RU2845938C1 - Cell-based assay for determining killing activity of tumour cells expressing chimeric immune cell antigens in vitro - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to immunology and medicine. What is presented is an in vitro method for determining the efficacy (for example, cytotoxicity) of an immune cell expressing a chimeric antigen receptor (CAR) molecule. In the test sample, CAR-expressing immune cells are incubated with target cells expressing an antigen that interacts with CAR. In the control sample, CAR-expressing immune cells are incubated with target cells and with an inhibiting molecule which blocks the interaction between CAR and target cells. Number of dead target cells is determined and compared in the test sample and a control sample.

EFFECT: disclosed analysis does not require use of simulated cells as controls, while reducing associated costs and complexity associated with use thereof.

33 cl, 1 dwg, 8 tbl, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/036,249, filed June 8, 2020, and U.S. Provisional Patent Application No. 63/125,173, filed December 14, 2020. The entire contents of the above-mentioned applications are hereby incorporated by reference in their entirety.

SEQUENCE LIST

The currently pending application contains a sequence listing provided in electronic form in ASCII format and incorporated herein by reference in its entirety. A copy of the ASCII listing created on June 4, 2021 is named JBI6329WOPCT1_SL.txt and is 28,710 bytes in size.

AREA OF TECHNOLOGY

The invention provides improved assays for determining the efficacy (e.g., cytotoxicity) of immune cells expressing chimeric antigen receptors. The improved assays avoid using a mock transfected immune cell as an assay control, instead using an inhibitory molecule that prevents the chimeric antigen receptor of the immune cell from interacting with the target cell as an assay control.

PREREQUISITES FOR THE CREATION OF THE INVENTION

Current methods for determining the in vitro specific cytotoxicity of chimeric antigen receptor T cells (CAR T cells) require the use of autologous, untransduced expanded T cells (surrogate cells) as background controls. These controls are used to calculate the specific cytotoxicity of transduced CAR T cells. However, generating untransduced expanded autologous or allogeneic control T cells (surrogate cells) is expensive and time-consuming, particularly since these cells are typically generated from the patient’s own T cells. Creating the supplicant cells also introduces potential for disruptions in their production, which may delay therapy or prevent proper dosing of CAR T cells during immunotherapy.

In vitro methods for determining CAR T cell cytotoxicity require the use of autologous, untransduced, expanded T cells (mock cells) as a background control. Background controls are used to calculate the percentage increase in CAR T cell-specific cytotoxicity (percentage CAR T killing). If autologous mock cells are not available, qualified lots of allergen-specific mock cells are used instead. However, the use of such qualified lots results in potency relative to allogeneic mock cells, which may not reflect the true potency of the CAR T cells. Alternatively, the background control is omitted and total cytotoxic activity is used. However, total cytotoxic activity does not indicate any CAR T cell-mediated enhancement of the cytotoxic activity of immune cell–target cell interactions. Total cytotoxic activity does not distinguish between contributions from the drug and from spontaneous death of the target cells themselves. Alternative assays such as cytokine ELISA have been used in place of functional assays as a surrogate for measuring activity, but such methods do not directly measure cytotoxicity.

Accordingly, there is a need for improved assay controls to simplify development and testing of CAR T cells while maintaining the accuracy of determining CAR T cell efficacy and reducing the associated costs and complexities associated with the use of mock cells and/or additional alternative assays when mock cells are not available. The subject matter described herein addresses this need by providing new assays that do not require the use of mock cells as controls.

SUMMARY OF THE INVENTION

In one aspect, an in vitro method for determining the efficacy of an immune cell expressing a chimeric antigen receptor (CAR) molecule is provided, comprising:

a) bringing CAR-expressing immune cells into contact with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR,

b) contacting CAR-expressing immune cells with target cells in a first control sample, wherein (i) said contacting is carried out in the presence of an inhibitory molecule or (ii) the CAR-expressing immune cells and/or target cells have been pre-incubated with an inhibitory molecule prior to said contacting, wherein the inhibitory molecule inhibits the interaction between the CAR and the target cells,

c) determination of the number of dead target cells in the test sample,

d) determining the number of dead target cells in the first control sample, and

e) determining the efficacy of the CAR expressing immune cells based on a comparison of the numbers of target cell deaths determined in steps (c) and (d),

wherein the duration of contact, the number of CAR expressing immune cells and the number of target cells are substantially the same in the test sample and the first control sample.

In some embodiments, the contacting steps (a) and (b) are performed simultaneously. In some embodiments, the determining steps (c) and (d) are performed simultaneously.

In some embodiments, in step (b)(i), the CAR expressing immune cells and/or target cells are pre-incubated with an inhibitory molecule prior to the contacting step.

In some embodiments, the method further comprises comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

In some embodiments, the method further comprises comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a third control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that causes target cell death. In certain embodiments, the detergent is Triton X-100.

In various embodiments, the target cells produce a detectable reporter signal upon death of said target cells, and step (c) comprises detecting the reporter signal in a test sample, step (d) comprises detecting the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals detected in steps (c) and (d).

In some embodiments, the reporter signal is luminescence. In some embodiments, the reporter signal is fluorescence. In some embodiments, the target cells express a reporter protein that generates a signal when the target cell undergoes cell death. In some embodiments, the reporter protein is beta-galactosidase, luciferase, or green fluorescent protein (GFP), or a variant or derivative thereof. In some embodiments, the inhibitory molecule specifically binds to an antigen on the target cells, wherein the antigen interacts with the CAR.

In some embodiments, the inhibitory molecule specifically binds to a CAR. In some embodiments, the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on target cells. In some embodiments, the inhibitory molecule is an antibody or antibody fragment. In certain embodiments, the antibody is an anti-idiotypic antibody. In some embodiments, the antibody fragment is a Fab, Fab', F(ab') ₂ , Fv or Fd fragment, single chain antibody (scFv), linear antibody, single domain antibody, heavy chain variable region (VH) domain, or light chain variable region (VL) domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen within an scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to a CDR within an scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to an antigen within a VH domain or a VL domain of a CAR. In some embodiments, the antibody or antibody fragment specifically binds to a CDR within a VH domain or a VL domain of a CAR. In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interacts with a CAR, or a functional fragment or derivative thereof.

In some embodiments, the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells. In some embodiments, the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cells comprise a BCMA receptor, and the inhibitory molecule is a soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In certain embodiments, the multiple myeloma cells are MM-1R cells.

In some embodiments, the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the CAR. In some embodiments, the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the GPRC5D receptor. In some embodiments, the target cells are multiple myeloma cells. In certain embodiments, the multiple myeloma cells are MM-1R cells.

In some embodiments, the CAR interacts with kallikerin 2 (KLK2), the target cells comprise KLK2, and the inhibitory molecule is a soluble KLK2 protein. In some embodiments, the target cells are prostate cells. In some embodiments, the prostate cancer cells are LNCaP cells. In various embodiments, the method is performed in a high-throughput format.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1A shows flow cytometry results demonstrating BCMA-specific competition of labeled BCMA protein on the surface of LCAR-B38M CAR T cells. Sample 1 is only FITC-BCMA labeled.

Figure 1B shows flow cytometry results demonstrating BCMA-specific competition of labeled BCMA protein on the surface of LCAR-B38M CAR T cells. Sample 6s represents competition of FITC-BCMA with unlabeled BCMA.

DETAILED DESCRIPTION

The described methods will be better understood with reference to the following detailed description in conjunction with the accompanying figures, which form a part of this specification. It should be understood that the described methods are not limited to the specific methods described and/or shown herein and that the terminology used herein is intended to describe specific embodiments by way of example only and is not intended to be limiting with respect to the claimed methods.

All patents, published patent applications and publications cited in this document are hereby incorporated by reference as if fully set forth herein.

When a list is presented, unless otherwise indicated, it should be understood that each individual element of the list and each combination of the list is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

Definitions

When used in the specification of this document, nouns may designate one or more items. When used in the claims of this document, nouns in combination with the word "comprising" may designate one or more than one.

The use of the term "or" in the claims is used with the meaning of "and/or" except in cases where it is clearly indicated that only alternatives are to be considered or the alternatives are mutually exclusive, however, the description supports a definition considering only alternatives and "and/or". As used herein, the term "another" means at least a second or more.

The term "about" means "within an acceptable range of error" for a particular value determined by one of ordinary skill in the art, where the error depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. Unless otherwise explicitly stated in the examples or elsewhere in the description in the context of a particular assay, result, or embodiment, "about" means in the range of 10% below the stated value to 10% above the stated value, such as 90 to 110 if the value is 100.

When used herein, the terms "encode" or "encoding" when referring to a nucleic acid are used to facilitate the understanding of the present invention by one of skill in the art; however, these terms may be used interchangeably with "include" and "including," respectively.

The term "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of binding to an antigen-binding domain or T cell receptor that is capable of mediating an immune response. Examples of immune responses include the production of antibodies and the activation of immune cells such as T cells, B cells, or NK cells. Antigens may be expressed by genes, synthesized, or purified from biological samples such as a tissue sample, tumor sample, cell or fluid containing other biological components, organisms, protein/antigen subunits, killed or inactivated whole cells, or lysates.

The term "antibodies" is intended in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, multispecific antibodies such as bispecific, trispecific, tetraspecific, etc., dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that contains an antigen-binding site of the desired specificity. "Full-length antibodies" consist of two heavy chains (HC) and two light chains (LC) joined together by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of the CH1, hinge, CH2 and CH3 domains). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) flanked by framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Based on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies of any vertebrate species can be classified into one of two clearly distinct types, namely kappa (κ) and lambda (λ), depending on the amino acid sequences of their constant domains.

The term "antibody fragment" refers to at least one portion of an intact antibody or recombinant variants thereof that retain the antigen-binding properties of the original full-length antibody. It refers to, for example, an antigen-binding domain, for example, an antigen determining variable region of an intact antibody, that is sufficient to recognize and bind, for example, specifically bind, the antibody fragment to a target such as an antigen. The term "antigen-binding fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments, single-chain antibodies (scFv), linear antibodies, single-domain antibodies such as sdAb (VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

The term "subject" is intended to refer to living organisms in which an immune response can be elicited (e.g., mammals such as humans). Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic strains of these. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

The term "chimeric antigen receptor" (CAR), as used herein, is defined as a cellular receptor that contains an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain in a combination that does not naturally occur on a single protein. This includes receptors in which the extracellular domain and intracellular signaling domain do not naturally occur on a single receptor protein. CARs are primarily intended for use with lymphocytes such as T cells and natural killer (NK) cells.

Complementarity-determining regions (CDRs) are the regions of antibodies that bind antigen. There are three CDRs in the VH region (HCDR1, HCDR2, HCDR3) and three CDRs in the VL region (LCDR1, LCDR2, LCDR3). CDR can be defined using various delimitation systems, such as Kabat (Wu et al. (1970) J Exp Med 132: 211–50; Kabat et al. Sequences of Proteins of Immunological interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901–17), FMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55–77), and AbM (Martin and Thornton J Bmol Biol 263: 800–15, 1996). The correspondence between the various delimitation systems and variable region numberings has been described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27: 55–77; Honegger and Pluckthun, J Mol Biol (2001) 309:657–70; International ImMunoGeneTics (FMGT) database; web resources, http://www_imgt_org). Available programs such as abYsis from UCL Business PLC can be used to annotate CDRs. As used in this document, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the methods described above, according to Kabat, Chothia, IMGT or AbM, unless explicitly stated otherwise in the description.

The terms "reduce," "attenuate," or "reduce" are used interchangeably herein and refer essentially to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) compared to the response mediated by a control sample or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or protein binding to its antigen or receptor, enhanced binding to Fey, or improved Fc effector functions such as improved ADCC, CDC, and/or ADCP. The decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle) or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 times or more, such as 500, 600, 700, 800, 900, 1000 times or more (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

The terms "enhance," "activate," "increase," "expand," or "improve" refer essentially to the ability of a test molecule to mediate a higher response (i.e., downstream effect) than the response mediated by a control sample or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or protein binding to its antigen or receptor, enhanced binding to Fey, or improved Fc effector functions such as improved ADCC, CDC, and/or ADCP. An enhancement may be a statistically significant difference in the measured response between the test molecule and a control (or vehicle) or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 times or more, such as 500, 600, 700, 800, 900, 1000 times or more (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

The term "dAb" or "dAb fragment" refers to a fragment of an antibody consisting of the VH domain (Ward et al. Nature 341:544 546 (1989)).

The term "Fab" or "Fab fragment" refers to an antibody fragment consisting of the VH, CH1, VL, and CL domains.

The term "F(ab') ₂ " or "F(ab') ₂ fragment" refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge at the hinge region.

The term "Fd" or "Fd fragment" refers to the fragment of an antibody consisting of the VH and CH1 domains.

The term "Fv" or "Fv fragment" refers to an antibody fragment consisting of the VH and VL domains from one arm of the antibody.

A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain consists of a heavy-chain variable domain (VH) and a heavy-chain constant domain consisting of the CH1, hinge, CH2, and CH3 subdomains. Each light chain consists of a light-chain variable domain (VL) and a light-chain constant domain (CL). VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), between which are framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The term "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from human immunoglobulin sequences. A humanized antibody may include substitutions in the framework regions such that the framework regions may not be an exact copy of the expressed human immunoglobulin or the germline gene sequences of the human immunoglobulin.

The term “intracellular signaling domain” or “cytoplasmic signaling domain” refers to the intracellular portion of the molecule. It is the functional portion of the protein that acts by transmitting information within the cell to regulate cellular activity through specific signaling pathways, generating second messengers or acting as effectors in response to such messengers. The intracellular signaling domain generates a signal that induces immune effector function in a cell containing a CAR, such as a CAR-T cell.

The term "isolated" refers to a homogeneous population of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially separated and/or purified from other components of the system in which the molecules were formed, such as a recombinant cell, as well as a protein that has undergone at least one purification or isolation step. The term "isolated" refers to an antibody that is substantially free of other cellular materials and/or chemicals, and encompasses molecules that are isolated to higher purities, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of the C-terminal lysine from the antibody heavy chain or post-translational modifications such as isomerization or deamidation of amino acids, oxidation of methionine or asparagine, or deamidation of glutamine. Monoclonal antibodies typically bind a single antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Within a population of antibodies, monoclonal antibodies may have heterogeneous glycosylation. A monoclonal antibody may be monospecific or multispecific, e.g., bispecific, monovalent, divalent, or multivalent.

The terms "natural killer cell" and "NK cell" are used interchangeably and are synonymous in this document. The term "NK cell" refers to a differentiated lymphocyte with a CD16 ⁺ CD56 ⁺ and/or CD57 ^{+ TCR-} phenotype. NK cells are characterized by the ability to bind to and kill cells that do not express "self" MHC/HLA antigens by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express ligand for NK cell-activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

The terms "protein" or "polypeptide" are used interchangeably herein and refer to a molecule that comprises one or more polypeptides, each of which consists of at least two amino acid residues linked by a peptide bond. The protein may be a monomer or a protein complex of two or more subunits, wherein the subunits are identical or different from each other. Small polypeptides containing less than 50 amino acid residues may be referred to as "peptides". The protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, PEGylation, or biotinylation. The protein may be recombinantly expressed.

The term "recombinant" refers to polynucleotides, polypeptides, vectors, viruses and other macromolecules that are produced, expressed, created or isolated by recombinant means. The term "recombinant antibody" refers to an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be understood as meaning an antibody that has been produced by synthesizing a DNA molecule encoding the antibody and which expresses the antibody protein, or an amino acid sequence defining the antibody, wherein the DNA or amino acid sequence was produced using recombinant DNA technology or an amino acid sequence that is available and known in the art.

The term "single chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and VH are continuously connected by a polypeptide linker, and which can be expressed as a single chain polypeptide. Unless otherwise specified, in the context of this document, an scFv can have the VL and VH variable regions in any order, for example, relative to the N-terminal and C-terminal ends of the polypeptide, an scFv can comprise a VL-linker-VH or can comprise a VH-linker-VL.

The terms "specifically binds", "specific binding", "specifically binds" or "binds" refer to the binding of a protein molecule to an antigen or an epitope within the antigen with greater affinity than other antigens. Typically, a protein molecule binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (K _D ) of about 1 x 10 ^-7 M or less, such as about 5 x 10 ^-8 M or less, about 1 x 10 ^-8 M or less, about 1 x 10 ^-9 M or less, about 1 x 10 ^-10 M or less, about 1 x 10 ^-11 M or less, or about 1 x 10 ^-12 M or less, typically with a K _D value that is at least one hundred times lower than its K _D value for binding to a non-specific antigen (e.g., BSA, casein).

The terms "T cell" and "T lymphocyte" are used interchangeably and are used synonymously herein. A T cell includes thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell may be a T helper (Th) cell, such as T helper 1 (Th1) or T helper 2 (Th2). A T cell may be a helper T cell (HTL; CD4 ⁺ T cell), a CD4 ⁺ T cell, a cytotoxic T cell (CTL; CD8 ⁺ T cell), a tumor-infiltrating cytotoxic T cell (TIL; CD8 ⁺ T cell), a CD4CD8 T cell, or any other T cell subset. Also included are “NKT cells,” which refer to a specialized population of T cells that express the semi-invariant αβ T cell receptor but also express a variety of molecular markers typically associated with Nk cells, such as NK1.1. NKT cells include NK1.1 ⁺ and ^NK1.1- , as well as CD4 ⁺ , CD4 ⁺ , CD8 ⁺ , and CD8 ⁺ cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have both protective and detrimental effects due to their ability to produce cytokines that promote inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)", which refer to a specialized population, namely a small subset of T cells, that have a distinct TCR on their surface, and unlike most T cells in which the TCR is composed of two glycoprotein chains, designated the TCR α and β chains, the T cell receptor in γδ T cells is composed of a γ chain and a δ chain. γδ T cells may play a role in immunosurveillance and immunoregulation, and have been found to be an important source of IL-17 and induce robust cytotoxic CD8 ⁺ T cell responses. Also included are “regulatory T cells” or “Tregs,” which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 ⁺ T cells and may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4 ⁺ T cells.

The term "tumor cell" or "cancer cell" refers to a cancerous, precancerous, or transformed cell, either in vivo or ex vivo or in tissue culture, that exhibits spontaneous or induced phenotypic changes. These changes do not necessarily involve the introduction of new genetic material. Although transformation may be induced by infection with a transforming virus and the introduction of new genomic nucleic acid, by the uptake of exogenous nucleic acid, or it may also occur spontaneously or following exposure to a carcinogen resulting in mutation of an endogenous gene. Transformation/cancer is manifested by morphological changes, cell immortalization, disruption of growth control, lesion formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasiveness, tumor growth in suitable host animals such as nude mice, etc., in vitro, in vivo, and ex vivo.

The terms "variant," "mutant," or "altered" refer to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

"Efficacy" of a cell (e.g., a CAR T cell) as discussed herein is an indicator or measure of its effectiveness or potential effectiveness in achieving a desired function. In the case of a CAR T cell, the desired function may be to target or kill another cell, such as a target cell (e.g., a tumor cell). Efficacy can be assessed directly by determining the effect of the cell on its target (e.g., the effect of a CAR T cell on a tumor cell in vitro or in vivo). Alternatively, efficacy can be measured indirectly, as in the various methods of the present invention. In particular, the efficacy of a CAR T cell can be assessed by determining the level of in vitro antigen-specific toxicity of the cell in an assay, such as described herein (relative to, for example, the cytotoxicity of an unstimulated CAR T cell, as described herein). Such a measure of efficacy can then be correlated with in vivo cell properties such as PK/PD parameters and thus considered predictive as described herein (e.g., CMAX, TAX, and AUC) that may be related to the cell's efficiency in killing its targets. As described further herein, efficacy can be expressed in terms of a cytotoxicity index that can be normalized based on the number of cells expressing the relevant CAR.

As used herein, the terms "reference" or "control" describe a standard or control against which a comparison is made. For example, in some embodiments, an analyte, animal, subject, population, sample, sequence, or value is compared to a reference or standard agent, animal, person, population, sample, sequence, or value. In some embodiments, the reference or control is tested and/or determined substantially simultaneously with the assay testing or determination. Typically, as will be appreciated by one of skill in the art, the reference or control is determined or characterized under comparable conditions or circumstances to those used to perform the assay. Those of skill in the art can determine when there are sufficient similarities to allow reliance on and/or comparison to a particular candidate reference or control.

The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a T cell, an NK cell, a B cell) that comprises a cytoplasmic signaling sequence that regulates activation of the immune cell in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal that is triggered, for example, by binding of a TCR/CD3 complex to a peptide-bearing MHC molecule, and that results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAM-containing cytoplasmic signal sequences include, but are not limited to, sequences derived from CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP1 0, and DAP12. In a CAR, the intracellular signaling domain may comprise an intracellular signaling sequence, such as the primary signaling sequence of CD3 zeta.

Other objects, features and advantages of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.

Chimeric antigen receptors

Immune cells (e.g., T cells) can be genetically modified to stably express a desired chimeric antigen receptor. A chimeric antigen receptor (CAR) is an engineered fusion protein or polypeptide comprising the antigen-binding domains of an antibody (scFv) linked to the signaling domains of an immune cell, such as a T cell. Characteristics of CARs may include their ability to redirect T cell specificity and reactivity to a chosen target in a non-MHC-restricted manner using the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor immune evasion. Moreover, when expressed in T cells, CARs do not preferentially dimerize with the endogenous alpha and beta chains of the T cell receptor (TCR).

The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. T cells expressing CARs are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. A cell can be genetically modified to stably express an antibody binding domain on its surface, conferring a novel antigen specificity that is independent of MHC.

In some cases, the T cell is genetically modified to stably express a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3 zeta chain or FcγRI protein into a single chimeric protein. In one embodiment, the stimulatory molecule is a zeta chain associated with a T cell receptor complex.

As used herein, the term "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. It is the functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity through specific signaling pathways, generating second messengers or acting as effectors in response to such messengers. The intracellular signaling domain generates a signal that induces an immune effector function in a cell containing a CAR, such as a CAR-T cell. Examples of immune effector function, such as in a CAR-T cell, include cytolytic activity and helper activity, including cytokine secretion.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include domains derived from molecules that are responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include domains derived from molecules that are responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence of a coreceptor or costimulatory molecule.

The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, derivatives of CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b, and CD66d of DAP10 and DAP12.

The term "zeta" or alternatively "zeta chain", "CD3 zeta" or "TCR zeta" is defined as the protein represented by GenBank accession number BAG36664.1, or equivalent residues, derived from a non-human species, such as a mouse, rabbit, primate, mouse, rodent, monkey, ape, etc., and the "zeta stimulatory domain", or alternatively "CD3 zeta stimulatory domain", or "TCR zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic zeta domain comprises residues 52 to 164 according to GenBank accession number BAG36664.1 or equivalent residues obtained from a non-human species, such as a mouse, rodent, monkey, ape, etc., that are functional orthologs thereof. In one aspect, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence set forth in SEQ ID NO: 10 below, or a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 10.

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 T cell response such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are essential for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecule, BTLA, and Toll receptor ligand, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137).

The costimulatory intracellular signaling domain may be an intracellular region of a costimulatory molecule. The costimulatory molecule may be present in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83-specific ligand, etc.

The intracellular signaling domain may comprise the entire intracellular region or the entire native intracellular signaling domain of the molecule from which it was derived, or a functional fragment thereof.

The term "4-1BB" or alternatively "CD137" refers to a member of the RFNO superfamily with the amino acid sequence provided by GenBank Accession Number AAA62478.2 or equivalent residues derived from a non-human species such as a mouse, rodent, monkey, ape, etc.; and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession Number: AAA62478.2 or equivalent residues derived from a non-human species such as a mouse, rodent, monkey, ape, etc. In one aspect, the "4-1BB costimulatory domain" or "CD137 costimulatory domain" is the sequence set forth in SEQ ID NO: 11 below, or equivalent residues derived from a non-human species, such as a mouse, rodent, monkey, ape, etc., or a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 11.

In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the CAR. In another embodiment, the transmembrane domain may be selected from or modified by amino acid substitution to avoid association of such domains with their transmembrane domains or various surface membrane proteins to minimize interactions with other members of the receptor complex. In one embodiment, the transmembrane domain comprises the hinge domain of CD8a.

In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined herein. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-zeta, and/or CD28. CD28 is a T cell marker important for T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and serves as a costimulatory immune checkpoint molecule. 4-1BB transmits a potent costimulatory signal to T cells, stimulating differentiation and enhancing long-term survival of T lymphocytes. CD3-zeta binds to the TCR to generate a signal and comprises immunoreceptor tyrosine-based activation motifs (ITAMs). In another embodiment, the costimulatory molecule is MyD88 or CD40.

In one embodiment, the CAR comprises an intracellular hinge domain comprising CD8a and an intracellular T cell receptor signaling domain comprising CD28, 4-1BB and CD3zeta. In another embodiment, the CAR comprises an intracellular hinge domain and an intracellular T cell receptor signaling domain comprising CD28, 4-1BB and CD3zeta, wherein the hinge domain comprises all or a portion of an extracellular region of CD8, CD4 or CD28; all or a portion of an antibody constant region; all or a portion of an FcγRIIIa receptor, all or an IgG hinge region, an IgM hinge region, an IgA hinge region, an IgD hinge region, an IgE hinge region or an Ig hinge region or portions of these regions. The hinge region of IgG can be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain"), comprising, for example, a functional signaling domain derived from a stimulatory molecule as defined below.

In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

CARs may be engineered to comprise the CD28 and/or 4-1BB signaling domain alone or combined with any other desired cytoplasmic domain(s) useful in the context of the CARs described herein. In one embodiment, the cytoplasmic domain of the CAR may further comprise the CD3-zeta signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, CD3-zeta, 4-1BB, and CD28 signaling modules, and combinations thereof.

In some embodiments, the CARs described herein comprise an extracellular antigen-binding domain that specifically binds a tumor antigen. Non-limiting examples of tumor antigens that can be recognized by the CARs described herein include BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

The invention further provides variants, such as functional variants, of the CARs, nucleic acids, polypeptides, and proteins described herein. The term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitutions, insertions, or deletions. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, polypeptide, or protein, wherein the functional variant retains the biological activity of the CAR, polypeptide, or protein of which it is a variant. Functional variants include, for example, those variants of a CAR, polypeptide, or protein described herein (a parent CAR, polypeptide, or protein) that retain the ability to recognize target cells (e.g., tumor cells) to a similar extent, to the same extent, or to a greater extent than the parent CAR, polypeptide, or protein. Relative to the parent CAR, polypeptide, or protein, a functional variant can be, for example, at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%), about 96%, about 97%, about 98%, about 99%, or more identical in amino acid sequence to the parent CAR, polypeptide, or protein.

A functional variant may, for example, comprise an amino acid sequence of a parent CAR, polypeptide, or protein with at least one conservative amino acid substitution. In another embodiment, functional variants may comprise an amino acid sequence of a parent CAR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, a non-conservative amino acid substitution may not interfere with or inhibit the biological activity of the functional variant. A non-conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent CAR, polypeptide, or protein.

Amino acid substitutions in the inventive CARs may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be an acidic amino acid substituted by another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain substituted by another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted by another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted by another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

The CAR, polypeptide or protein may consist essentially of the amino acid sequence or sequences described herein such that other components, such as other amino acids, do not substantially alter the biological activity of the functional variant.

The CARs, polypeptides and proteins of embodiments of the invention (including functional regions, functional variants) may be of any length, i.e. may contain any number of amino acids, as long as the CARs, polypeptides or proteins (or functional regions thereof, or functional variants thereof) retain their biological activity, such as the ability to specifically bind to an antigen, detect diseased cells (e.g., cancer cells) in a host organism, or treat or prevent a disease in a host organism, etc. For example, a polypeptide can have a length of from about 50 to about 5,000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000 or more amino acids. The polypeptides described herein also include oligopeptides.

CARs, polypeptides and proteins used in aspects and embodiments of the present invention (including functional regions and functional variants of CARs) may comprise synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide and α-tert-butylglycine.

CARs, polypeptides and proteins used in aspects and embodiments of the present invention (including functional regions and functional variants) may be subject to post-translational modifications. They may be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via, for example, a disulfide bridge, or converted into an acid addition salt. In some embodiments, they are dimerized or polymerized or conjugated.

CARs, polypeptides, and/or proteins used in aspects and embodiments of the present invention (including functional portions and functional variants thereof) can be prepared by methods known in the art. Suitable methods for de novo synthesizing polypeptides and proteins are described in publications such as Chan et al. Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; and Epitope Mapping, ed. Westwood et al. Oxford University Press, Oxford, United Kingdom, 2001. In addition, the polypeptides and proteins can be produced recombinantly using the nucleic acids described herein using standard recombinant techniques. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Some of the CARs, polypeptides, and proteins described herein (including functional portions and functional variants thereof) may be further isolated and/or purified from a source such as a plant, bacterium, insect, mammal, etc. Methods for isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and/or proteins described herein (including functional portions and functional variants thereof) may be commercially synthesized. In this regard, the CARs, polypeptides, and proteins may be synthetic, recombinant, isolated, and/or purified.

Examples of modified nucleotides that can be used to produce recombinant nucleic acids used to produce the polypeptides described herein include, without limitation, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N ⁶ -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5''-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N ⁶ -isopentenyladenine, uracil-5-hydroxyacetic acid (v), vibutoxysine, pseudouracil, queuosine, beta-B-galactosylqueosine, inosine, N ⁶ -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine.

The nucleic acid may comprise any isolated or purified nucleotide sequence that encodes any of the CARs, polypeptides, or proteins, or functional portions thereof, or functional variants thereof. Alternatively, the nucleotide sequence may comprise a sequence degenerate into any of the sequences or a combination of degenerate sequences. The nucleic acid molecules may be inserted into a recombinant expression vector. Recombinant expression vectors comprising one or more nucleic acids may be used. As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that provides for expression of an mRNA, protein, polypeptide, or peptide by a host cell, wherein the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient for expression of the mRNA, protein, polypeptide, or peptide within the cell. The vectors described herein do not occur in nature as a whole; however, portions of the vectors may be of natural origin. The recombinant expression vectors described herein may contain any type of nucleotides, including, without limitation, DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially derived from natural sources, and which may contain natural, non-natural, or altered nucleotides. The recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both. Non-natural or altered nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

The recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD, USA), the pBluescript series (Stratagene, La Jolla, CA, USA), the pET series (Novagen, Madison, WI, USA), the pGEX series (Pharmacia Biotech, Uppsala, Sweden) and the pEX series (Clontech, Palo Alto, CA, USA). Bacteriophage vectors such as λGT10, λGT11, λEMBL4 and λNM1149, λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBH01.3 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector, such as a gamma retroviral vector.

Recombinant expression vectors are prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., supra, and Ausubel et al., supra. Expression vector constructs can be prepared that are circular or linear so that they contain a replication system that functions in a prokaryotic or eukaryotic host cell. Replication systems can be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, etc.

A recombinant expression vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the host type (e.g., bacteria, plants, fungi, or animals) into which the vector is to be introduced, if necessary, taking into account whether the vector is DNA or RNA based.

The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host organisms. Marker genes include biocide resistance genes, such as antibiotic resistance genes, heavy metal resistance genes, etc., complementation in an auxotrophic host to ensure prototrophy, etc. Suitable marker genes for the described expression vectors include, for example, neomycin/G418 resistance genes, histidinol X resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector may comprise a native or normal promoter operably linked to a nucleotide sequence encoding a CAR, a polypeptide or a protein (including functional portions thereof and functional variants thereof), or to a nucleotide sequence that is complementary to or that hybridizes with a nucleotide sequence encoding a CAR, a polypeptide or a protein. The selection of promoters, such as strong, weak, tissue-specific, inducible and development-specific promoters, is within the skill of a person of ordinary skill in the art. Likewise, combining a nucleotide sequence with a promoter is also within the skill of a person of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter or a promoter present in the long terminal repeat of a mouse stem cell virus.

Recombinant expression vectors can be designed for either transient expression, stable expression, or both. In addition, recombinant expression vectors can be designed for constitutive expression or inducible expression.

Additionally, recombinant expression vectors can be created that include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes a cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, such as a drug, to a cell in which the gene is expressed, and causes the cell to die when the cell is contacted or exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

The inhibitory molecule may be an antibody (e.g., a monoclonal antibody), or an antigen-binding portion thereof, or a soluble antigen, or a functional portion or functional variant thereof, that binds, e.g., specifically binds, to an epitope of a CAR immune cell. The antibody may be any type of immunoglobulin known in the art. Immunoglobulins may belong to five major classes - IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further divided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies in vertebrates can be classified into one of two types, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. An antibody may belong to any class or isotype.

Antibodies used in the methods described herein may include immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomeric, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site of the desired specificity. The antibody may be a natural antibody, such as an antibody isolated and/or purified from a mammal, such as primates, mice, rabbits, goats, horses, chickens, hamsters, humans, etc. Alternatively, the antibody may be an engineered (e.g., genetically engineered) antibody.

Humanized antibodies have antigen-binding sites derived from non-human species and variable region framework regions derived from human immunoglobulin sequences. The term "human antibodies" refers to antibodies having heavy and light chain variable regions in which both the framework regions and antigen-binding sites are derived from sequences of human origin.

In addition, the antibody can have any level of affinity or avidity for the functional region of the CAR. In some embodiments, the antibody can bind the hK2 antigen over a range of affinities (K _D ). In various embodiments, the antibody binds the hK2 antigen with high affinity, such as a K _D value of about 10 M or less, such as, but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, 10 ^-13 M, 10 ^-14 M, 10 ^-15 M, or any range or value therein, as determined by surface plasmon resonance or Kinexa assays, as used by those skilled in the art. In one example, the affinity is equal to or less than 1×10 ^-8 M. In another example, the affinity is equal to or less than 1×10 ^-9 M.

Methods for testing antibodies for the ability to bind to any functional region of a CAR are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays.

Suitable methods for producing antibodies are known in the art. For example, standard hybridoma methods are described in publications such as Köhler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). Alternatively, other methods, such as EBV hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al. Methods Enzymol., 121, 140-67 (1986)), and bacteriophage-based expression vector systems (see, e.g., Huse et al. Science, 246, 1275-81 (1989)) are known in the art. In addition, methods for producing antibodies in non-human animals are described, e.g., in U.S. Patent Nos. 5,545,806, 5,569,825, and 5,714,352 and U.S. Patent Application Publication No. 2002/0197266 A1).

Phage displays can also be used to produce antibodies useful in any of the methods described herein. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al., supra, and Ausubel et al., supra). A phage encoding a variable region with the desired specificity is selected to specifically bind to the desired antigen (i.e., hK2), and a full or partial antibody containing the selected variable domain is recovered. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell, that is used to produce a hybridoma, so that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Patent No. 6,265,150).

Antibodies can be produced using transgenic mice that are transgenic for specific immunoglobulin heavy and light chain genes. Such methods are known in the art and are described, for example, in U.S. Patent Nos. 5,545,806 and 5,569,825 and Janeway et al., supra.

Methods for making humanized antibodies are known in the art and are described, for example, in Janeway et al., supra, U.S. Patent Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400 B1 and British Patent No. 2,188,638. Humanized antibodies can also be made using the antibody resurfacing technology described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

The antibodies used herein may be multi-chain or single-chain or intact immunoglobulins and may be obtained from natural sources or from recombinant sources. The antibodies may be tetramers of immunoglobulin molecules.

Additionally, antigen-binding portions of any of the antibodies described herein are provided. The antigen-binding portion can be any portion that has at least one antigen-binding site, such as Fab, F(ab') ₂ , dsFv, sFv, diabodies, and triabodies. In some embodiments, the antigen-binding fragments are heavy chain complementarity determining regions (HCDRs) 1, 2, and/or 3, light chain complementarity determining regions (LCDRs) 1, 2, and/or 3, a heavy chain variable region (VH), or a light chain variable region (VL), Fab, F(ab') ₂ , Fd, and Fv fragments, and domain antibodies (dAbs) comprising (e.g., consisting of) either one VH domain or one VL domain. The VH and VL domains can be linked together by a linker, such as a synthetic linker.

In addition, the antibody or its antigen-binding portion can be modified to contain a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

The present invention also provides a nucleic acid comprising a nucleotide sequence encoding any of the CARs, polypeptides or proteins described herein (including functional regions thereof and functional variants thereof).

The antibody or antibody fragment portion of a CAR may exist in a variety of forms where the antigen-binding domain is expressed as a portion of a continuous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), an scFv, and a chimeric or humanized human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of a CAR composition comprises an antibody fragment. In one aspect, the CAR comprises an antibody fragment that comprises an scFv.

In one embodiment, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide between the light chain variable region and the heavy chain variable region.

In recombinant expression systems, the linker is a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be of sufficient length to link VH and VL so that they form the correct conformation relative to each other so that they retain the desired activity, such as binding to hK2.

The linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that can be used are Gly-rich linkers containing Gly and Ser, Gly and Ala linkers containing Ala and Ser linkers, and other flexible linkers.

In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. The signal polypeptide can be located at the N-terminus of the extracellular antigen-binding domain, which binds to hK2. The signal polypeptide can be further cleaved from the extracellular antigen-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of various signal polypeptides known to one of skill in the art can be used as the signal polypeptide. Non-limiting examples of peptides from which signal polypeptides can be derived include FcsR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8a, or any of various other proteins secreted by T cells. In various embodiments, the signal polypeptide is compatible with the secretory pathway of a T cell.

In one aspect, the description provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a component of the TNF receptor superfamily member 9 (CD 137), a component of the CD3 zeta chain (CD3z) of the T cell surface glycoprotein, a component of the cluster of differentiation (CD27), a member of the differentiation superfamily (such as, for example, CD28 or a component of the inducible T cell costimulator (ICOS)) and a combination thereof. In one embodiment, the transmembrane domain comprises a CD8a transmembrane region polypeptide (CD8a-TM). In one embodiment, the transmembrane domain comprises at least the transmembrane region(s) of a T cell receptor α, β, or ζ chain, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, CD154. In another embodiment, the transmembrane domain comprises at least the transmembrane domain of ζη or FcεRlγ and -β, MB1 (Igα), B29 or CD3-γ, ζ, or η. In another embodiment, the transmembrane domain is synthetic, for example, comprising predominantly hydrophobic residues such as leucine and valine, a phenylalanine triplet, or tryptophan.

In one embodiment, the CAR further comprises a hinge region linking the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region.

In one aspect, the present invention provides isolated immunoresponsive cells comprising CARs as described herein. In some embodiments, the isolated immunoresponsive cell is transduced with a CAR, e.g., the CAR is constitutively expressed on the surface of the immunoresponsive cell. In certain embodiments, the isolated immunoresponsive cell is further transduced with at least one costimulatory ligand, such that the immunoresponsive cell expresses the at least one costimulatory ligand. In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD48, CD70, CD80, CD86, OX40L, TNFRSF14, and combinations thereof. In certain embodiments, the isolated immunoresponsive cell is further transduced with at least one cytokine, such that the immunoresponsive cell secretes the at least one cytokine. In certain embodiments, at least the cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, TL-15, IL-17, IL-21, and combinations thereof. In some embodiments, the isolated immunoresponsive cell is selected from the group consisting of a T lymphocyte (T cell), a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, a lymphoid progenitor cell, a T cell progenitor cell, and a pluripotent stem cell from which lymphoid cells can be differentiated.

In one embodiment, immune cells expressing CAR-T cells as described can be obtained by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-hK2, a hinge and transmembrane domain of CD8α, and the 4-1BB and CD3-zeta signaling domains of human, into the cells. Immune cells expressing CAR-T cells of the invention are capable of replicating in vivo, resulting in long-term persistence, which can lead to durable tumor control.

Any CAR and inhibitory molecules may be expressed in host cells containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector. The host cell may be a eukaryotic cell, such as a plant, animal, algae, or fungus, or may be a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism, such as a human. The host cell may be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, E. coli DH5α cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. For producing a recombinant CAR, polypeptide, or protein, the host cell can be a mammalian cell. The host cell can be a human cell. Although the host cell can be any cell type, can be from any tissue type, and can be at any stage of development, the host cell can be a peripheral blood lymphocyte (PBL). The host cell can be a T cell.

For the purposes of this invention, the T cell may be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTl, etc., or a T cell obtained from a mammal. If the T cell is obtained from a mammal, it may be obtained from a variety of sources, including, but not limited to, bone marrow, blood, lymph node, thymus, or other tissues or fluids. T cells may also be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be any type of T cell and any developmental stage, including, but not limited to, CD4 ⁺ /CD8 ⁺ double positive T cells, CD8 ⁺ T cells (e.g., cytotoxic T cells), CD4 ⁺ T helper cells such as Th ₁ and Th ₂ cells, peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells, memory T cells, naive T cells, etc. The T cell may be a CD8 ⁺ T cell or a CD4 ⁺ T cell.

Additionally, a population of cells is provided comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising a host cell comprising any of the recombinant expression vectors described in addition to at least one other cell, such as a host cell (e.g., a T cell) that does not comprise any of the recombinant expression vectors or a cell other than a T cell, such as a B cell, a macrophage, a erythrocyte, a neutrophil, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a substantially homogeneous population in which the population primarily comprises (e.g., consists primarily of) host cells comprising a recombinant expression vector. The population may also be a clonal population of cells, in which all cells in the population are clones of a single host cell containing the recombinant expression vector, such that all cells in the population contain the recombinant expression vector. In one embodiment, the population of cells is a clonal population comprising host cells containing the recombinant expression vector as described herein.

Inhibitors that bind to CAR

In some embodiments, the inhibitory molecules used in the methods described herein can be monoclonal antibodies that specifically bind to a CAR polypeptide. For example, a monoclonal antibody can specifically bind to a constant domain of a CAR polypeptide described herein, such as a CAR polypeptide expressed on a CAR T cell. Alternatively, the antibody can bind to an antigen recognition domain of a CAR polypeptide (e.g., a CD-19-binding CAR polypeptide). The antibody can inhibit the ability of a CAR T cell to bind to a target cell (e.g., a tumor cell). Without being limited by theory, the antibody can prevent a CAR T cell from binding to a target cell (e.g., a tumor cell).

In various embodiments, a monoclonal antibody is used that specifically binds to a BCMA-targeted CAR polypeptide. In some aspects, the monoclonal antibody binds to a BCMA-specific CAR polypeptide and competes for binding of the polypeptide to a target cell - a multiple myeloma cell, or any other cell expressing BCMA. The monoclonal antibody can be an anti-idiotypic antibody. Anti-idiotypic antibodies are specific antibodies that can bind to CDR sequences in specific antibodies. The monoclonal antibody can be a type 1 anti-idiotypic antibody that binds to the CDR regions of the variable region of the target antibody in such a way as to inhibit, impair or neutralize the activity of the target antibody, i.e., its ability to bind an antigen.

The inhibitory molecule may be an anti-idiotypic peptide. In some embodiments, the anti-idiotypic peptide binds an antigen-binding receptor of one or more additional cellular therapeutic agents (e.g., an scFv on a CAR T cell). In some embodiments, the anti-idiotypic polypeptide binds an antigen-binding receptor of one or more CDRs of an antigen-binding receptor (e.g., an scFv on a CAR T cell). In various embodiments, the anti-idiotypic antibody or peptide (e.g., an scFv) binds to a B-cell-specific marker antigen-binding region (e.g., a CAR that binds CD19, CD20, CD21, CD22, CD24, CD79a, CD79b, ROR1, or BCMA) on a CAR T cell. Additionally, for example, in some embodiments, an anti-idiotypic antibody or fragment (e.g., an scFv) binds an anti-CD19 antibody or fragment (e.g., an anti-CD19 antibody (e.g., an anti-CD19 scFv) expressed by a CAR T cell).

Inhibitory molecules are also provided that comprise all or part of a heavy chain variable region of a monoclonal antibody that specifically binds to a BCMA-targeted CAR polypeptide. Such inhibitory molecules may also specifically bind to a BCMA-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise all or part of a light chain variable region of a monoclonal antibody that specifically binds to a BCMA-targeted CAR polypeptide. Such inhibitory molecules may also specifically bind to a BCMA-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise one, two, three, four, five or six complementarity determining regions (CDRs) from a light variable and/or heavy variable chain of a monoclonal antibody that binds to a BCMA-specific CAR polypeptide.

In certain embodiments, a monoclonal antibody is used that specifically binds to a GPRC5D-targeted CAR polypeptide. In some aspects, the monoclonal antibody binds to a GPRCSD-specific CAR polypeptide and competes for binding of the polypeptide to a target cell - a multiple myeloma cell, or any other cell expressing GPRC5D. The monoclonal antibody can be an anti-idiotypic antibody. Inhibitory molecules are also provided that comprise all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to a GPRC5D-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a GPRC5D-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise all or part of a light chain variable region of a monoclonal antibody that specifically binds to a GPRC5D-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a GPRC5D-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise one, two, three, four, five or six complementarity determining regions (CDRs) from a light variable and/or heavy variable chain of a monoclonal antibody that binds to a GPRC5D-specific CAR polypeptide.

In certain embodiments, a monoclonal antibody is used that specifically binds to a CD79-targeted CAR polypeptide. In some aspects, the monoclonal antibody binds to a CD79-specific CAR polypeptide and competes for binding of the polypeptide to a target cell - a multiple myeloma cell, or any other cell expressing CD79. The monoclonal antibody can be an anti-idiotypic antibody. Inhibitory molecules are also provided that comprise all or a portion of a heavy chain variable region of a monoclonal antibody that specifically binds to a CD79-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a CD79-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise all or a portion of a light chain variable region of a monoclonal antibody that specifically binds to a CD79-targeted CAR polypeptide. Such inhibitory molecules may also specifically bind to a CD79-targeted CAR polypeptide. In addition, inhibitory molecules are provided that comprise one, two, three, four, five or six complementarity determining regions (CDRs) from a light variable and/or heavy variable chain of a monoclonal antibody that binds to a CD79-specific CAR polypeptide.

In certain embodiments, a monoclonal antibody is used that specifically binds to a KLK2-targeted CAR polypeptide. In some aspects, the monoclonal antibody binds to a KLK2-specific CAR polypeptide and competes for binding of the polypeptide to a target cell - a multiple myeloma cell, or any other cell expressing KLK2. The monoclonal antibody can be an anti-idiotypic antibody. Inhibitory molecules are also provided that comprise all or a portion of a heavy chain variable region of a monoclonal antibody that specifically binds to a KLK2-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a KLK2-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise all or a portion of a light chain variable region of a monoclonal antibody that specifically binds to a KLK2-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a KLK2-targeted CAR polypeptide. In addition, inhibitory molecules are provided that comprise one, two, three, four, five or six complementarity determining regions (CDRs) from a light variable and/or heavy variable chain of a monoclonal antibody that binds to a KLK2-specific CAR polypeptide. In certain embodiments, a monoclonal antibody is used that specifically binds to a CD19-targeted CAR polypeptide. In some aspects, the monoclonal antibody binds to a CD19-specific CAR polypeptide and competes for binding of the polypeptide to a target cell - a multiple myeloma cell, or any other cell expressing CD19. The monoclonal antibody can be an anti-idiotypic antibody. Inhibitory molecules are also provided that comprise all or part of a heavy chain variable region of a monoclonal antibody that specifically binds to a CD19-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a CD19-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise all or part of a light chain variable region of a monoclonal antibody that specifically binds to a CD19-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a CD19-targeted CAR polypeptide. Inhibitory molecules are also provided that comprise one, two, three, four, five or six complementarity determining regions (CDRs) from a light variable and/or heavy variable chain of a monoclonal antibody that binds to a CD19-specific CAR polypeptide.

In various embodiments, an inhibitory molecule, such as a monoclonal antibody, a fragment of a monoclonal antibody, or a derivative of a monoclonal antibody, is capable of transgene-specific propagation. Anti-idiotypic antibodies, including antigen-binding fragments thereof, specifically recognize, specifically target, and/or specifically bind to an idiotope of an antibody or an antigen-binding fragment thereof, such as an antigen-binding domain of a recombinant receptor, such as a chimeric antigen receptor (CAR). An idiotope is any single antigenic determinant or epitope in the variable region of an antibody. Anti-idiotypic antibodies or antigen-binding fragments thereof may be agonists and/or exhibit specific activity for stimulating cells expressing a particular antibody, including conjugates or recombinant receptors comprising the latter or their antigen-binding fragments (see, e.g., U.S. Patent Publication Nos. US 2016/0096902; US 2016/0068601; US 2014/0322183; US 2015/0175711; US 2015/283178; U.S. Pat. No. 9,102,760; Jena et al. PloS one (2013) 8(3):e57838; Long et al., Nature Medicine (2015) 21(6):581-590; Lee et al., The Lancet (2015) 385(9967):517-528; Zhao et al., PloS One (2014) 9(5):e96697; Leung et al., M.A.bs. (2015) 7(l):66-76).

In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interacts with the CAR, or a functional fragment or derivative thereof. For example, the soluble antigen can be a soluble form of BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, or FLT3, or a functional fragment or derivative thereof.

Exposing CAR T cells to an inhibitor

Described herein are various methods comprising blocking or modifying the activity of a specific CAR T cell (e.g., a T cell expressing a BCMA-binding CAR) by contacting the cell with a monoclonal antibody, or soluble antigen, that binds to the antigen recognition domain of the CAR.

In one aspect, an in vitro method is provided for determining the cytotoxicity of an immune cell expressing a chimeric antigen receptor (CAR) molecule, comprising:

a) incubating CAR-expressing immune cells with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR,

b) incubating CAR expressing immune cells in contact with target cells in a first control sample, wherein said incubation is carried out in the presence of an inhibitory molecule, wherein the inhibitory molecule reduces, inhibits, blocks and/or prevents interaction between the CAR and the target cells,

c) determination of the number of dead target cells in the test sample,

d) determining the number of dead target cells in the first control sample, and

e) determining the cytotoxicity of CAR expressing immune cells based on a comparison of the number of target cell deaths determined in steps (c) and (d),

wherein the incubation time, the number of CAR expressing immune cells and the number of target cells are substantially the same in the test sample and the first control sample.

In some embodiments, the incubation time of the test sample is 85-115%, 90-110%, or 95-105% of the incubation time of the first control sample. In some embodiments, the number of CAR-expressing immune cells is 85-115%, 90-110%, or 95-105% of the number of target cells.

In some embodiments, incubation steps (a) and (b) are performed simultaneously. Simultaneity may allow for some difference in the start and end times of these steps, such as one hour, 30 minutes, or 15 minutes. Simultaneous performance of steps (a) and (b) may provide conditions in which the incubation time, the number of CAR-expressing immune cells, and the number of target cells are substantially the same. In some embodiments, determining steps (c) and (d) are performed simultaneously. Simultaneity may allow for some difference in the start and end times of these steps, such as one hour, 30 minutes, or 15 minutes. Simultaneous performance of steps (c) and (d) may provide conditions in which the incubation time, the number of CAR-expressing immune cells, and the number of target cells are substantially the same.

In some embodiments, the CAR expressing immune cells and/or target cells are pre-incubated with an inhibitory molecule prior to said contacting step.

In some embodiments, the method further comprises comparing the number of dead target cells (e.g., tumor cells) determined in step (c) with the number of dead target cells determined in a second control sample in which the target cells are incubated in the absence of CAR-expressing immune cells.

In some embodiments, the method further comprises comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a third control sample in which the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that causes target cell death. In certain embodiments, the detergent is Triton X-100.

In various embodiments, the target cells (e.g., tumor cells) generate a detectable reporter signal upon death of said target cells, and step (c) comprises detecting the reporter signal in a test sample, step (d) comprises detecting the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals detected in steps (c) and (d). In some embodiments, the target cells express a reporter protein that generates a signal when the target cell undergoes cell death. Examples of reporter proteins suitable for use in the methods disclosed herein include, but are not limited to, beta-galactosidase, luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), and variants or derivatives thereof.

In some embodiments, the reporter signal is luminescence. A protein that can generate a reporter signal (e.g., luminescence) can be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into an environment where the protein can generate a luminescent signal, such as by enzymatic action on some agent to generate luminescence. Examples of cells that can be used in this manner include KILR® target cells. The target cells can be engineered to express an antigen that interacts with a chimeric antigen receptor. In certain embodiments, target cells such as KILR® MM-1R multiple myeloma cells are used. The target cells can also stably express a protein tagged with a tag or enzyme. When a target cell line is used in a cytotoxicity assay and its membrane is disrupted by cell death, the target cell line may release labeled protein into the medium. The labeled protein can be detected by adding reagents to the medium that are substrates for an enzymatic label on the protein. For example, the enzyme beta-galactosidase can hydrolyze the substrate to produce a chemiluminescent product. The luminescence can be quantified using a plate reader capable of measuring chemiluminescence. Alternatively, the labeled protein can be detected using label detection assays.

In some embodiments, the reporter signal is fluorescence. A protein that can generate a reporter signal (e.g., fluorescence) can be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into an environment where the protein can generate a fluorescent signal.

In some embodiments, the inhibitory molecule specifically binds to an antigen on target cells that interacts with the CAR. Without being limited by theory, the use of an inhibitory molecule that binds to an antigen allows for a suitable control in a cytotoxicity assay or other related assay for CAR efficacy, such that the need to use untransfected or mock-transfected immune cells as a control is eliminated. This eliminates the need to produce mock CAR T cells in parallel with the drug. The production of mock CAR T cells can impact the production of the drug in several ways. Eliminating the need to use mock cell controls simplifies the manufacturing process, ensures that the desired dosages are obtained for the patient while reducing the collection of autologous CAR T cells from the patient, and overall reduces the cost of CAR T cell therapy. The methods described herein reduce or eliminate testing delays caused by interruptions in the production of mock cells. The method also reduces testing errors by using a more simplified format. Reducing testing delays and reducing errors also helps avoid delays in product development and/or patient administration.

In some embodiments, the inhibitory molecule specifically binds to the CAR. In some embodiments, the inhibitory molecule specifically binds to a region of the CAR that specifically binds to an antigen expressed on target cells that interacts with the CAR. By binding to the CAR, in particular to a region of the CAR that specifically binds to an antigen (e.g., comprising one or more CDR sequences, an epitope or portions thereof), the inhibitory molecule can block the interaction of the CAR T cell with the target cell. Such blocking can prevent the CAR T cell from killing the target cell. Thus, instead of using a mock transfected immune cell, a patient's own CAR T cells with the addition of an inhibitory molecule can be used as an acceptable control.

In some embodiments, the inhibitory molecule is an antibody. In certain embodiments, the antibody is an anti-idiotypic antibody. The anti-idiotypic antibody can compete with an antigen on a host cell for binding to a chimeric antigen receptor. The anti-idiotypic antibody can share some structural elements with the antigen.

In some embodiments, the antibody or antibody fragment specifically binds to an antigen in an scFv domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in an scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen in a Fab domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in a Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen in a VH or VL domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in a VH or VL domain.

In some embodiments, the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells. In some embodiments, the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cells comprise a BCMA receptor, and the inhibitory molecule is a soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In certain embodiments, the multiple myeloma cells are MM-1R cells.

In various other embodiments, the CAR interacts with tumor and disease antigens, which include, but are not limited to, BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

In some embodiments, the CAR interacts with the tumor antigen GPRC5D. In some embodiments, the GPRC5D receptor is expressed by target cells. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the CAR. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the GPRC5D receptor. As a non-limiting example, the target cells are multiple myeloma cells. A non-limiting example of multiple myeloma cells are MM-1R cells.

In some embodiments, the CAR interacts with a KLK2 tumor antigen. In some embodiments, the KLK2 antigen is expressed by target cells. In some embodiments, the inhibitory molecule is a soluble KLK2 protein. As a non-limiting example, the target cells are prostate cancer cells. A non-limiting example of prostate cancer cells are LNCaP cells.

In various embodiments, the method is carried out in a format that provides high throughput.

Host cells

The inhibitory molecules described herein can be expressed in a cell, such as an immune effector cell (e.g., a population of cells, such as a population of immune effector cells), comprising a nucleic acid molecule, a CAR polypeptide molecule, or a vector as described herein. The immune effector cell can be, for example, a T cell or an NK cell. The inhibitory molecules can be expressed in various mammalian cell types (e.g., Chinese hamster ovary cells), and subsequently purified prior to use in any of the assays described herein.

The CAR T molecules described herein can be expressed in immune effector cells, such as T cells or NK cells. The immune effector cells can be obtained from a unit of blood collected from a patient using any technique known to those skilled in the art, such as Ficoll™ separation. Cells can be obtained from the subject's circulating blood by apheresis. The apheresis product essentially comprises lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction, and the cells can subsequently be placed in an appropriate buffer or medium for subsequent processing. The cells can be washed with phosphate-buffered saline (PBS). The wash solution may be calcium-free, magnesium-free, and/or all divalent cations-free.

The methods described herein may include, for example, selecting a specific subpopulation of immune effector cells, such as T cells, that is a population with a reduced content of T regulatory cells, without CD25+ cells, using, for example, negative selection techniques, such as those described herein. Preferably, the population with a reduced content of T regulatory cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

Efficiency analyses

Methods for characterizing the efficacy of chimeric antigen receptor-expressing T cells (CAR T cells) are described herein. The methods comprise (a) stimulating a CAR T cell in an antigen-specific manner (e.g., via a CAR in a CAR T cell) and (b) determining the level of antigen-specific cytotoxicity of the stimulated cell.

In one aspect, an in vitro method for determining the efficacy of an immune cell expressing a chimeric antigen receptor (CAR) molecule is provided, comprising:

a) incubating CAR-expressing immune cells with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR,

b) incubating CAR expressing immune cells in contact with target cells in a first control sample, wherein said incubation is carried out in the presence of an inhibitory molecule, wherein the inhibitory molecule reduces, inhibits, blocks and/or prevents interaction between the CAR and the target cells,

c) determining the amount of interaction between CAR expressing immune cells and target cells in the test sample,

d) determining the amount of interaction between CAR expressing immune cells and target cells in the first control sample, and

e) determining the efficacy of the CAR expressing immune cells based on a comparison of the amount of interaction determined in steps (c) and (d),

wherein the incubation time, the number of CAR expressing immune cells and the number of target cells are substantially the same in the test sample and the first control sample.

The interaction between CAR-expressing immune cells and target cells can be measured directly, for example by assessing the binding of CAR-expressing immune cells to target cells. The interaction between CAR-expressing immune cells and target cells can be measured indirectly, for example by assessing cell death, apoptosis, necrosis, cytokine release, changes in cell morphology, etc.

In some embodiments, the incubation time of the test sample is 85-115%, 90-110%, or 95-105% of the incubation time of the first control sample. In some embodiments, the number of CAR-expressing immune cells is 85-115%, 90-110%, or 95-105% of the number of target cells.

In some embodiments, incubation steps (a) and (b) are performed simultaneously. Simultaneity may allow for some difference in the start and end times of these steps, such as one hour, 30 minutes, or 15 minutes. Simultaneous performance of steps (a) and (b) may result in conditions in which the incubation time, the number of CAR-expressing immune cells, and the number of target cells are substantially the same. In some embodiments, determining steps (c) and (d) are performed simultaneously. Simultaneity may allow for some difference in the start and end times of these steps, such as one hour, 30 minutes, or 15 minutes. Simultaneous performance of steps (c) and (d) may result in conditions in which the incubation time, the number of CAR-expressing immune cells, and the number of target cells are substantially the same.

In some embodiments, the method further comprises comparing the amount of interaction between CAR-expressing immune cells and target cells in step (c) with the amount of dead target cells determined in a second control sample in which the target cells are incubated in the absence of CAR-expressing immune cells.

In some embodiments, the method further comprises comparing the amount of interaction between the CAR-expressing immune cells and the target cells in step (c) with the amount of target cell death determined in a third control sample in which the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that causes target cell death. In certain embodiments, the detergent is Triton X-100.

In various embodiments, the target cells produce a detectable reporter signal upon death of said target cells, and step (c) comprises detecting the reporter signal in a test sample, step (d) comprises detecting the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals detected in steps (c) and (d). In some embodiments, the target cells express a reporter protein that generates a signal when the target cell interacts with CAR-expressing immune cells.

In some embodiments, the reporter signal is luminescence. A protein that can generate a reporter signal (e.g., luminescence) can be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into an environment where the protein can generate a luminescent signal, such as by enzymatic action on some agent to generate luminescence. Examples of cells that can be used in this manner include KILR® target cells. The target cells can be engineered to express an antigen that interacts with a chimeric antigen receptor. In certain embodiments, target cells such as KILR® MM-1R multiple myeloma cells are used. The target cells can also stably express a protein tagged with a tag or enzyme. When a target cell line is used in a cytotoxicity assay and its membrane is disrupted by cell death, the target cell line may release labeled protein into the medium. The labeled protein can be detected by adding reagents to the medium that are substrates for an enzymatic label on the protein. For example, the enzyme beta-galactosidase can hydrolyze the substrate to produce a chemiluminescent product. The luminescence can be quantified using a plate reader capable of measuring chemiluminescence. Alternatively, the labeled protein can be detected using label detection assays.

In some embodiments, the reporter signal is fluorescence. A protein that can generate a reporter signal (e.g., fluorescence) can be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into an environment where the protein can generate a fluorescent signal.

In some embodiments, the inhibitory molecule specifically binds to an antigen on target cells (e.g., tumor cells) that interacts with the CAR. Without being limited by theory, the use of an inhibitory molecule that binds to an antigen allows for a suitable control in a cytotoxicity assay or other related assay for CAR efficacy such that untransfected or mock-transfected immune cells are not required as controls. This eliminates the need to produce mock CAR T cells in parallel with the drug. The production of mock CAR T cells can impact drug production in several ways. Eliminating the need to use mock cell controls simplifies the manufacturing process, ensures that the dosages required by the patient are obtained while reducing the collection of autologous CAR T cells from the patient, and overall reduces the cost of CAR T cell therapy. The methods described herein reduce or eliminate testing delays caused by interruptions in the production of mock cells. The method also reduces testing errors by using a more simplified format. Reducing testing delays and reducing errors also avoids delays in product development and/or patient administration.

In some embodiments, the inhibitory molecule specifically binds to the CAR. In some embodiments, the inhibitory molecule specifically binds to a region of the CAR that specifically binds to an antigen expressed on target cells that interacts with the CAR. By binding to the CAR, in particular to a region of the CAR that specifically binds to an antigen (e.g., comprising one or more CDR sequences, an epitope or portions thereof), the inhibitory molecule can block the interaction of the CAR T cell with the target cell. Such blocking can prevent the interaction of the CAR T cell with the target cell. Thus, instead of using a mock transfected immune cell, a patient's own CAR T cells with the addition of an inhibitory molecule can be used as an acceptable control.

In some embodiments, the inhibitory molecule is an antibody. In certain embodiments, the antibody is an anti-idiotypic antibody. The anti-idiotypic antibody can compete with an antigen on a host cell for binding to a chimeric antigen receptor. The anti-idiotypic antibody can share some structural elements with the antigen.

In some embodiments, the antibody or antibody fragment specifically binds to an antigen in an scFv domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in an scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen in a Fab domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in a Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen in a VH or VL domain of a chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to a CDR in a VH or VL domain.

In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interacts with a CAR, or a functional fragment or derivative thereof.

In some embodiments, the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells. In some embodiments, the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cells comprise a BCMA receptor, and the inhibitory molecule is a soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In certain embodiments, the multiple myeloma cells are MM-1R cells.

In various other embodiments, the CAR interacts with tumor and disease antigens, which include, but are not limited to, BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

In some embodiments, the CAR interacts with the tumor antigen GPRC5D. In some embodiments, the GPRC5D receptor is expressed by target cells. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the CAR. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the GPRC5D receptor. As a non-limiting example, the target cells are multiple myeloma cells. A non-limiting example of multiple myeloma cells are MM-1R cells.

In some embodiments, the CAR interacts with a KLK2 tumor antigen. In some embodiments, the KLK2 antigen is expressed by target cells. In some embodiments, the inhibitory molecule is a soluble KLK2 protein. As a non-limiting example, the target cells are prostate cancer cells. A non-limiting example of prostate cancer cells are LNCaP cells.

Instead of using mock-transfected CAR T cells as a control for determining the level of antigen-specific cytotoxicity, the same CAR T cells tested in the assay can be treated with an inhibitory molecule, as described herein. Treatment with an inhibitory molecule (e.g., a monoclonal antibody or soluble antigen to which the CAR specifically binds) can block the CAR T cell from binding to the antigen and causing cytotoxicity. Detection of an increase in the level of cytotoxicity of an antigen-specifically stimulated cell, compared to the same CAR T cell treated with an inhibitory molecule or a non-specifically stimulated CAR T cell (i.e., a stimulated CAR T cell that is not stimulated in an antigen-specific manner), can be used as an indication for the use of the stimulated CAR T cell for therapeutic purposes. The methods can be performed in vitro.

In various embodiments, the inhibitory molecule (e.g., a tumor antigen or an anti-idiotypic antibody) is not effective in stimulating a CAR T cell that may be stimulated by an antigen (e.g., a tumor antigen) to which the CAR on the CAR T cell is specific. Such embodiments may provide the advantage of not activating an efficacy parameter that increases with activation of the CAR T cell.

In addition, methods for determining the potency and cytotoxic function of CAR T cells are provided. In essence, the methods include antigen-specific stimulation of a CAR on a CAR T cell followed by quantitative determination of the antigen-specific cytotoxicity of the CAR T cell. A measure of CAR T cell potency can be used as an in vitro proxy for the expected in vivo pharmacokinetics of a CAR T cell therapeutic product. CAR T cell potency assays can further be used to determine the potential suitability of a CAR T cell product for clinical use, to assess the potential efficacy of a CAR T cell product, to determine CAR T cell dosages to administer, and/or to characterize new approaches to manufacturing CAR T cell therapeutic products.

The efficacy of a CAR T cell therapeutic product may be expressed in terms that reflect the level of antigen-specific cytotoxicity of the product. The level of cytotoxicity may be compared, for example, to the level of cytotoxicity of a control sample of the CAR T cell therapeutic product that is exposed to both antigen-specific stimulation and an inhibitory molecule described herein. In addition, the results of the calculations may be normalized based on, for example, the number of cells in the test samples that express the CAR. Based on this information, the cytotoxicity index (CI), defined according to the following expression, may be used as a measure of the efficacy of CAR T cell therapeutic products:

CI=[(cytotoxicity in stimulated group) - (cytotoxicity in control group)] / % of CAR-expressing cells.

To determine the proportion of cells expressing CAR (e.g., the level of transduction), methods known in the art can be used for normalization. For example, in flow cytometry assays, CAR antibodies can be included in the assay and used to quantify the level of CAR expressing cells relative to the total number of T cells.

The level of antigen-specific in vitro cytotoxicity of CAR T cell therapeutic products can correlate with the in vivo pharmacokinetics (PK) and pharmacodynamics (PD) properties of the CAR T cell products. Potential PK/PD parameters of a CAR T cell preparation of the invention can include, for example, Cmax, Tmax, and area under the curve (AUC), which can be determined in clinical samples using standard methods in the art. The relationship between the in vitro cytotoxicity of a CAR T cell therapeutic product, as reflected, for example, in the cytotoxicity index described above, and the in vivo PK/PD characteristics of the product can be demonstrated using standard methods, such as, for example, the Spearman correlation coefficient method, which can be used to assess linear relationships between such characteristics. The various methods described herein can provide a basis for predicting PK/PD parameters from antigen-specific in vitro cytotoxicity, as demonstrated, for example, by determining the cytotoxicity index CI.

Sets

Any of the compositions described herein may be included in a kit. In some embodiments, the kit includes CAR binding antibodies, and the kit may also include suitable components for cell expansion, such as media, APC, growth factors, antigens, other antibodies (e.g., for sorting or characterizing CAR T cells), and/or plasmids encoding a CAR or transposase.

In a non-limiting example, the kit includes a CAR binding antibody, a chimeric receptor expression construct (or reagents for producing a chimeric receptor expression construct), reagents for transfecting the expression construct, and/or one or more tools for producing allogeneic T cells for transfection with the expression construct (such a tool may be a syringe, a pipette, tweezers, and/or any such device approved for use in medical purposes). In some aspects of the invention, the kit includes reagents or devices for electroporating cells.

The kit may include target cells expressing an antigen that specifically interacts with a CAR binding antibody, reagents for transfecting an expression construct encoding the antigen, and/or one or more instruments for producing allogeneic T cells for transfection with the expression construct (such an instrument may be a syringe, a pipette, tweezers, and/or any such device approved for use in medical purposes). In some aspects of the invention, the kit includes reagents or devices for electroporating cells.

The kits may contain one or more suitably aliquoted compositions of the present invention or reagents for preparing the compositions of the invention. The components of the kits may be packaged in either an aqueous medium or in lyophilized form. The containers in the kits may include at least one vial, test tube, flask, bottle, syringe or other container into which a component can be placed and, preferably, suitably aliquoted. If the kit contains more than one component, such a kit will typically also contain a second, third or other additional container into which additional components can be separately placed. However, a vial may contain various combinations of components. The kits of the present invention typically also include containers for housing the chimeric receptor expression construct and any other containers for reagents in close proximity for commercial sale. Such containers may include, for example, plastic containers manufactured by injection or blow molding, in which the desired vials are located.

IMPLEMENTATION OPTIONS

1. An in vitro method for determining the efficacy of an immune cell expressing a chimeric antigen receptor (CAR) molecule, comprising:

a) bringing CAR-expressing immune cells into contact with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR,

b) contacting CAR-expressing immune cells with target cells in a first control sample, wherein (i) said contacting is carried out in the presence of an inhibitory molecule or (ii) the CAR-expressing immune cells and/or target cells have been pre-incubated with an inhibitory molecule prior to said contacting, wherein the inhibitory molecule inhibits the interaction between the CAR and the target cells,

c) determination of the number of dead target cells in the test sample,

d) determining the number of dead target cells in the first control sample, and

e) determining the efficacy of the CAR expressing immune cells based on a comparison of the numbers of target cell deaths determined in steps (c) and (d),

wherein the duration of contact, the number of CAR expressing immune cells and the number of target cells are substantially the same in the test sample and the first control sample.

2. The method according to embodiment 1, wherein the steps (a) and (b) of bringing into contact are carried out simultaneously.

3. The method according to embodiment 1 or embodiment 2, wherein the steps of determining (c) and (d) are carried out simultaneously.

4. The method according to any one of embodiments 1-3, wherein in step (b)(i), the CAR expressing immune cells and/or target cells are pre-incubated with an inhibitory molecule prior to the contacting step.

5. The method according to any one of embodiments 1-4, further comprising comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

6. The method according to any one of embodiments 1-5, further comprising comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a third control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that causes target cell death.

7. The method of embodiment 6, wherein the detergent is Triton X-100.

8. The method according to any one of embodiments 1-7, wherein the target cells generate a detectable reporter signal upon death of said target cells, and step (c) comprises detecting the reporter signal in a test sample, step (d) comprises detecting the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals detected in steps (c) and (d).

9. The method of embodiment 8, wherein the reporter signal is luminescence.

10. The method of embodiment 8, wherein the reporter signal is fluorescence.

11. The method of any one of embodiments 8-10, wherein the target cells express a reporter protein that generates a signal when the target cell undergoes cell death.

12. The method of embodiment 11, wherein the reporter protein is beta-galactosidase, luciferase, or green fluorescent protein (GFP), or a variant or derivative thereof.

13. The method of any one of embodiments 1-12, wherein the inhibitory molecule specifically binds to an antigen on target cells, wherein the antigen interacts with the CAR.

14. The method according to any one of embodiments 1-13, wherein the inhibitory molecule specifically binds to the CAR.

15. The method of embodiment 14, wherein the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on target cells.

16. The method of embodiments 13, 14 or 15, wherein the inhibitory molecule is an antibody or antibody fragment.

17. The method of embodiment 16, wherein the antibody is an anti-idiotypic antibody.

18. The method of embodiments 16 or 17, wherein the antibody fragment is a Fab, Fab', F(ab') ₂ , Fv or Fd fragment, single chain antibody (scFv), linear antibody, single domain antibody, heavy chain variable region (VH) domain or light chain variable region (VL) domain.

19. The method of embodiments 16, 17, or 18, wherein the antibody or antibody fragment specifically binds to an antigen within a domain of the scFv CAR.

20. The method of embodiment 19, wherein the antibody or antibody fragment specifically binds to a complementarity determining region (CDR) within the scFv CAR domain.

21. The method of embodiments 16, 17, or 18, wherein the antibody or antibody fragment specifically binds to an antigen within the VH domain or VL domain of the CAR.

22. The method of embodiment 21, wherein the antibody or antibody fragment specifically binds to a CDR within the VH domain or VL domain of the CAR.

23. The method of embodiment 14 or 15, wherein the inhibitory molecule is a soluble form of an antigen expressed on target cells that interacts with a CAR, or a functional fragment or derivative thereof.

24. The method according to any one of embodiments 1-23, wherein the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs) and natural killer (NK) cells.

25. The method of any one of embodiments 1-24, wherein the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cells comprise a BCMA receptor, and the inhibitory molecule is a soluble cytoplasmic domain of BCMA.

26. The method of embodiment 25, wherein the target cells are multiple myeloma cells.

27. The method of embodiment 26, wherein the multiple myeloma cells are MM-1R cells.

28. The method of any one of embodiments 1-24, wherein the CAR interacts with a G protein-coupled receptor class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to the CAR.

29. The method of any one of embodiments 1-24, wherein the CAR interacts with a G protein-coupled receptor class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to a GPRC5D receptor.

30. The method of embodiment 28 or 29, wherein the target cells are multiple myeloma cells.

31. The method of embodiment 30, wherein the regulatory T cells are MM-1R+ T cells.

32. The method of any one of embodiments 1-24, wherein the CAR interacts with kallikerin 2 (KLK2), the target cells comprise KLK2, and the inhibitory molecule is a soluble KLK2 protein.

33. The method of embodiment 32, wherein the target cells are prostate cancer cells.

34. The method of embodiment 33, wherein the prostate cancer cells are LNCaP cells.

35. The method according to any one of embodiments 1-34, wherein the method is carried out in a format that provides high throughput.

EXAMPLES

The present invention is also described and its operation demonstrated by means of the following examples. However, the use of these and other examples anywhere in the description is illustrative only and in no way limits the scope and meaning of the invention or any term cited. Likewise, the invention is not limited to any specific preferred embodiments described herein. Moreover, upon reading the present description, many modifications and variations of the present invention may be apparent to those skilled in the art, and such variations may be made without departing from the present invention both in spirit and scope. Accordingly, the invention should be limited only by the appended claims and the full scope of similar modifications to which these claims pertain.

Example 1

The following materials were used in this example. The test material was a CAR T cell therapeutic product (DP) sample, with KILR® MM1-R® reporter cells (Cat. #97-1045P052, Eurofins Discoverx Corp., Fremont, CA, USA) as the target cell line. KILR® MM-1R® cells express an enhanced Prolabel-tagged (ePL) housekeeping gene. Upon cell lysis, the ePL-tagged protein is released into the medium. Addition of an enzymatic acceptor will cause the assembly of the β-galactosidase enzyme fragments, EA and ePL. The resulting functional enzyme will hydrolyze the substrate, generating a chemiluminescent signal.

KILR MM-1R® cells were grown in RPMI 1640 (ATCC formulation) medium (Gibco cat.#A10491-01, ThermoFisher, Waltham, MA, USA) with 10%HI-FBS (6140-071, Life Technologies, Carlsbad, CA, USA) and 250 μg/ml G418 sulfate (Corning cat.#30-234-CR, ThermoFisher, Waltham, MA, USA). Assays were performed in assay medium consisting of RPMI 1640 medium with L-glutamine and 25 mM HEPES (Corning cat. no. 10-041-CV, ThermoFisher, Waltham, MA, USA) and 10% HI-FBS (6140-071, Life Technologies, Carlsbad, CA, USA).

Soluble human BCMA (sBCMA) (BCA-H522y, Aero Biosystems, Newark, DE, USA), a blocking protein, was added to the assay medium. A 10% TritonX-100 solution (Cat.#93443-100ML, SigmaAldrich Co., St. Louis, MO, USA) was used to provide a complete cell killing control. Cell cytotoxicity was determined using the KILR® Detection™ kit (Cat.#97-001, Eurofins Discovers Corp., Fremont, CA, USA).

In this example, the ability of a CAR T cell therapeutic (DP) to kill multiple myeloma target cells expressing a relevant antigen was measured. Target cells were used that expressed reporter genes, which produced a measurable signal (e.g., luminescence) when the cells were killed by binding to effector CAR T cells. The result was reported as an activity measurement, which was used to determine whether the DP therapeutic product demonstrated the appropriate level of activity to release the reporter.

The assay included four components used in a total of 16 wells. The four components used were (i) CAR T cell drug with target cells (total activity), (ii) CAR T cell drug cells blocked with blocking reagent with target cells (background control), (iii) target cells with medium (no cell death control), and (iv) target cells with 0.1% TitonX-100 (total cell death control). All four assay components were run in quadruplicate as shown in the plate layout in Table 1A below. A detailed description of the samples in the 96-well plate is provided in Table 1B. One 96-well plate was used to run six assays, one of which was the CAR T cell quality control assay used to determine system suitability and method performance trends. The CAR T cells for quality control were prequalified for their activity.

The assay was performed with LCAR-B38M CAR T cell therapeutic product cells and KILR® MM-1R multiple myeloma target cells (Eurofins Discoverx Corp., Fremont, CA, USA). RPMI1640 and 10% HI-FBS were used as assay medium. KILR® MM-1R cells were grown according to the manufacturer's instructions. Background controls were generated by blocking cells with LCAR-B38M CAR T cell therapeutic product and soluble cytoplasmic domain of B cell maturation antigen (sBCMA). The KILR® Detection™ kit (Eurofins Discoverx Corp., Fremont, CA, USA) was used as the assay detection reagent. Optimization was performed for the effector cell (DP) to target cell (e.g., KILR® MM-1R) ratio (E:T) and the amount of blocking reagent required to completely block the interaction between CAR T cells (DP) and their target cells. The E:T ratio and blocking reagent concentration were optimized for each drug and its corresponding target cell line. Once determined, the E:T ratio was fixed during assay run and remained fixed for the entire drug run. The blocking reagent was lot-qualified and used at the level found for drug testing. Detection conditions can be further optimized based on the reporter system used.

The LCAR-B38M CAR T cell therapeutic product assay was performed in 96-well opaque TC-treated assay plates as follows. Each assay plate could accommodate up to 6 assays as described in Table 1A above. For each assay, 25 µl of blocking reagent was added to each well of the assay plate containing blocked CAR T cells (background control), followed by the addition of 25 µl of assay medium to the wells containing the CAR T cells to be tested. Subsequently, 25 µl of CAR T cell therapeutic product cells at 8 x ¹⁰⁵ live cells/mL (for a total of 2 x ¹⁰⁴ live cells per well) were added to the wells containing the CAR T cells to be tested and to the background control wells. 50 µl of the assay medium were added only to the wells with KILR MM-1R cells (no cell death). The well contents were gently mixed and incubated for 10 min (±5 min) at 37°C, 5% CO ₂ , with humidification.

After incubation was complete, 50 µl of target cells at 8 × 10 ⁴ viable cells/mL (total 4 × 10 ³ viable cells/well) were added to all assay wells, giving a final E:T ratio of 5:1. The last reagent added in each assay was 0.1% Triton X-100 (50 µl/well) to the cell death control wells. The wells were incubated at 37°C in 5% CO ₂ with humidification for 22 h (±1 h). After co-culture incubation was complete, the assay plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min (±5 min). In parallel, the detection reagent was thawed and subsequently allowed to equilibrate at room temperature for 30 min. After equilibration, 100 µl of KILR® Detection Reagent was added to the wells and incubated protected from light for 50 min (±5 min). The plate was read on a Molecular Devices Paradigm plate reader in chemiluminescence mode after shaking for 10 seconds.

The data are presented in Tables 2 and 3 below. The data in Table 2 show a lot-by-lot titration of sBCMA protein demonstrating blockade of BCMA killing by LCAR-B38M cells of the multiple myeloma target KILR MM-1R cells in the range of 50-400 μg/mL. The data in Table 3 show the specificity of sBCMA blockade for multiple myeloma target cells (RPMI8226_Luc) compared to non-B cell target cells (K562_luc cells).

Example 2

GPRC5D CAR T cells were tested using an anti-GO antibody to CAR T cells. The assay conditions for GPRC5D CAR T cells were similar to those used for LCAR-B38M cells in Example 1 above. For GPRC5D CAR T cells, the other multiple myeloma target cells, the same KILR MM-1R target cells were used. The assay media and detection reagents were the same as those used in Example 1. The same 5:1 E:T cell ratio and seeding densities were used as in Example 1. The blocking reagent was GCPR5D anti-idiotype antibody to GPRC5D CAR. Examples of GCPR5D anti-idiotype antibodies for use in the assay include GP5B337, GP5B332, GP5B324, and GP5B206. The heavy chain and light chain sequences of these anti-idiotype antibodies are provided in Table 5. Incubation times were as used in Example 1 above. All other reagents were as used in Example 1. The results of the primary titration assay presented in Table 4 below indicate blocking of GPRC5D CAR T cells by the anti-ID antibody (GP5B337).

Example 3

GPRC5D CAR T cells were tested using the Fab fragment of the anti-ID antibody to CAR T cells. The assay conditions for GPRC5D CAR T cells were similar to those used for LCAR-B38M cells in Example 1 above. For GPRC5D CAR T cells, the other multiple myeloma target cells, the same KILR MM-1R target cells were used. The assay media and detection reagents were the same as those used in Examples 1 and 2. The same 5:1 E:T cell ratio and seeding densities were used as in Examples 1 and 2. The blocking reagent was the Fab fragment of the GCPR5D anti-idiotypic antibody (GP5B337) to GPRC5D CAR. Incubation times were as used in Examples 1 and 2 described above. All other reagents were as used in Examples 1 and 2. The results of the primary titration study presented in Table 6 below indicate blockade of GPRC5D CAR T cells by the Fab fragment of the anti-idiotypic antibody (GP5B337).

The anti-GPRC5D anti-idiotypic antibody Fab fragment was generated from the full-length anti-GPRC5D CAR antibody using the Pierce Fab Preparation Kit (ThermoFisher, Cat#: VF299292) according to the kit instructions with minor adjustments. Briefly, VirH phosphate-buffered saline (PBS) and digestion buffer were prepared. IgG was removed from the refrigerator, diluted with the prepared PBS, and passed through desiccation columns. The eluent was collected and transferred to the prepared papain digestion columns for digestion at 37°C. After approximately 5 hours of digestion and rotation at 37°C, the tubes were removed from the incubator and centrifuged to collect the sample. Protein A columns from the kit were prepared, sample was loaded and the columns were spun overnight at 2-8°C for approximately 20 hours. The Protein A columns were centrifuged to collect the sample in fractions. The first and second fractions were collected, pooled and saved. Previous work such as A280 and 1D silver stains showed that these fractions contain the most useful fragment. Fresh (not previously used) Protein A columns were prepared, sample was loaded again for a second spin at 2-8°C for approximately 1 hour. The result of the second incubation on the Protein A column was loaded to further clean up and remove any traces of the Fc region that were thought to interfere with the assay. The fragments were eluted, concentrated in 10K Amicon tubes (Cat. UFC501008) and stored at 2-8°C.

Example 4

GPRC5D CAR T cells were tested using an anti-GPRCSD antibody or its Fab fragment to the CAR T cell antigen (GPRC5D receptor). The assay conditions for GPRC5D CAR T cells were similar to those used for the GPRC5D CAR T cell therapeutic product in Example 2 described above. For GPRC5D CAR T cells, the other multiple myeloma target cells, the same KILR MM-1R target cells were used. The assay media and detection reagents were the same as those used in Examples 2 and 3. The same 5:1 E:T cell ratio and seeding densities were used as in Examples 2 and 3. The blocking reagent was an anti-GCPRSD antibody or its Fab fragment. These blocking reagents bind to GPRC5D receptors on KILR MM-1R target cells and inhibit the ability of CAR T cells to bind their target. The Fab fragment of the anti-GPRCSD antibody was generated from the full-length anti-GPRCSD antibody using standard methods as detailed above. Incubation times were as used in Examples 2 and 3. All other reagents were as used in Examples 2 and 3. The results of the primary titration study presented in Table 7 below indicate blocking of KILR MM-1R cells by the anti-GPRCSD antibody or its Fab fragment.

Example 5

KLK2 CAR T cells were tested using human soluble KLK2 protein to CAR T cells. The assay conditions for KLK2 CAR T cells were similar to those used for LCAR-B38M cells in Example 1 above. The target of KLK2 CAR T cells is kallikerin 2 (KLK2), a molecule expressed on malignant luminal prostate. A reporter cell line overexpressing KLK2 was generated using LNcaP cells (ATCC, CRL1740) and the DiscoverX Killing Immune-Lysis Reaction (KILR) reporter. The killing mechanism is the same as described in Example 1. These cells express elevated Prolabel-tagged (ePL) housekeeping gene, and upon cell lysis, the ePL-tagged reporter protein was released into the medium. Addition of the enzymatic acceptor caused the assembly of the β-galactosidase enzyme fragments, EA and ePL. The resulting functional enzyme hydrolyzed its substrate to generate a chemiluminescent signal. The assay media, detection reagents and target seeding densities were the same as in Example 1, but the E:T cell ratio was 10:1. The blocking reagent was soluble KLK2 CAR protein. The amino acid sequence of this soluble KLK2 protein with a His ₆ tag at the C-terminus (SEQ ID NO: 12) is shown below (the underlined sequence is the signal peptide):

KILR LNcaP-KLK2 cells were grown in RPMI 1640 medium (Gibco cat.#11875-093, ThermoFisher, Waltham, MA, USA) with 10% FBS (97068-085, VWR, Radnor, PA, USA) and 250 μg/ml G418 sulfate (Corning cat.#30-234-CR, Corning, Tewksbury, MA, USA). Assays were performed in assay medium consisting of RPMI 1640 medium with L-glutamine (Corning cat.#10-040-CV, Corning, Tewksbury, MA, USA) and 10% FBS (97068-085, VWR, Radnor, PA, USA).

Although the target cell line in this example was different from that used in Examples 1 and 2, the strategy and parameters were the same as those used in Examples 1 and 2.

The following conditions were used to perform the assay with the KLK2 CAR T cell therapeutic product and KILR LNcaP-KLK2 target cells. The assay medium was RPMI1640 and 10% HI-FBS. KILR LNcaP-KLK2 cells were grown in RPMI1640 and 10% FBS, 1x nonessential amino acids, 2.5 μg/mL puromycin, and 500 μg/mL G418 sulfate. Background controls and assay optimization were as described in Example 1. Background controls were generated by blocking the KLK2 CAR T cell therapeutic product cells with soluble human KLK2 protein (sKLK2). The KILR® Detection™ kit (Eurofins Discoverx Corp., Fremont, CA, USA) was used as the assay detection reagent. Optimization was performed for the effector cell (DP) to target cell (e.g., KILR LNcaP-KLK2) ratio (E:T) and the amount of blocking reagent required to completely block the interaction between CAR T cells (therapeutic product) and their target cells. The E:T ratio and blocking reagent concentration were optimized for each drug and its corresponding target cell line. Once matched, the E:T ratio was fixed in the assay and remained fixed for all drug testing. The blocking reagent was lot-qualified and used at that level for drug testing. In addition, detection conditions were optimized based on the reporter system used.

The KLK2 CAR T cell therapeutic product assay was performed as in Example 1 in 96-well opaque TC-treated assay plates. The assay steps are described below, with each assay plate capable of holding up to 6 assays as described in Table 1A, Example 1. For each assay, 25 μl of blocking reagent was added to each well of the assay plate containing blocked CAR T cells (background control), followed by the addition of 25 μl of assay medium to the wells containing the CAR T cells to be tested. Subsequently, 25 μl of CAR T cell therapeutic product cells at 1.6 x 10 ⁶ live cells/mL (for a total of 4 x 10 ⁴ live cells per well) were added to the wells containing the CAR T cells to be tested and to the background control wells. Fifty microliters of assay medium were added only to wells containing KILR LNcaP-KLK2 cells (no cell death). The well contents were gently mixed and incubated for 15 min (±5 min) at 37°C, 5% CO ₂ , and humidified.

After incubation, 50 µl of target cells at 8 × 10 ⁴ viable cells/mL (total 4 × 10 ³ viable cells per well) were added to each assay well, yielding a final E:T ratio of 10:1. The wells were incubated at 37°C, 5% CO ₂ , with humidification for 20 h (±2 h). After co-culture incubation, the assay plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min (±5 min). In parallel, the detection reagent was defrosted and subsequently allowed to equilibrate at room temperature for 30 min. For each assay, after equilibration, 0.1% Triton X-100 (50 µl/well) was added to total cell death control wells, followed by the addition of 100 µl of KILR® Detection Reagent to each well and incubation protected from light for 50 minutes (±5 minutes). The plate was read on a Molecular Devices Paradigm plate reader in chemiluminescence mode after shaking for 10 seconds.

The data obtained are presented in Table 8. The data presented show a titration across batches of sKLK2 protein demonstrating blockade of KLK2 killing by KLK2 CAR T cell therapeutic product of KILR LNcaP-KLK2 target cells in the range of 10-500 μg/mL.

Incubation times were as used in Example 1 above. All other reagents were as used in Example 1. The results of the primary titration assay presented in Table 8 below indicate blockade of KLK2 CAR T cells by soluble KLK2 protein. (Int., KL2W12.009).

The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to be included within the scope of the appended claims. It should also be understood that all values are approximate and are provided for the purpose of description.

The disclosures of all patents, patent applications, publications, product descriptions and protocols that are referenced in this application are hereby incorporated by reference in their entirety for all purposes.

--->

SEQUENCE LIST

<110> JANSSEN BIOTEC, INC.

<120> CELLULAR ASSAY FOR DETERMINING TUMOR CELL KILLING ACTIVITY

CELLS EXPRESSING CHIMERIC ANTIGENS OF IMMUNE CELLS IN VITRO

<130> JBI6329WOPCT1

<140>

<141>

<150> 63/125,173

<151> December 14, 2020

<150> 63/036,249

<151> June 08, 2020

<160> 12

<170> PatentIn, version 3.5

<210> 1

<211> 447

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Thr Ser Val Glu Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu

115 120 125

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

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser

145 150 155 160

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

165 170 175

Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp

180 185 190

Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr

195 200 205

Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys

210 215 220

Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser

245 250 255

Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp

260 265 270

Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

275 280 285

Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser

290 295 300

Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

305 310 315 320

Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile

325 330 335

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

340 345 350

Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met

355 360 365

Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn

370 375 380

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

385 390 395 400

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

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Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu

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His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

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

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

20 25 30

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

35 40 45

Tyr Ile Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 3

<211> 452

<212> PROTEIN

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Phe Pro Trp Asp Leu Ala Tyr Ala Leu Asp Tyr Trp

100 105 110

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

115 120 125

Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser

130 135 140

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

145 150 155 160

Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His

195 200 205

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

210 215 220

Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu

225 230 235 240

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

245 250 255

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

260 265 270

Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

275 280 285

Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

290 295 300

Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val

370 375 380

Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu

385 390 395 400

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

405 410 415

Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val

420 425 430

Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg

435 440 445

Thr Pro Gly Lys

450

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<212> PROTEIN

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<220>

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Phe Pro Phe

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 5

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

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synthetic polypeptide"

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Asn Tyr Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys His Gly Ala Phe Ser Ser Tyr Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr

130 135 140

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

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser

180 185 190

Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile

210 215 220

Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile

245 250 255

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

260 265 270

Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His

275 280 285

Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu

355 360 365

Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp

370 375 380

Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val

385 390 395 400

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

405 410 415

Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His

420 425 430

Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro

435 440 445

Gly Lys

450

<210> 6

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ala Asp Phe

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Arg Ala Pro Phe

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 7

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

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synthetic polypeptide"

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Ala Leu Lys

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Arg Glu Asp Ser Tyr Gly Asp Leu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr

115 120 125

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

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp

145 150 155 160

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

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser

180 185 190

Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys

210 215 220

Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser

245 250 255

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

260 265 270

Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr

275 280 285

Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val

290 295 300

Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr

355 360 365

Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr

370 375 380

Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu

385 390 395 400

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

405 410 415

Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu

420 425 430

Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly

435 440 445

Lys

<210> 8

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note = "description of artificial sequence:

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<400> 8

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Thr Asn Thr Trp Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 9

<211> 267

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note = "description of artificial sequence:

synthetic polypeptide"

<400> 9

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

1 5 10 15

Ala Val Pro Leu Ile Glu Gly Arg Ile Val Gly Gly Trp Glu Cys Glu

20 25 30

Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala

35 40 45

His Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala

50 55 60

His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu

65 70 75 80

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

85 90 95

Pro His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg

100 105 110

Pro Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu

165 170 175

His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val

180 185 190

Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Thr

195 200 205

Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln

210 215 220

Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro

225 230 235 240

Ala Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr

245 250 255

Ile Ala Ala Asn Pro His His His His His

260 265

<210> 10

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note = "description of artificial sequence:

synthetic polypeptide"

<400> 10

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> 11

<211> 42

<212> PROTEIN

<213> Unknown

<220>

<221> source

<223> /note="description of unknowns: Sequence 4-1BB

costimulatory domain or CD137 costimulatory domain

<400> 11

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> 12

<211> 6

<212> PROTEIN

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His His His His His

1 5

<---

Claims (40)

1. An in vitro method for determining the cytotoxicity of an immune cell expressing a chimeric antigen receptor (CAR) molecule, comprising: a) contacting CAR-expressing immune cells with target cells in a test sample comprising CAR-expressing immune cells and target cells, wherein the target cells express an antigen that interacts with the CAR, b) contacting CAR-expressing immune cells with target cells in a control sample comprising CAR-expressing immune cells, target cells and an inhibitory molecule, wherein (i) said contacting is carried out in the presence of an inhibitory molecule or (ii) the CAR-expressing immune cells and/or target cells have been pre-incubated with an inhibitory molecule prior to said contacting, wherein the inhibitory molecule specifically binds to an antigen on the target cells, wherein the antigen interacts with the CAR or wherein the inhibitory molecule specifically binds to the CAR, and the inhibitory molecule inhibits the interaction between the CAR and target cells, c) determination of the number of dead target cells in the test sample, d) determination of the number of dead target cells in the control sample, and e) determining the cytotoxicity of CAR expressing immune cells based on a comparison of the numbers of target cell deaths determined in steps (c) and (d), wherein the duration of contact, the number of CAR expressing immune cells and the number of target cells are substantially the same in the test sample and the control sample. 2. The method according to claim 1, wherein the steps (a) and (b) of bringing into contact are performed simultaneously. 3. The method according to claim 1, wherein the steps (c) and (d) of determining are performed simultaneously. 4. The method of claim 1, wherein in step (b)(i), the CAR expressing immune cells and/or target cells are pre-incubated with an inhibitory molecule prior to the contacting step. 5. The method of claim 1, further comprising comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in the second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells. 6. The method of claim 1, further comprising comparing the number of dead target cells determined in step (c) with the number of dead target cells determined in a third control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that causes target cell death. 7. The method of claim 6, wherein the detergent is Triton X-100. 8. The method of claim 1, wherein the target cells generate a detectable reporter signal upon death of said target cells, and step (c) comprises detecting the reporter signal in a test sample, step (d) comprises detecting the reporter signal in a control sample, and step (e) comprises comparing the reporter signals detected in steps (c) and (d). 9. The method according to claim 8, wherein the reporter signal is luminescence. 10. The method of claim 8, wherein the reporter signal is fluorescence. 11. The method of claim 8, wherein the target cells express a reporter protein that generates a signal when the target cells undergo cell death. 12. The method of claim 11, wherein the reporter protein is beta-galactosidase, luciferase, or green fluorescent protein (GFP), or a variant or derivative thereof. 13. The method of claim 1, wherein the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on target cells. 14. The method of claim 1, wherein the inhibitory molecule is an antibody or antibody fragment. 15. The method of claim 14, wherein the antibody is an anti-idiotypic antibody. 16. The method of claim 14, wherein the antibody fragment is a Fab, Fab', F(ab') ₂ , Fv or Fd fragment, single chain antibody (scFv), linear antibody, single domain antibody, heavy chain variable region (VH) domain or light chain variable region (VL) domain. 17. The method of claim 14, wherein the antibody or antibody fragment specifically binds to an antigen within the scFv CAR domain. 18. The method of claim 17, wherein the antibody or antibody fragment specifically binds to a complementarity determining region (CDR) within the scFv CAR domain. 19. The method of claim 14, wherein the antibody or antibody fragment specifically binds to an antigen within the VH domain or VL domain of the CAR. 20. The method of claim 19, wherein the antibody or antibody fragment specifically binds to a CDR within the VH domain or VL domain of a CAR. 21. The method of claim 1, wherein the inhibitory molecule is a soluble form of an antigen expressed on target cells that interacts with a CAR, or a functional fragment or derivative thereof. 22. The method according to claim 1, wherein the immune cells are selected from T cells, induced pluripotent stem cells (iPSC) and natural killer cells (NK). 23. The method of claim 1, wherein the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cells comprise a BCMA receptor, and the inhibitory molecule is a soluble extracellular domain of BCMA. 24. The method of claim 23, wherein the target cells are multiple myeloma cells. 25. The method of claim 24, wherein the multiple myeloma cells are MM-1R cells. 26. The method of claim 1, wherein the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or an antibody fragment to the CAR. 27. The method of claim 1, wherein the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cells comprise a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment to a GPRC5D receptor. 28. The method of claim 26, wherein the target cells are multiple myeloma cells. 29. The method of claim 28, wherein the multiple myeloma cells are MM-1R cells. 30. The method of claim 1, wherein the CAR interacts with kallikerin 2 (KLK2), the target cells comprise KLK2, and the inhibitory molecule is a soluble KLK2 protein. 31. The method of claim 30, wherein the target cells are prostate cancer cells. 32. The method of claim 31, wherein the prostate cancer cells are LNCaP cells. 33. The method according to item 1, carried out in a format that ensures high productivity.