-4 (369606, Biolegend).

Analysis of cross-reactivity of HLA alleles. 0.25 x 10 ⁶ Tregs expressing CARs according to the present invention, or CARs including VH and VL antibodies BB7.2 (mA2 CAR), were incubated with a panel of FlowPRA® single antigen antibody beads (FL1HD01, FL1HD02, FL1HD03, FL1HD04 FL1HD06 and FL1HD08, One Lambda) and fixable viability dye (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 using a fixable viability dye. Single antigen beads were then gated after excluding dead cells and doublets. The number of beads per HLA antigen was then determined by their corresponding peak PE intensity. 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 Tregs compared to the control (non-CAR-expressing cells) was determined by subtracting the number of beads in the CAR-Treg from the number of beads in the control sample, then dividing by the average number of beads in the non-CAR-expressing control, and multiplying by 100.

Results:

Generation of humanized HLA-A2-specific CARs

1 humanized heavy chain and 3 different humanized light chains were produced, and a total of 3 different chimeric antigen receptors were produced by combining the humanized heavy and light chains.

Expression and proper folding of CAR were tested using Jurkat NFAT reporter cell lines.

Cross-reactivity of HLA-A2-CAR constructs to other HLA class I alleles

There are many different HLA alleles that have evolved over time from a smaller number of ancestral alleles. Consequently, there are families of alleles that may differ by only a few amino acids, and a single anti-HLA antibody can recognize multiple alleles in an evolutionarily related family. The mouse monoclonal antibody (BB7.2) is known to be cross-reactive to additional HLA-A alleles (Hilton & Parham, 2013). Specifically, when tested in an enzyme-linked assay, BB7.2 was found to recognize five HLA-A2 subtypes (*02:01, *02:02, *02:03, *02:05, *02:06) and cross-reference 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). To evaluate the cross-reactivity of the humanized anti-HLA-A2 antibodies of the invention and compare with the antibody containing VH and VL antibodies BB7.2, we adapted the ONE Lambda solid-phase assay, which is designed to measure anti-HLA antibody in serum, by measuring bead binding, HLA-coated, with humanized CARs of interest. GFP+ Tregs expressing the indicated humanized CARs were incubated with Flow Panel Reactive Single Antigen beads, with binding to one class of beads quantified as loss of signal at the bead gate in the forward light scatter/side scatter plot. 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 by the ratio of the amount of binding to GFP+ control Tregs to the number of negative beads in the GFP sample multiplied by the number of negative beads in the GFP sample.

The results are shown in Figure 1 for the scFv-containing construct having the sequence SEQ ID NO: 72). This HLA-A2-CAR construct significantly binds HLA-A*02:01 ( Figure 1A ). Surprisingly, for the CAR construct of the invention, we did not observe any statistically significant binding to any HLA-A or B alleles other than A*02:01 tested. (Figure 1A-B).

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LIST OF SEQUENCES

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<223> X is G or A

<400> 10

Asp Xaa Val Met Thr Gln Xaa Pro Xaa Xaa Leu Xaa Val Xaa Xaa Gly

1 5 10 15

Xaa Xaa Xaa Xaa Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Xaa Gln Lys Pro Gly Gln Xaa

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Xaa Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Xaa Ile

65 70 75 80

Ser Arg Xaa Glu Xaa Glu Asp Xaa Xaa 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

Arg

<210> 11

<211> 113

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 11

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Glu 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 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 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

Arg

<210> 12

<211> 113

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 12

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Thr Leu Gly

1 5 10 15

Asp Arg 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 Gln Gln Lys 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 Thr Ile

65 70 75 80

Ser Arg Val Glu Pro 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

Arg

<210> 13

<211> 113

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 13

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr 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 Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Leu Glu Pro Glu Asp Phe Ala 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

Arg

<210> 14

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> G3S linker

<400> 14

Gly Gly Gly Ser

1

<210> 15

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> G3S linker

<400> 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> G4S linker

<400> 16

Gly Gly Gly Gly Gly Ser

15

<210> 17

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> G4S linker

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 18

<211> 15

<212>PRT

<213> Artificial Sequence

<220>

<223> G4S linker

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

<211> 20

<212>PRT

<213> Artificial Sequence

<220>

<223> G4S linker

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 20

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 20

Ala Gly Ser Ser Ser Ser Gly Gly Ser Thr Thr Gly Gly Ser Thr Thr

1 5 10 15

<210> 21

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 21

Gly Thr Thr Ala Ala Ser Gly Ser Ser Gly Gly Ser Ser Ser Gly Ala

1 5 10 15

<210> 22

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 22

Ser Ser Ala Thr Ala Thr Ala Gly Thr Gly Ser Ser Thr Gly Ser Thr

1 5 10 15

<210> 23

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 23

Thr Ser Gly Ser Thr Gly Thr Ala Ala Ser Ser Thr Ser Thr Ser Thr

1 5 10 15

<210> 24

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 24

ggtggcggag gttctggagg tggaggttcc 30

<210> 25

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Hinge domain

<400> 25

Lys Ile Arg Arg Asp Ser Ser

15

<210> 26

<211> 45

<212>PRT

<213> Artificial Sequence

<220>

<223> CD8 Hinge domain

<400> 26

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> 27

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 Hinge domain

<400> 27

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 28

<211> 230

<212>PRT

<213> Artificial Sequence

<220>

<223> IgG4 Hinge domain

<400> 28

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

1 5 10 15

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

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

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

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 29

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG4 Hinge domain

<400> 29

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaagatg 690

<210> 30

<211> 282

<212>PRT

<213> Artificial Sequence

<220>

<223> IgD Hinge domain

<400> 30

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

1 5 10 15

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

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

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

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 31

<211> 847

<212> DNA

<213> Artificial Sequence

<220>

<223> IgD Hinge domain

<400> 31

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca 60

gaaggcagcc tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc 120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc 180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag 240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag 300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga 420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca 480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat 540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc 600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt 720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc 780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact 840

gaccatt 847

<210> 32

<211> 39

<212>PRT

<213> Artificial Sequence

<220>

<223> CD28 Hinge domain

<400> 32

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 33

<211> 117

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 Hinge domain

<400> 33

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117

<210> 34

<211> 24

<212>PRT

<213> Artificial Sequence

<220>

<223> CD8 transmembrane domain

<400> 34

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

20

<210> 35

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 transmembrane domain

<400> 35

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 36

<211> 27

<212>PRT

<213> Artificial Sequence

<220>

<223> CD28 transmembrane domain

<400> 36

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> 37

<211> 81

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 transmembrane domain

<400> 37

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 38

<211> 112

<212>PRT

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 38

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 39

<211> 112

<212>PRT

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 39

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> 40

<211> 113

<212>PRT

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 40

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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 Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

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

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 41

<211> 113

<212>PRT

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 41

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 Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

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

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 42

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 42

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 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> 43

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223>CD3zeta

<400> 43

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 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> 44

<211> 42

<212>PRT

<213> Artificial Sequence

<220>

<223> 4-1BB intracellular domain

<400> 44

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> 45

<211> 48

<212>PRT

<213> Artificial Sequence

<220>

<223> CD27 intracellular domain

<400> 45

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

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

35 40 45

<210> 46

<211> 41

<212>PRT

<213> Artificial Sequence

<220>

<223> CD28 intracellular domain

<400> 46

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> 47

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> 4-1BB intracellular domain

<400> 47

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 48

<211> 144

<212> DNA

<213> Artificial Sequence

<220>

<223> CD27 intracellular domain

<400> 48

caacgaagga aatatagatc aaacaaagga gaaagtcctg tggagcctgc agagccttgt 60

cgttacagct gccccaggga ggagaggagc agcaccatcc ccatccagga ggattaccga 120

aaaccggagc ctgcctgctc cccc 144

<210> 49

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 intracellular domain

<400> 49

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 50

<211> 21

<212>PRT

<213> Artificial Sequence

<220>

<223>CD8 leader sequence

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 51

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Tag

<400> 51

Asn Trp Ser His Pro Gln Phe Glu Lys

15

<210> 52

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> Tag

<400> 52

Ser Ala Trp Ser His Pro Gln Phe Glu Lys

1 5 10

<210> 53

<211> 222

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 53

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

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

35 40 45

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

50 55 60

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

65 70 75 80

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

85 90 95

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

100 105 110

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

115 120 125

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

130 135 140

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

145 150 155 160

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

165 170 175

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

180 185 190

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

195 200 205

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 54

<211> 223

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 54

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

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

35 40 45

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

50 55 60

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

65 70 75 80

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

85 90 95

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

100 105 110

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

115 120 125

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

130 135 140

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

145 150 155 160

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

165 170 175

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

180 185 190

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

195 200 205

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 55

<211> 223

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 55

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 Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

65 70 75 80

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

85 90 95

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

100 105 110

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

115 120 125

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

130 135 140

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

145 150 155 160

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

165 170 175

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

180 185 190

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

195 200 205

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 56

<211> 236

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 56

Asn Trp Ser His Pro Gln Phe Glu Lys Met His Thr Thr Thr Pro Ala

1 5 10 15

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

20 25 30

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

35 40 45

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

50 55 60

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

65 70 75 80

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

85 90 95

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

100 105 110

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Arg Arg Val Lys Phe

115 120 125

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

130 135 140

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

145 150 155 160

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

165 170 175

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

180 185 190

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

195 200 205

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

210 215 220

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

225 230 235

<210> 57

<211> 224

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 57

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 Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

65 70 75 80

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

85 90 95

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

100 105 110

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

115 120 125

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

130 135 140

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

145 150 155 160

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

165 170 175

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

180 185 190

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

195 200 205

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 58

<211> 237

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 58

Asn Trp Ser His Pro Gln Phe Glu Lys Met His Thr Thr Thr Pro Ala

1 5 10 15

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

20 25 30

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

35 40 45

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

50 55 60

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

65 70 75 80

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

85 90 95

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

100 105 110

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Arg Arg Val Lys Phe

115 120 125

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

130 135 140

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

145 150 155 160

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg

165 170 175

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

180 185 190

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

195 200 205

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

210 215 220

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

225 230 235

<210> 59

<211> 226

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 59

Met His Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu His

65 70 75 80

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

85 90 95

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

100 105 110

Thr Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

115 120 125

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

130 135 140

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

145 150 155 160

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

165 170 175

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

180 185 190

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

195 200 205

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

210 215 220

Pro Arg

225

<210> 60

<211> 227

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 60

Met His Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu His

65 70 75 80

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

85 90 95

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

100 105 110

Thr Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

115 120 125

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

130 135 140

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

145 150 155 160

Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

165 170 175

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

180 185 190

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

195 200 205

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

210 215 220

Pro Pro Arg

225

<210> 61

<211> 24

<212>PRT

<213> Artificial Sequence

<220>

<223> P2A ribosomal cleavage site

<400> 61

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

1 5 10 15

Asp Val Glu Glu Asn Pro Gly Pro

20

<210> 62

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR1 of BB7.2

<400> 62

Ser Tyr His Ile Gln

15

<210> 63

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR2 of BB7.2

<400> 63

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

1 5 10 15

Gly

<210> 64

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR3 of BB7.2

<400> 64

Glu Gly Thr Tyr Tyr Ala Met Asp Tyr

15

<210> 65

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR1 of BB7.2

<400> 65

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

1 5 10 15

<210> 66

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR2 of BB7.2

<400> 66

Lys Val Ser Asn Arg Phe Ser

15

<210> 67

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR3 of BB7.2

<400> 67

Phe Gln Gly Ser His Val Pro Arg Thr

15

<210> 68

<211> 239

<212>PRT

<213> Artificial Sequence

<220>

<223>eGFP

<400> 68

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

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

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

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

100 105 110

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

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

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

180 185 190

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

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 69

<211> 246

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<220>

<221> SITE

<222> 135

<223> X is for V or I

<220>

<221> SITE

<222> 140

<223> X is for T or S

<220>

<221> SITE

<222> 142

<223> X is for L or S or A

<220>

<221> SITE

<222> 143

<223> X is for S or T

<220>

<221> SITE

<222> 145

<223> X is for P or S

<220>

<221> SITE

<222> 147

<223> X is for T or S

<220>

<221> SITE

<222> 148

<223> X is for L or P

<220>

<221> SITE

<222> 150

<223> X is for E or D

<220>

<221> SITE

<222> 151

<223> X is for P or R

<220>

<221> SITE

<222> 152

<223> X is for A or V

<220>

<221> SITE

<222> 153

<223> X is for S or T

<220>

<221> SITE

<222> 175

<223> X is for L or Q

<220>

<221> SITE

<222> 181

<223> X is for S or A

<220>

<221> SITE

<222> 196

<223> X is for V or I

<220>

<221> SITE

<222> 212

<223> X is for K or T

<220>

<221> SITE

<222> 216

<223> X is for V or L

<220>

<221> SITE

<222> 218

<223> X is for A or P

<220>

<221> SITE

<222> 221

<223> X is for L or F

<220>

<221> SITE

<222> 222

<223> X is for G or A

<400> 69

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr 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

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Xaa Val Met Thr Gln Xaa Pro Xaa Xaa Leu

130 135 140

Xaa Val Xaa Xaa Gly Xaa Xaa Xaa Xaa 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 Xaa Gln

165 170 175

Lys Pro Gly Gln Xaa Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

180 185 190

Phe Ser Gly Xaa Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

195 200 205

Phe Thr Leu Xaa Ile Ser Arg Xaa Glu Xaa Glu Asp Xaa Xaa 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 Leu Glu Ile Lys Arg

245

<210> 70

<211> 246

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 70

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr 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

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu

130 135 140

Pro Val Thr Leu Gly Glu 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 Arg 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 Leu 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 Leu Glu Ile Lys Arg

245

<210> 71

<211> 246

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 71

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr 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

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu

130 135 140

Ser Val Thr Leu Gly Asp Arg Val 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 Gln Gln

165 170 175

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

245

<210> 72

<211> 246

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 72

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr 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

Ser 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 Ser Pro Ala Thr Leu

130 135 140

Ser Val Ser Pro Gly Glu Arg Ala Thr 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 Gln Gln

165 170 175

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

180 185 190

Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

195 200 205

Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 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 Leu Glu Ile Lys Arg

245

<210> 73

<211> 247

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<220>

<221> SITE

<222> 136

<223> X is V or I

<220>

<221> SITE

<222> 141

<223> X is for T or S

<220>

<221> SITE

<222> 143

<223> X is for L or S or A

<220>

<221> SITE

<222> 144

<223> X is for S or T

<220>

<221> SITE

<222> 146

<223> X is for P or S

<220>

<221> SITE

<222> 148

<223> X is for T or S

<220>

<221> SITE

<222> 149

<223> X is for L or P

<220>

<221> SITE

<222> 151

<223> X is for E or D

<220>

<221> SITE

<222> 152

<223> X is for P or R

<220>

<221> SITE

<222> 153

<223> X is for A or V

<220>

<221> SITE

<222> 154

<223> X is for S or T

<220>

<221> SITE

<222> 176

<223> X is for L or Q

<220>

<221> SITE

<222> 182

<223> X is for S or A

<220>

<221> SITE

<222> 197

<223> X is for V or I

<220>

<221> SITE

<222> 213

<223> X is for K or T

<220>

<221> SITE

<222> 217

<223> X is for V or L

<220>

<221> SITE

<222> 219

<223> X is for A or P

<220>

<221> SITE

<222> 222

<223> X is for L or F

<220>

<221> SITE

<222> 223

<223> X is for G or A

<400> 73

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Xaa Val Met Thr Gln Xaa Pro Xaa Xaa

130 135 140

Leu Xaa Val Xaa Xaa Gly Xaa Xaa Xaa Xaa Ile Ser Cys Arg Ser Ser

145 150 155 160

Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Xaa

165 170 175

Gln Lys Pro Gly Gln Xaa Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Xaa Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Xaa Ile Ser Arg Xaa Glu Xaa Glu Asp Xaa Xaa Val

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 74

<211> 247

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 74

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser

130 135 140

Leu Pro Val Thr Leu Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

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

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 75

<211> 247

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 75

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Val Thr Leu Gly Asp Arg Val Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

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

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 76

<211> 247

<212>PRT

<213> Artificial Sequence

<220>

<223> scFv

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

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

130 135 140

Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 77

<211> 471

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<220>

<221> SITE

<222> 136

<223> X is for V or I

<220>

<221> SITE

<222> 141

<223> X is for T or S

<220>

<221> SITE

<222> 143

<223> X is for L or S or A

<220>

<221> SITE

<222> 144

<223> X is for S or T

<220>

<221> SITE

<222> 146

<223> X is for P or S

<220>

<221> SITE

<222> 148

<223> X is for T or S

<220>

<221> SITE

<222> 149

<223> X is for L or P

<220>

<221> SITE

<222> 151

<223> X is for E or D

<220>

<221> SITE

<222> 152

<223> X is for P or R

<220>

<221> SITE

<222> 153

<223> X is for A or V

<220>

<221> SITE

<222> 154

<223> X is for S or T

<220>

<221> SITE

<222> 176

<223> X is for L or Q

<220>

<221> SITE

<222> 182

<223> X is for S or A

<220>

<221> SITE

<222> 197

<223> X is for V or I

<220>

<221> SITE

<222> 213

<223> X is for K or T

<220>

<221> SITE

<222> 217

<223> X is for V or L

<220>

<221> SITE

<222> 219

<223> X is for A or P

<220>

<221> SITE

<222> 222

<223> X is for L or F

<220>

<221> SITE

<222> 223

<223> X is for G or A

<400> 77

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Xaa Val Met Thr Gln Xaa Pro Xaa Xaa

130 135 140

Leu Xaa Val Xaa Xaa Gly Xaa Xaa Xaa Xaa Ile Ser Cys Arg Ser Ser

145 150 155 160

Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Xaa

165 170 175

Gln Lys Pro Gly Gln Xaa Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Xaa Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Xaa Ile Ser Arg Xaa Glu Xaa Glu Asp Xaa Xaa Val

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 78

<211> 471

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 78

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser

130 135 140

Leu Pro Val Thr Leu Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

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

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 79

<211> 471

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 79

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Val Thr Leu Gly Asp Arg Val Ser Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn

180 185 190

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

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

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 80

<211> 471

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 80

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

65 70 75 80

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

115 120 125

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

130 135 140

Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Ile Ser Cys Arg Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 81

<211> 492

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<220>

<221> SITE

<222> 157

<223> X is for V or I

<220>

<221> SITE

<222> 162

<223> X is for T or S

<220>

<221> SITE

<222> 164

<223> X is for L or S or A

<220>

<221> SITE

<222> 165

<223> X is for S or T

<220>

<221> SITE

<222> 167

<223> X is for P or S

<220>

<221> SITE

<222> 169

<223> X is for T or S

<220>

<221> SITE

<222> 170

<223> X is for L or P

<220>

<221> SITE

<222> 172

<223> X is for E or D

<220>

<221> SITE

<222> 173

<223> X is for P or R

<220>

<221> SITE

<222> 174

<223> X is for A or V

<220>

<221> SITE

<222> 175

<223> X is for S or T

<220>

<221> SITE

<222> 197

<223> X is for L or Q

<220>

<221> SITE

<222> 203

<223> X is for S or A

<220>

<221> SITE

<222> 218

<223> X is for V or I

<220>

<221> SITE

<222> 234

<223> X is for Kor T

<220>

<221> SITE

<222> 238

<223> X is for V or L

<220>

<221> SITE

<222> 240

<223> X is for A or P

<220>

<221> SITE

<222> 243

<223> X is for L or F

<220>

<221> SITE

<222> 244

<223> X is for G or A

<400> 81

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Xaa Val Met Thr

145 150 155 160

Gln Xaa Pro Xaa Xaa Leu Xaa Val Xaa Xaa Gly Xaa Xaa Xaa Xaa Ile

165 170 175

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

180 185 190

Leu Glu Trp Tyr Xaa Gln Lys Pro Gly Gln Xaa Pro Arg Leu Leu Ile

195 200 205

Tyr Lys Val Ser Asn Arg Phe Ser Gly Xaa Pro Asp Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Xaa Ile Ser Arg Xaa Glu Xaa

225 230 235 240

Glu Asp Xaa Xaa Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

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

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 82

<211> 492

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 82

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr

145 150 155 160

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

165 170 175

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

180 185 190

Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

195 200 205

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

210 215 220

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

225 230 235 240

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

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

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

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 83

<211> 492

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 83

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Val Thr Leu Gly Asp Arg Val Ser Ile

165 170 175

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

180 185 190

Leu Glu Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

195 200 205

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

210 215 220

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

225 230 235 240

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

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

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

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 84

<211> 492

<212>PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 84

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Ser Tyr His Ile Gln Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr

145 150 155 160

Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Ile

165 170 175

Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

180 185 190

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

195 200 205

Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

225 230 235 240

Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

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

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 85

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> LAGLIDADG motif

<400> 85

Leu Ala Gly Leu Ile Asp Ala Asp Gly

15

<210> 86

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223>VH

<400> 86

caggtccagc tagtacaaag cggccctgaa gtaaagaaac ctggtgcctc tgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctaccaca tccagtgggt tcgacaggcc 120

cctggacagg gactagagtg gatcggctgg atctatcctg gcgacggcag cacccagtac 180

aacgagaagt tcaagggcag agttaccatc accgccgaca agagcaccag cacagcctat 240

atggagctga gcagcctgac cagcgaggac acagctgttt actattgtgc cagagaggc 300

acctactacg caatggatta ttggggccag gggaccagcg tgaccgtttc ttct 354

<210> 87

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223>VL

<400> 87

gatgttgtaa tgacccagac acctctgagc ctgcctgtga ccctgggaga accagcatcc 60

atcagctgtc ggagcagcca gagcatcgtt cacagcaacg gcaacaccta cctggaatgg 120

tatctacaga agcccggaca gagccccagg ctgctgatct acaaggtgtc caaccgcttc 180

agtggtgtgc ccgatagatt ttctggcagc ggctctggca ccgacttcac cctgaagatc 240

tccagagtgg aagccgagga cctgggcgtg tactactgct tccaaggcag ccatgtgcca 300

agaacctttg gtggaggcac aaagctggaa atcaagcgg 339

<210> 88

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223>VL

<400> 88

gatgttgtaa tgacccagag ccctagtagc ctgtctgtga ccctgggaga tcgagtatcc 60

atcagctgtc ggagcagcca gagcatcgtt cacagcaacg gcaacaccta cctggaatgg 120

tatcaacaga agcccggaca gagccccagg ctgctgatct acaaggtgtc caaccgcttc 180

agtggtgtgc ccgatagatt ttctggcagc ggctctggca ccgacttcac cctgacgatc 240

tccagagtgg aaccagagga cctgggcgtg tactactgct tccaaggcag ccatgtgcca 300

agaacctttg gtggaggcac aaagctggaa atcaagcgg 339

<210> 89

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223>VL

<400> 89

gatattgtaa tgacccagag ccctgcaaca ctgtctgtgt cccctggaga acgagcaaca 60

atcagctgtc ggagcagcca gagcatcgtt cacagcaacg gcaacaccta cctggaatgg 120

tatcaacaga agcccggaca ggcccccagg ctgctgatct acaaggtgtc caaccgcttc 180

agtggaatac ccgatagatt ttctggcagc ggctctggca ccgacttcac cctgacgatc 240

tccagattag aaccagagga ctttgcagtg tactactgct tccaaggcag ccatgtgcca 300

agaacctttg gtggaggcac aaagctggaa atcaagcgg 339

<---

Claims (31)

1. A humanized antibody capable of binding to HLA-A2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of VH includes SEQ ID NO:9 and the amino acid sequence of VL includes SEQ ID NO:13. 2. A humanized antibody capable of binding to HLA-A2 according to claim 1, wherein said antibody is a full-length antibody, a single chain antibody, a dimeric single chain antibody, an Fv, a scFv, a Fab, an F(ab)'2, a defucosylated antibody, or a unitibody. 3. A humanized antibody capable of binding to HLA-A2 according to claim 1, wherein said antibody is a scFv, preferably a scFv having the sequence SEQ ID NO: 72. 4. Chimeric antigen receptor (CAR), specifically binding to HLA-A2, containing: - an extracellular domain containing a humanized antibody capable of binding to HLA-A2 according to claim 1, - transmembrane domain and - cytoplasmic domain, including an intracellular signaling domain. 5. Nucleic acid encoding CAR according to claim 4. 6. An expression vector containing the nucleic acid according to claim 5, encoding the CAR according to claim 4. 7. A regulatory immune cell for inducing immune tolerance in a subject in need thereof, comprising a CAR according to claim 4, or a nucleic acid according to claim 5, or an expression vector according to claim 6, wherein said regulatory immune cell is selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory γδ T cell, a regulatory DN T cell, a regulatory B cell, a regulatory NK cell, a regulatory macrophage, regulatory dendritic cell, or a combination thereof. 8. The regulatory immune cell of claim 7, wherein said regulatory immune cell is a regulatory T cell. 9. A regulatory immune cell for the treatment of organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof, comprising the CAR of claim 4, or the nucleic acid of claim 5, or the expression vector of claim 6, wherein said regulatory immune cell is selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory γδ T cell, a regulatory DN T cell, a regulatory B cell, a regulatory NK cell, a regulatory macrophage, regulatory dendritic cell, or a combination thereof. 10. The regulatory immune cell of claim 9, wherein said regulatory immune cell is a regulatory T cell. 11. A composition for inducing immune tolerance in a subject in need thereof, comprising a therapeutically effective amount of regulatory immune cells according to claim 7 or 8, wherein said composition is preferably a pharmaceutical composition and further includes a pharmaceutically acceptable excipient. 12. A composition for treating organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof, comprising a therapeutically effective amount of regulatory immune cells according to claim 9 or 10, wherein said composition is preferably a pharmaceutical composition and further includes a pharmaceutical composition acceptable filler. 13. A method of inducing immune tolerance in a subject in need thereof, comprising administering a regulatory immune cell according to claim 7 or 8 to the subject, wherein said tolerance is preferably tolerance to a transplanted organ or tissue. 14. A method of treating organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof, comprising administering to the subject a regulatory immune cell according to claim 9 or claim 10. 15. The method of claim 13 or 14, further comprising administering to the subject at least one immunosuppressive agent. 16. Use of a regulatory immune cell according to claim 7 or 8 for the induction of immune tolerance in a subject in need thereof. 17. Use of a regulatory immune cell according to claim 9 or 10 for the treatment of organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof. 18. Use according to claim 16 or 17, wherein the subject additionally receives an immunosuppressive agent. 19. Use of a combination of a regulatory immune cell according to claim 7 or 8 with at least one immunosuppressive agent to induce immune tolerance in a subject in need thereof. 20. Use of a combination of a regulatory immune cell according to claim 9 or 10 with at least one immunosuppressive agent for the treatment of organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof. 21. A kit for inducing immune tolerance in a subject in need thereof, comprising a first part containing a regulatory immune cell according to claim 7 or 8, and a second part containing at least one immunosuppressive agent. 22. A kit for the treatment of organ or tissue transplant rejection or graft-versus-host disease in a subject in need thereof, comprising a first part containing a regulatory immune cell according to claim 9 or 10, and a second part containing at least one immunosuppressive agent.