Cell-based assay for determining the in vitro tumor-killing activity of chimeric antigen-expressing immune cells

Abstract

The present disclosure provides an in vitro method for determining the efficacy (e.g., cytotoxicity) of immune cells expressing a chimeric antigen receptor (CAR) molecule. In a test sample, the CAR-expressing immune cells are incubated with target cells expressing an antigen that interacts with the CAR. In a control sample, the CAR-expressing immune cells are incubated with the target cells and an inhibitory molecule that prevents interaction between the CAR and the target cells. The amount of target cell death is determined in both the test sample and the control sample and compared.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Application No. 63 / 036,249, filed on 8 June 2020, and U.S. Provisional Application No. 63 / 125,173, filed on 14 December 2020. The entire contents of the aforementioned applications are incorporated herein by reference.

[0002] (Sequence Listing) This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy was created on 04 June 2021, named JBI6329WOPCT1_SL.txt, and has a size of 28,710 bytes.

[0003] (Field of Invention) The present invention provides an improved assay for determining the potency (e.g., cytotoxicity) of immune cells expressing chimeric antigen receptors. The improved assay avoids the use of mock-transfected immune cells as assay controls by instead using inhibitory molecules that prevent the chimeric antigen receptors of immune cells from interacting with their target cells as assay controls. [Background technology]

[0004] Current methods for determining the specific in vitro cytotoxicity of T cells expressing chimeric antigen receptors (CAR-T cells) involve using untransduced, proliferated autologous T cells (mock cells) as a baseline control. These controls are used to calculate the specific cytotoxicity of transduced CAR-T cells. However, the generation of untransduced, proliferated autologous T cells or allogeneic control T cells (mock cells) is costly and time-consuming, especially since these cells are usually generated from the patient's own T cells. In addition, mock cell generation is prone to production failure, which can delay treatment or interfere with the proper dosing of CAR-T cells during immunotherapy.

[0005] A method for determining the in vitro cytotoxicity of CAR-T cells involves using non-transduced expanded autologous T cells (mock) as a baseline control. The baseline control is used to calculate the increase rate of cytotoxicity specific to CAR-T cells (CAR-T killing rate). If autologous mock cells are not available, a qualified lot of allogeneic mock is used instead. However, the use of such a qualified lot results in potency compared to an allogeneic mock that may not reflect the true potency of the CAR-T cells. Alternatively, the baseline control is omitted and total cytotoxic activity is used. However, total cytotoxic activity does not indicate whether there was any enhancement of the cytotoxic activity of immune cell / target cell interactions by CAR-T cells. Total cytotoxic activity does not distinguish contributions from the formulation or spontaneous death of the target cells themselves. Alternative assays, such as cytokine ELISA, have been used in place of functional assays as surrogates to measure activity, but these methods are not direct measurements of cytotoxicity. SUMMARY OF THE INVENTION

[0006] Accordingly, there is a need for improved assay controls to simplify the production and testing of CAR-T cells while preserving the accuracy of CAR-T cell potency and reducing the associated high costs and complexity associated with using mock cells and / or additional alternative assays when mock cells are not available. The subject matter described throughout this application fulfills this need by providing novel assays that do not require the use of mock cells as controls.

[0007] In one aspect, provided is an in vitro method for determining the potency of an immune cell expressing a chimeric antigen receptor (CAR) molecule, the method comprising a) contacting CAR-expressing immune cells with target cells in a test sample, wherein the target cells express an antigen that interacts with the CAR, and b) contacting CAR-expressing immune cells with target cells in a first control sample, wherein (i) the contacting is performed in the presence of an inhibitory molecule, or (ii) the CAR-expressing immune cells and / or the target cells are pre-incubated with the inhibitory molecule prior to the contacting, and the inhibitory molecule inhibits the interaction between the CAR and the target cells; c) determining the amount of target cell death in the test sample; d) determining the amount of target cell death in the first control sample; e) determining the potency of the CAR-expressing immune cells based on comparing the amounts of target cell death determined in steps (c) and (d). Provided is the method, wherein the contacting time, the amount of CAR-expressing immune cells and the amount of target cells in the test sample and the first control sample are substantially the same.

[0008] In some embodiments, the contacting steps (a) and (b) are performed simultaneously. In some embodiments, the determining steps (c) and (d) are performed simultaneously.

[0009] In some embodiments, in step (b)(i), the CAR-expressing immune cells and / or the target cells are pre-incubated with the inhibitory molecule prior to the contacting step.

[0010] In some embodiments, the method further comprises comparing the amount of target cell death determined in step (c) with the amount of target cell death determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

[0011] In some embodiments, the method further comprises comparing the amount of target cell death determined in step (c) with the amount of target cell death 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 surfactant that causes target cell death. In a specific embodiment, the surfactant is Triton X-100.

[0012] In various embodiments, the target cells produce a detectable reporter signal upon death of the target cells, step (c) comprises determining the reporter signal in a test sample, step (d) comprises determining the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals determined in steps (c) and (d).

[0013] In some embodiments, the reporter signal is luminescence. In some embodiments, the reporter signal is fluorescence. In some embodiments, the target cell expresses a reporter protein that produces a signal when the target cell undergoes cell death. In some embodiments, the reporter protein is β-galactosidase, luciferase, or green fluorescent protein (GFP), or their varians or derivatives. In some embodiments, the inhibitory molecule specifically binds to an antigen on the target cell, causing the antigen to interact with the CAR.

[0014] In some embodiments, the inhibitory molecule specifically binds to the CAR. In some embodiments, the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on the target cell. In some embodiments, the inhibitory molecule comprises an antibody or antibody fragment. In certain embodiments, the antibody is an anti-idiotype antibody. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, Fv or Fd fragment, a single-chain antibody (scFv), a linear antibody, a single-domain antibody, a heavy chain variable region (VH) domain, or a light chain variable region (VL) domain. In some embodiments, the antibody or antibody fragment specifically binds to an antigen within the scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to a CDR within the scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to an antigen within the VH domain or VL domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to a CDR within the VH domain or VL domain of the CAR. In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interact with CAR, or a functional fragment or derivative thereof.

[0015] 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 the B-cell maturation antigen (BCMA) receptor, the target cells contain the BCMA receptor, and the inhibitory molecule is the soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In one embodiment, the multiple myeloma cells are MM-1R cells.

[0016] In some embodiments, CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cells contain the GPRC5D receptor, and the inhibitory molecule is an anti-idiotype antibody or antibody fragment against CAR. In some embodiments, the target cells are multiple myeloma cells. In one embodiment, the multiple myeloma cells are MM-1R cells.

[0017] In some embodiments, CAR interacts with kallikerin 2 (KLK2), the target cells contain KLK2, and the inhibitory molecule is the soluble KLK2 protein. In some embodiments, the target cells are prostate cancer cells. In some embodiments, the prostate cancer cells are LNCaP cells.

[0018] In various embodiments, this method is performed in a high-throughput format. [Brief explanation of the drawing]

[0019] [Figure 1] 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 contains only labeled FITC-BCMA. 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 shows FITC-BCMA competition with unlabeled BCMA. [Modes for carrying out the invention]

[0020] The methods of this disclosure can be more readily understood by referring to the following detailed description, which is interpreted in relation to the accompanying drawings, which form part of this disclosure. It should be understood that the methods of this disclosure are not limited to any particular methods described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate specific embodiments by example and are not intended to limit them to the methods described in the claims.

[0021] All published patent applications and publications referenced herein are incorporated by reference as if they were described in their entirety herein.

[0022] Where a list is presented, please understand that, unless otherwise specified, each individual element of that list and all combinations of that list represent a distinct 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".

[0023] definition As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more.

[0024] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes, or unless the substitutes are mutually exclusive; however, this disclosure supports the definition that refers only to substitutes and “and / or.” As used herein, “another” may mean at least a second or more.

[0025] "Approximately" means within the acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on how that value is measured or determined, i.e., the limitations of the measuring system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the example or elsewhere in this specification, "approximately" means within a range of 10% below the value and 10% above the value, for example, if the value is 100, then 90 to 110.

[0026] As used herein, the terms “encode” or “encode” relating to nucleic acids are used so that those skilled in the art can easily understand the present invention; however, these terms may be used interchangeably with “comprise” or “comprising,” respectively.

[0027] An "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, a part thereof, or a combination thereof) that can mediate an immune response or to which a T cell receptor can bind. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens may be expressed by genes, synthesized from, or purified from biological samples such as tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or lysates.

[0028] The term "antibody" has a broad meaning and includes monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; polyspecific antibodies such as bispecific, triplicate, and quadruplicate antibodies; dimers, tetramers, or multimers; single-chain antibodies; domain antibodies; and immunoglobulin molecules, including any other modified forms of immunoglobulin molecules containing antigen-binding sites of the required specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and a multimer thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). 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 classified into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0029] The term "antibody fragment" refers to at least one portion of an intact antibody or its recombinant variant that retains the antigen-binding properties of the parent full-length antibody. This refers to, for example, an antigen-binding domain (e.g., the antigen-determining variable region of an intact antibody) that is sufficient to confer recognition and binding (e.g., specific binding of the antibody fragment to a target such as an antigen). "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 (either VL or VH) such as sdAb, camel VHH domains, and multispecific antibodies formed from antibody fragments.

[0030] The term "subject" is intended to include organisms (e.g., mammals, e.g., humans) from which an immune response can be induced. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors.

[0031] As used herein, the term “chimeric antigen receptor” (CAR) is defined as a cell surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain—all combinations not found together in a single protein in nature. This includes receptors where the extracellular domain and intracellular signaling domain are not found together in a single receptor protein in nature. CARs are primarily intended for use in lymphocytes such as T cells and natural killer (NK) cells.

[0032] The "complementarity-determining region (CDR)" is the region of an antibody that binds to an antigen. There are three CDRs in the VH (Very High Hormone) (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (Very Low Hormone) (LCDR1, LCDR2, LCDR3). CDR can be defined using various descriptions, including the following: 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), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). Correspondence between various descriptions and variable area numbering is described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77, Honegger and Pluckthun, J Mol Biol (2001) 309:657-70, the International ImMunoGeneTics (IMGT) database, web resources, http: / / www_imgt_org). CDRs can be depicted using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs as defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0033] The terms “decrease” and “reduce” are used interchangeably herein and generally refer to the ability of a test molecule to mediate a reduced response (i.e., a downstream effect) compared to a response mediated by a control or vehicle. Illustrative responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or a decrease of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, for example, 1.5, 1.6, 1.7, 1.8, etc.).

[0034] The terms “enhance,” “promote,” “increase,” “expand,” or “improve” generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) compared to a response mediated by a control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. Enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, for example, 1.5, 1.6, 1.7, 1.8, etc.).

[0035] "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)).

[0036] "Fab" or "Fab fragment" refers to an antibody fragment composed of VH, CH1, VL, and CL domains.

[0037] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide crosslinks within a hinge region.

[0038] "Fd" or "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains.

[0039] "Fv" or "Fv fragment" refers to an antibody fragment consisting of a VH domain and a VL domain derived from a single arm of the antibody.

[0040] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as a polymer of these (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0041] A "humanized antibody" means an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0042] The "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. This is the functional part of a protein that acts by transmitting information within the cell to regulate cellular activity via signaling pathways defined by generating second messengers, or by functioning as an effector in response to such messengers. Intracellular signaling domains generate signals that enhance the immune effector function of CAR-containing cells (e.g., CAR-T cells).

[0043] "Isolated" refers to a homogeneous collection of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system in which molecules are produced, such as in recombinant cells, as well as proteins subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free from other cellular materials and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0044] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules (i.e., individual antibodies within the population are identical except for possible known modifications such as removal of C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or amidogenesis, methionine oxidation, or asparagine or glutamine amidogenesis). Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0045] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. As used herein, NK cells refer to CD16 + CD56 + and / or CD57 + TCR - NK cells refer to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0046] In this specification, the terms “protein” and “polypeptide,” used interchangeably, refer to molecules containing one or more polypeptides, each composed of at least two amino acid residues linked by peptide bonds. Proteins may be monomers or protein complexes of two or more identical or different subunits. Small polypeptide molecules consisting of fewer than 50 amino acids may be referred to as “peptides.” Proteins may be heterofusion proteins, glycoproteins, or proteins modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation. Proteins may be recombinantly expressed.

[0047] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, produced, or isolated by recombinant means. The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by bacteriophages or yeast expression systems. The term should be interpreted as meaning an antibody produced by the synthesis of a DNA molecule encoding the antibody, where this DNA molecule expresses an antibody protein or an amino acid sequence that identifies the antibody, in which case the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology that is available and known in the art.

[0048] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein the VL and VH are sequentially linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified, when used herein, scFv may have the VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may contain VL-linker-VH or VH-linker-VL.

[0049] "Specifically binds", "specific binding", "specifically bound", or "binding" refers to that a proteinaceous molecule binds to an antigen or an epitope within the antigen with a higher affinity than its affinity for other antigens. Typically, the proteinaceous molecule has an affinity of about 1×10 -7 M or less, for example about 5×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less, and binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K D ), typically, K D is at least 100 times smaller than the K for binding to non-specific antigens (e.g., BSA, casein). D

[0050] "T cell", "T-cell" and "T lymphocyte" are interchangeable and are used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, for example, a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL, CD4 + T cell), CD4 + T cell, cytotoxic T cell (CTL, CD8 + T cell), tumor infiltrating cytotoxic T cell (TIL, CD8 + T cell), CD4 + CD8 + T cell, or any other subset of T cells. It also includes "NKT cells", which refer to a special population of T cells that not only express a semi-invariant αβ T cell receptor, but also express various 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 - The cells included are NKT cells, which are unique in that their TCR recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)," which refer to a special population of a small subset of T cells that have different TCRs on their surface. Unlike most T cells, whose TCR consists of two glycoprotein chains denoted as α and β-TCR chains, the TCR in γδ T cells consists of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immunomodulation, are an important source of IL-17, and are potent CD8 + It has been found that it induces a cytotoxic T cell response. It also includes "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 IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0051] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, ex vivo, or tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can be induced by infection with transforming viruses and the incorporation of new genomic nucleic acids, or by the uptake of exogenous nucleic acids, and may occur spontaneously or after exposure to carcinogens, thereby resulting in mutations of endogenous genes. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and ex vivo.

[0052] "Variant," "mutant," or "altered" means a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0053] The “potency” of cells (e.g., CAR-T cells) as referred to herein is an indicator or measure of their effectiveness or potential effectiveness in achieving a desired function. In the case of CAR-T cells, the desired function may be to target or kill another cell, such as a target cell (e.g., tumor cell). Potency can be directly assessed by determining the effect of the cell on its target (e.g., the effect of CAR-T cells on tumor cells in vitro or in vivo). Alternatively, potency can be measured indirectly, as in the various methods of the present invention. In particular, the potency of CAR-T cells can be assessed, for example, by determining the in vitro level of antigen-specific cytotoxicity of the cell (e.g., compared to the cytotoxicity of unstimulated CAR-T cells as described herein) in the assay described herein. This measure of potency can then be associated with in vivo properties of the cell, such as PK / PD parameters (e.g., CMAX, TAX, and AUC) as described herein, which may be related to the effectiveness of the cell in killing its target, and can therefore be considered a predictor thereof. As further described herein, efficacy may be expressed in terms of a cytotoxicity index that can be normalized based on the number of cells expressing the relevant CAR.

[0054] Where used herein, “reference” or “control” refers to a standard or control on which comparison is made. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to the drug, animal, individual, population, sample, sequence, or value of reference or control. In some embodiments, the reference or control is tested and / or determined substantially concurrently with the test or determination of interest. Typically, as will be understood by those skilled in the art, the reference or control is determined or characterized under conditions or environments comparable to those under evaluation. Those skilled in the art will understand that there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0055] The term “stimulating molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides cytoplasmic signaling sequences that modulate the activation of immune cells in a stimulating manner in at least some aspects of immune cell signaling pathways. In one aspect, the signal is a primary signal initiated, for example, by the binding of a TCR / CD3 complex to a peptide-loaded MHC molecule, and results in mediating a T cell response including, but not limited to, proliferation, activation, and differentiation. A stimulating primary cytoplasmic signaling sequence (also called a “primary signaling domain”) may contain a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1 G), FcγRIIa, FcRβ (FcεR1 b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In CAR, the intracellular signaling domain may include an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ.

[0056] Other objects, features, and advantages of the present invention will become apparent from the following "Modes for Carrying Out the Invention." However, while the modes for carrying out the invention and specific examples illustrate preferred embodiments of the present invention, it should be understood that they are given only as examples, as various modifications and improvements within the spirit and scope of this disclosure will become apparent to those skilled in the art through these modes for carrying out the invention.

[0057] Chimeric antigen receptor 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 artificially constructed hybrid protein or polypeptide containing the antigen-binding domain of an antibody (scFv) bound to an immune cell, such as a T cell signaling domain. Characteristics of CARs include the ability to redirect T cell specificity and reactivity toward non-MHC restricted targets and to utilize the antigen-binding properties of monoclonal antibodies. Due to non-MHC restricted antigen recognition, T cells expressing CARs can recognize antigens independently of antigen processing, thus bypassing the main mechanism of tumor evasion. Furthermore, when expressed in T cells, CARs do not favorably dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0058] 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 the "cytoplasmic signaling domain") which includes a functional signaling domain derived from a stimulating molecule as defined below. T cells expressing CARs are referred herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cells can be genetically modified to stably express an antibody-binding domain on their surface, conferring novel MHC-independent antigen specificity.

[0059] In some cases, T cells are genetically modified to stably express a CAR that combines the antigen-recognition domain of a specific antibody with the intracellular domain of a CD3-ζ chain or an FcγRI protein to form a single chimeric protein. In one embodiment, the stimulating molecule is a ζ chain associated with the T cell receptor complex.

[0060] As used herein, “intracellular signaling domain” or “cytoplasmic signaling domain” refers to the intracellular portion of a molecule. This is the functional part of a protein that acts by transmitting information within the cell to regulate cellular activity via signaling pathways defined by the generation of second messengers, or by acting as an effector in response to such messengers. Intracellular signaling domains generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). Examples of immune effector functions in CAR-T cells include cytolytic activity and helper activity (including cytokine secretion).

[0061] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain may be derived from a molecule involved in primary stimulation or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. An example of a co-stimulatory intracellular signaling domain may be derived from a molecule involved in co-stimulatory signaling or antigen-independent stimulation. For example, in the case of CAR-T cells, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0062] The primary intracellular signaling domain may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[0063] The terms "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" are defined as the protein provided as GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rabbits, primates, rodents, monkeys, and apes, and the "ζ stimulating domain" or alternatively "CD3-ζ stimulating domain" or "TCR-ζ stimulating domain" are defined as amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signals necessary for T cell activation. In one embodiment, the cytoplasmic domain of ζ includes residues 52-164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, and apes that are functional orthologues thereof. In one embodiment, the "ζ-stimulating domain" or "CD3-ζ-stimulating domain" is the sequence provided as Sequence ID No. 10 below, or a sequence having 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% sequence identity with Sequence ID No. 10.

[0064] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 10)

[0065] The term "costimulatory molecule" refers to an allobinding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells (e.g., proliferation, but not limited to T cell proliferation). Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0066] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to 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.

[0067] An intracellular signaling domain may include the entire intracellular portion of the molecule from which it originates, the entire intrinsic intracellular signaling domain, or functional fragments thereof.

[0068] The term "4-1BB" or alternatively "CD137" refers to the amino acid sequence provided as GenBank accession number AAA62478.2, or a member of the TNFR superfamily having equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. The "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. In one embodiment, the "4-1BB costimulatory domain" or the "CD137 costimulatory domain" is the sequence provided as Sequence ID No. 11, or an equivalent residue from a non-human species, such as a mouse, rodent, monkey, or ape, or a sequence having 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% sequence identity with Sequence ID No. 11.

[0069] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 11)

[0070] In one embodiment, a transmembrane domain naturally associated with one of the domains within the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex. In one example embodiment, the transmembrane domain includes a CD8α hinge domain.

[0071] 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-ζ, and / or CD28. CD28 is an important T cell marker in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and functions as a costimulatory immune checkpoint molecule. 4-1BB transmits potent costimulatory signals to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3-ζ associates with the TCR to generate a signal and contains an immune receptor tyrosine-based activation motif (ITAM). In another embodiment, the costimulatory molecule is MyD88 or CD40.

[0072] In one embodiment, the CAR comprises an intracellular hinge domain containing CD8 and an intracellular T cell receptor signaling domain containing CD28, 4-1BB, and CD3-ζ. In another embodiment, the CAR comprises an intracellular hinge domain and an intracellular T cell receptor signaling domain containing CD28, 4-1BB, and CD3-ζ, wherein the hinge domain comprises all or part of the extracellular region of CD8, CD4, or CD28, all or part of the antibody constant region, the FcyRIIIA receptor, IgG hinge, IgM hinge, IgA hinge, IgD hinge, IgE hinge, or all or part of the Ig hinge. The IgG hinge may be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0073] 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 the "cytoplasmic signaling domain") which includes a functional signaling domain derived from, for example, a stimulating molecule as defined below.

[0074] In one embodiment, 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 stimulating molecule. In one embodiment, 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 co-stimulatory molecule and a functional signaling domain derived from a stimulating molecule. In one embodiment, 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 co-stimulatory molecules and a functional signaling domain derived from a stimulating molecule.

[0075] The CAR may be designed to contain only the CD28 and / or 4-1BB signaling domain, or to be combined with any other desired cytoplasmic domain useful in the context of the CAR described herein. In one embodiment, the cytoplasmic domain of the CAR may further include the CD3-ζ signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, the CD3-ζ, 4-1BB, and CD28 signaling modules, or combinations thereof.

[0076] In some embodiments, the CARs described herein include an extracellular antigen-binding domain that specifically binds to a tumor antigen. Non-exclusive examples of tumor antigens that can be recognized by the CARs described herein include BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0077] This disclosure further provides variants (e.g., functional variants) of the CARs, nucleic acids, polypeptides, and proteins described herein. A “variant” means a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitution, insertion, or deletion. As used herein, the term “functional variant” means a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent CAR, polypeptide, or protein, and these functional variants retain the biological activity of the variant CAR, polypeptide, or protein. Functional variants include, for example, variants of the CARs, polypeptides, or proteins (parent CARs, polypeptides, or proteins) described herein, which retain the ability to recognize target cells (tumor cells) to the same degree, the same degree, or a higher degree than the parent CAR, polypeptide, or protein. With respect to the parent CAR, polypeptide, or protein, the functional variant may have an amino acid sequence that is identical to, for example, the parent CAR, polypeptide, or protein by 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.

[0078] A functional variant may include, for example, an amino acid sequence of a parent CAR, polypeptide, or protein having at least one conserved amino acid substitution. In another embodiment, a functional variant may include an amino acid sequence of a parent CAR, polypeptide, or protein having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution cannot suppress or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the parent CAR, polypeptide, or protein.

[0079] The amino acid substitutions of CARs in the present invention 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 a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions may include acidic amino acids substituted with another acidic amino acid (e.g., Asp or Glu), amino acids with nonpolar side chains substituted with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids substituted with another basic amino acid (e.g., Lys, Arg), and amino acids with polar side chains substituted with another amino acid having a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0080] CARs, polypeptides, or proteins may essentially consist of a specific amino acid sequence or a sequence described herein, and as a result, other components, such as other amino acids, do not substantially alter the biological activity of the functional variant.

[0081] The CARs, polypeptides, and proteins (including functional portions and functional variants) of embodiments of this disclosure may be of any length, i.e., may contain any number of amino acids, provided that the CARs, polypeptides, or proteins (or functional portions or their functional variants) retain their biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells (e.g., cancer cells) in a host, or the ability to treat or prevent disease in a host. For example, polypeptides may have amino acid lengths of approximately 50 to approximately 5000, for example, approximately 50, approximately 70, approximately 75, approximately 100, approximately 125, approximately 150, approximately 175, approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 525, approximately 550, approximately 575, approximately 600, approximately 625, approximately 650, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, approximately 800, approximately 825, approximately 850, approximately 875, approximately 900, approximately 925, approximately 950, approximately 975, approximately 1000 or more. The polypeptides of the present invention also include oligopeptides.

[0082] CARs, polypeptides, and proteins (including functional portions and functional variants of CARs) used in the aspects and embodiments of this specification may include synthetic amino acids instead of one or more naturally occurring 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-acetylaminomethyl-cysteine, 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- Examples include hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 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.

[0083] CARs, polypeptides, and proteins (including functional moieties and functional variants) used in the embodiments and aspects of this specification may undergo post-translational modifications. These can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via disulfide crosslinks, for example, or converted to acid addition salts. In some embodiments, these are dimerized, polymerized, or conjugated.

[0084] The CARs, polypeptides, and / or proteins (including their functional portions and functional variants) used in the aspects and embodiments of this specification can be obtained by methods known in the art. Suitable methods for the de novo synthesis of polypeptides and proteins are described in reference to 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. Polypeptides and proteins can also be produced by recombination using the nucleic acids described herein with standard recombination methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some of the CARs, polypeptides, and proteins (including their functional parts and functional variants) described herein can be isolated and / or purified from sources such as plants, bacteria, insects, and mammals. Methods of isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and / or proteins (including their functional parts and functional variants) described herein can be commercially synthesized. In this regard, CARs, polypeptides, and proteins can be synthesized, recombinant, isolated, and / or purified.

[0085] Examples of modified nucleotides that can be used to generate recombinant nucleic acids used to produce the polypeptides described herein include 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, β-D-mannosylquosin, 5″-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, quosin, β-D-galactosylquosin, inosine, N 6 Examples include, but are not limited to, isopentenyl adenine, 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-oxyacetate methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0086] Nucleic acids may comprise any isolated or purified nucleotide sequence encoding a CAR, polypeptide, or protein, or a functional portion or a functional variant thereof. Alternatively, a nucleotide sequence may comprise a degenerate nucleotide sequence or a combination of degenerate sequences. Nucleic acids can be incorporated into recombinant expression vectors. 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 enables the expression of mRNA, protein, polypeptide, or peptide by a host cell, in which case the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is brought into contact with the cell under conditions sufficient to express mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein as a whole do not exist in nature. However, parts of the vectors may exist in nature. The recombinant expression vectors described may contain any type of nucleotide, including but not limited to DNA and RNA (which may be single-stranded or double-stranded, synthesized, or partially obtained from natural sources, and may contain natural, unnatural, or modified nucleotides). Recombinant expression vectors may contain naturally occurring nucleotide linkages, non-natural nucleotide linkages, or both types of linkages. Non-natural or modified nucleotides or nucleotide linkages do not inhibit the transcription or replication of the vector.

[0087] The recombinant expression vector may be any suitable recombinant expression vector that can be used to transform or transfect any suitable host. Suitable vectors include those designed for the reproduction and proliferation of plasmids and viruses, or for expression, or both. Vectors may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors, such as gamma retroviral vectors.

[0088] Recombinant expression vectors are prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. (above) and Ausubel et al. (above). The circular or linear expression vector constructs may be prepared to include a replication system that functions in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0089] Recombinant expression vectors may, as appropriate, include regulatory sequences such as transcription and translation start and stop codons that are specific to the type of host into which the vector is introduced (e.g., bacteria, plants, fungi, or animals), taking into consideration whether the vector is DNA-based or RNA-based.

[0090] Recombinant expression vectors may contain one or more marker genes that enable selection of transformed or transfected hosts. Marker genes may include those for biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.) or for supplementation to provide protonutrient in nutritionally dependent hosts. 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.

[0091] Recombinant expression vectors may include a native or normative promoter operably ligated to a nucleotide sequence encoding a CAR, polypeptide, or protein (including functional portions and functional variants thereof), or to a nucleotide sequence complementary to or hybridizing with a CAR, polypeptide, or protein-encoding nucleotide sequence. The choice of promoter (e.g., strong, weak, tissue-specific, inducible, and developmentally specific) is within the scope of the art. Similarly, combining nucleotide sequences with promoters is also within the scope of the art. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, RSV promoters, SV40 promoters, or promoters found in long terminal repeats of murine stem cell viruses.

[0092] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0093] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to a drug (e.g., a pharmacokinetic) to cells expressing the gene, causing the cells to die when they come into contact with or are exposed to the drug. 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.

[0094] The inhibitory molecule may be an antibody (e.g., a monoclonal antibody) or its antigen-binding moiety, or a soluble antigen, or its functional moiety or functional variant, that binds, for example, specifically, to the epitope of the CAR of an immune cell. The antibody may be any type of immunoglobulin known in the art. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chains of vertebrate species may be assigned to one of two types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The antibody may be of any class or isotype.

[0095] The antibodies used in the methods described herein may include immunoglobulin molecules, including monoclonal antibodies, such as mouse, human, humanized, and chimeric monoclonal antibodies; polyclonal antibodies; antigen-binding fragments; bispecific or multispecific antibodies; monomeric, dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; domain antibodies; and immunoglobulin molecules of any other modified conformation containing antigen-binding sites of the required specificity. The antibodies may be naturally occurring antibodies, such as antibodies isolated and / or purified from mammals (e.g., rats, primates, mice, rabbits, goats, horses, chickens, hamsters, humans, etc.). Alternatively, the antibodies may be genetically engineered antibodies.

[0096] Humanized antibodies have antigen-binding sites derived from non-human species, and their variable region framework is derived from human immunoglobulin sequences. Human antibodies have heavy and light chain variable regions in which both the framework and antigen-binding sites are derived from human sequences.

[0097] Furthermore, antibodies may have any level of affinity or binding activity to the functional portion of CAR. In some embodiments, antibodies have an affinity (K) within a certain range. D ) can bind to the hK2 antigen. In various embodiments, the antibody has high affinity, for example, about 10, as determined by surface plasmon resonance or Kinexa method performed by those skilled in the art. -7 For KD below M, for example, although not limited to, 1 to 9.9 (or any range or value that falls within this range, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 It binds to the hK2 antigen with a KD of M, or any range or value within this range. An example of affinity is 1 × 10⁻⁶. -8 It is less than or equal to M. Another example of affinity is 1 × 10 -9 It is M or less.

[0098] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assay.

[0099] Suitable methods for producing antibodies are known in the art. For example, the standard hybridoma method is described, for instance, in Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, for example, Huse et al., Science, 246, 1275-81 (1989)). Furthermore, methods for producing antibodies in non-human animals are described, for example, in U.S. Patents No. 5,545,806, No. 5,569,825, and No. 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266 (A1)).

[0100] Antibodies used in any of the methods described herein can also be generated using phage display. In this regard, phage libraries encoding the antigen-binding variable (V) domain of an antibody can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al. (above) and Ausubel et al. (above)). Phages encoding a variable region with the desired specificity are selected for specific binding to the desired antigen (i.e., hK2), and a complete or partial antibody containing the selected variable domain is reconstituted. The nucleic acid sequence encoding the reconstituted antibody is introduced into a suitable cell line, such as myeloma cells used for hybridoma production, resulting in the secretion of an antibody having the characteristics of a monoclonal antibody by the cells (see, e.g., Janeway et al. (above), Huse et al. (above), and U.S. Patent No. 6,265,150).

[0101] Antibodies can be produced by transgenic mice that are transgenic of specific heavy and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Patents No. 5,545,806 and No. 5,569,825, and by Janeway et al., as described above.

[0102] Methods for generating humanized antibodies are known in the art and are described, for example, by Janeway et al., U.S. Patents No. 5,225,539, 5,585,089, and 5,693,761, European Patent No. 0239400(B1), and British Patent No. 2188638. Humanized antibodies can also be generated using the antibody resurfacing technique described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

[0103] When used herein, antibodies may be multiple or single chains, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0104] Antigen-binding moieties of any of the antibodies described herein are also provided. The antigen-binding moiety may be any moiety having at least one antigen-binding site, such as Fab, F(ab')2, dsFv, sFv, diabody, and triabody. In some embodiments, the antigen-binding fragment is a domain antibody (dAb) comprising (e.g., consisting of) heavy chain complementarity determining regions (HCDR) 1, 2, and / or 3, light chain complementarity determining regions (LCDR) 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 either one VH domain or one VL domain. The VH domain and VL domain may be linked together via a linker, such as a synthetic linker.

[0105] Furthermore, antibodies or their antigen-binding moieties may be modified to include detectable labels such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0106] This disclosure provides nucleic acids comprising nucleotide sequences encoding any of the CARs, polypeptides, or proteins (including functional portions and functional variants thereof) described herein.

[0107] The portion of the CAR containing an antibody or an antibody fragment may exist in various forms, in which case the antigen-binding domain is expressed as part of a continuous polypeptide chain containing, for example, a single-domain antibody fragment (sdAb), an scFv, and a human chimeric antibody or humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988; Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition contains an antibody fragment. In one embodiment, the CAR contains an antibody fragment containing an scFv.

[0108] In one embodiment, the extracellular antigen-binding domain includes an scFv. In some embodiments, the scFv includes a linker polypeptide between the light chain variable region and the heavy chain variable region.

[0109] In recombinant expression systems, the linker is a peptide linker and may contain 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 needs to be of an appropriate length to link VH and VL in such a way that they form precise higher-order structures relative to each other, in order to maintain desired activities such as binding to hK2.

[0110] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker is approximately 10 to 40 amino acids long. In some embodiments, the linker is approximately 10 to 35 amino acids long. In some embodiments, the linker is approximately 10 to 30 amino acids long. In some embodiments, the linker is approximately 10 to 25 amino acids long. In some embodiments, the linker is approximately 10 to 20 amino acids long. In some embodiments, the linker is approximately 15 to 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 acid lengths. In some embodiments, the linker is 16 amino acid lengths. In some embodiments, the linker is 17 amino acid lengths. In some embodiments, the linker is 18 amino acid lengths. In some embodiments, the linker is 19 amino acid lengths. In some embodiments, the linker is 20 amino acid lengths. In some embodiments, the linker is 21 amino acid lengths. In some embodiments, the linker is 22 amino acid lengths. In some embodiments, the linker is 23 amino acid lengths. In some embodiments, the linker is 24 amino acid lengths. In some embodiments, the linker is 25 amino acid lengths. In some embodiments, the linker is 26 amino acid lengths. In some embodiments, the linker is 27 amino acid lengths. In some embodiments, the linker is 28 amino acid lengths. In some embodiments, the linker is 29 amino acid lengths.In some embodiments, the linker is 30 amino acid lengths. In some embodiments, the linker is 31 amino acid lengths. In some embodiments, the linker is 32 amino acid lengths. In some embodiments, the linker is 33 amino acid lengths. In some embodiments, the linker is 34 amino acid lengths. In some embodiments, the linker is 35 amino acid lengths. In some embodiments, the linker is 36 amino acid lengths. In some embodiments, the linker is 37 amino acid lengths. In some embodiments, the linker is 38 amino acid lengths. In some embodiments, the linker is 39 amino acid lengths. In some embodiments, the linker is 40 amino acid lengths. Exemplary linkers that can be used are glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and other flexible linkers.

[0111] In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. The signal polypeptide may be located at the N-terminus of the extracellular antigen-binding domain that binds to hK2. The signal polypeptide may be optionally cleaved from the extracellular antigen-binding domain during cell processing and localization of CAR to the cell membrane. Any of the various signal polypeptides known to those skilled in the art may be used as the signal polypeptide. Non-limiting examples of peptides from which the signal polypeptide may be derived include FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of the various other proteins secreted by T cells. In various embodiments, the signal polypeptide is adapted to the secretory pathway of T cells.

[0112] In one embodiment, the disclosure 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 polypeptide components selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3ζ chain (CD3z) component, a differentiation cluster (CD27) component, a differentiation cluster superfamily member (e.g., CD28 or an inducible T-cell co-stimulator, ICOS) component, and combinations thereof. In one embodiment, the transmembrane domain comprises a CD8a transmembrane domain (CD8A-TM) polypeptide. In one embodiment, the transmembrane domain includes at least (multiple) transmembrane regions of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. In another embodiment, the transmembrane domain includes at least the ζ, η, or FcεR1γ and β, MB1(Igα), B29, or CD3-γ, ζ, or η transmembrane domain. In yet another embodiment, the transmembrane domain is synthetic and includes mainly hydrophobic residues such as a triplet of leucine and valine, phenylalanine, or tryptophan.

[0113] In one embodiment, the CAR further includes a hinge region that links the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region.

[0114] In one embodiment, the present disclosure provides isolated immune-responsive cells containing CARs as described herein. In some embodiments, isolated immune-responsive cells are transduced with a CAR, for example, constitutively expressed on the surface of the immune-responsive cells. In certain embodiments, isolated immune-responsive cells are further transduced with at least one costimulatory ligand to cause the immune-responsive cells to express 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, isolated immune-responsive cells are further transduced with at least one cytokine to cause the immune-responsive cells to secrete 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, IL-15, IL-17, IL-21, and combinations thereof. In some embodiments, the isolated immune-responsive cells are selected from a group consisting of T lymphocytes (T cells), natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, lymphoid progenitor cells, T cell progenitor cells, and pluripotent stem cells that can differentiate into lymphoid cells.

[0115] In one embodiment, CAR T-expressing immune cells of the present disclosure can be generated by introducing a lentiviral vector containing a desired CAR, for example, an anti-hK2, CD8α hinge and transmembrane domain, and a human 4-1BB and CD3-ζ signaling domain, into cells. The CAR T-expressing immune cells of the present invention can replicate in vivo, resulting in long-term persistence that may lead to sustained tumor control.

[0116] Both CARs and inhibitory molecules can 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 may contain a recombinant expression vector. A host cell may be a eukaryotic cell, e.g., a plant, animal, or algae or fungus, or a prokaryotic cell, e.g., a bacterium or protist. A host cell may be a cultured cell or a primary cell, i.e., it may be directly isolated from an organism, e.g., a human. A host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5α cell. For the purpose of producing recombinant CARs, polypeptides, or proteins, the host cell may be a mammalian cell. A host cell may be a human cell. The host cell can be any cell type, originate from any tissue type, and be at any developmental stage, but the host cell may be a peripheral blood lymphocyte (PBL). The host cell may be a T cell.

[0117] For the purposes of this specification, T cells may be any T cells, such as cultured T cells (e.g., primary T cells), or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. When obtained from mammals, T cells may be obtained from a number of sources (including, but not limited to, bone marrow, blood, lymph nodes, thymus, or other tissues or body fluids). T cells may also be concentrated or purified. T cells may be human T cells. T cells may be T cells isolated from humans. T cells may be CD4 + / CD8 + Double positive T cells, CD8 + CD4 cells such as T cells (e.g., cytotoxic T cells), Th1 and Th2 cells +This includes, but is not limited to, helper T cells, peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells, memory T cells, and naive T cells; it may be any type of T cell at any developmental stage. T cells are CD8 + T cells or CD4 + It could be a T cell.

[0118] A cell population comprising at least one host cell described herein is also provided. The cell population may be a heterogeneous population comprising at least one other cell, e.g., a host cell not comprising any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell (e.g., a B cell, macrophage, erythrocyte, neutrophil, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc.), in addition to a host cell comprising any of the recombinant expression vectors described herein. Alternatively, the cell population may be a substantially homogeneous population in which the population mainly comprises (e.g., essentially consists of) host cells comprising recombinant expression vectors. The population may also be a clonal population of cells in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, and therefore all cells of the population contain the recombinant expression vector. In one embodiment, the cell population is a clonal population comprising host cells comprising the recombinant expression vectors described herein.

[0119] CAR-binding inhibitors In some embodiments, the inhibitory molecule used in the methods herein may be a monoclonal antibody that specifically binds to a CAR polypeptide. For example, a monoclonal antibody can specifically bind to the constant domain of a CAR polypeptide described herein, e.g., a CAR polypeptide expressed on CAR-T cells. Alternatively, the antibody can bind to the antigen-recognition domain of a CAR polypeptide (e.g., a CD-19-conjugated CAR polypeptide). The antibody can interfere with the ability of CAR-T cells to bind to target cells (e.g., tumor cells). While we do not wish to be bound by theory, antibodies can prevent CAR-T cells from binding to target cells (e.g., tumor cells).

[0120] In various embodiments, monoclonal antibodies that specifically bind to BCMA-targeted CAR polypeptides are used. In some embodiments, the monoclonal antibody binds to the BCMA-specific CAR polypeptide and competes for polypeptide binding to multiple myeloma target cells or any other cells expressing BCMA. The monoclonal antibody may be an anti-idiotype antibody. An anti-idiotype antibody is a specific antibody that can bind to a CDR sequence within a specific antibody. The monoclonal antibody may be a type 1 anti-idiotype antibody that binds to the CDR of the target antibody's variable domain in a manner that inhibits, disrupts, or neutralizes the activity of the target antibody, i.e., its ability to bind to the antigen.

[0121] The inhibitory molecule may be an anti-idiotype peptide. In some embodiments, the anti-idiotype peptide binds to the antigen-binding receptor of one or more additional cell therapeutics (e.g., the scFv of a CAR-T cell). In some embodiments, the anti-idiotype peptide binds to the antigen-binding receptor of one or more CDRs (e.g., the scFv of a CAR-T cell). In various embodiments, the anti-idiotype antibody or peptide (e.g., scFv) binds to the B cell-specific marker antigen-binding moiety of a CAR-T cell (e.g., a CAR that binds to CD19, CD20, CD21, CD22, CD24, CD79a, CD79b, ROR1, or BCMA). In addition, for example, in some embodiments, the anti-idiotype antibody or fragment (e.g., scFv) binds to an anti-CD19 antibody or fragment (e.g., an anti-CD19 antibody expressed by a CAR-T cell (e.g., anti-CD19 scFv)).

[0122] Inhibitory molecules are also provided that include all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to a BCMA-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a BCMA-targeted CAR polypeptide. Inhibitory molecules are also provided that include all or part of the light chain variable region of a monoclonal antibody that specifically binds to a BCMA-targeted CAR polypeptide. Such inhibitory molecules can also specifically bind to a BCMA-targeted CAR polypeptide. Inhibitory molecules are also provided that include one, two, three, four, five, or six complementarity-determining regions (CDRs) derived from the variable light chain and / or variable heavy chain of a monoclonal antibody that binds to a BCMA-specific CAR polypeptide.

[0123] In certain embodiments, a monoclonal antibody that specifically binds to a GPRC5D-targeted CAR polypeptide is used. In some embodiments, the monoclonal antibody binds to the GPRC5D-specific CAR polypeptide and competes for polypeptide binding with multiple myeloma target cells (e.g., multiple myeloma tumor cells) or any other cells expressing GPRC5D. The monoclonal antibody may be an anti-idiotype antibody. Inhibitory molecules comprising all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to a GPRC5D-targeted CAR polypeptide are also provided. Such inhibitory molecules can also specifically bind to a GPRC5D-targeted CAR polypeptide. Inhibitory molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to a GPRC5D-targeted CAR polypeptide are also provided. Such inhibitory molecules can also specifically bind to a GPRC5D-targeted CAR polypeptide. Inhibitory molecules are also provided that contain one, two, three, four, five, or six complementarity-determining regions (CDRs) derived from the variable light chain and / or variable heavy chain of a monoclonal antibody that binds to a GPRC5D-specific CAR polypeptide.

[0124] In certain embodiments, a monoclonal antibody that specifically binds to a CD79-targeted CAR polypeptide is used. In some embodiments, the monoclonal antibody binds to the CD79-specific CAR polypeptide and competes for polypeptide binding to multiple myeloma target cells or any other cells expressing CD79. The monoclonal antibody may be an anti-idiotype antibody. Inhibitor molecules are also provided that include all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to a CD79-targeted CAR polypeptide. Such inhibitor molecules can also specifically bind to a CD79-targeted CAR polypeptide. Inhibitor molecules are also provided that include all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CD79-targeted CAR polypeptide. Such inhibitor molecules can also specifically bind to a CD79-targeted CAR polypeptide. Inhibitor molecules are also provided that include one, two, three, four, five, or six complementarity-determining regions (CDRs) derived from the variable light chain and / or variable heavy chain of a monoclonal antibody that binds to a CD79-specific CAR polypeptide.

[0125] In certain embodiments, monoclonal antibodies that specifically bind to KLK2-targeted CAR polypeptides are used. In some embodiments, the monoclonal antibody binds to the KLK2-specific CAR polypeptide and competes for polypeptide binding to multiple myeloma target cells or any other cells expressing KLK2. The monoclonal antibody may be an anti-idiotype antibody. Inhibitor molecules comprising all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to KLK2-targeted CAR polypeptides are also provided. Such inhibitor molecules can also specifically bind to KLK2-targeted CAR polypeptides. Inhibitor molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to KLK2-targeted CAR polypeptides are also provided. Such inhibitor molecules can also specifically bind to KLK2-targeted CAR polypeptides. Inhibitor molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) derived from the variable light chain and / or variable heavy chain of a monoclonal antibody that binds to KLK2-specific CAR polypeptides are also provided. In certain embodiments, monoclonal antibodies that specifically bind to CD19-targeted CAR polypeptides are used. In some embodiments, the monoclonal antibody binds to the CD19-specific CAR polypeptide and competes for polypeptide binding to multiple myeloma target cells or any other cells expressing CD19. The monoclonal antibody may be an anti-idiotype antibody. Inhibitor molecules comprising all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to CD19-targeted CAR polypeptides are also provided. Such inhibitor molecules can also specifically bind to CD19-targeted CAR polypeptides. Inhibitor molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to CD19-targeted CAR polypeptides are also provided. Such inhibitor molecules can also specifically bind to CD19-targeted CAR polypeptides. Inhibitor molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) derived from the variable light chain and / or variable heavy chain of a monoclonal antibody that binds to CD19-specific CAR polypeptides are also provided.

[0126] In various embodiments, inhibitory molecules, such as monoclonal antibodies, fragments of monoclonal antibodies, or derivatives of monoclonal antibodies, are capable of transgene-specific expansion. An anti-idiotype antibody containing its antigen-binding fragment specifically recognizes, specifically targets, and / or specifically binds to an idiotope of the antibody or its antigen-binding fragment, such as the antigen-binding domain of a recombinant receptor, including a chimeric antigen receptor (CAR). The idiotope is any single antigenic determinant or epitope within the variable region of the antibody. An anti-idiotype antibody or its antigen-binding fragment may be an agonist and / or may exhibit specific activity that stimulates cells expressing a particular antibody, and may include a conjugate or recombinant receptor containing the antibody or its antigen-binding fragment (e.g., U.S. Patent Application Publication 2016 / 0096902, U.S. Patent Application Publication 2016 / 0068601, U.S. Patent Application Publication 2014 / 0322183, U.S. Patent Application Publication 2015 / 0175711, U.S. Patent Application Publication 2015 / 283178, U.S. Patent 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 See Lancet (2015) 385(9967):517-528, Zhao et al., PloS One (2014) 9(5):e96697, and Leung et al., MAbs. (2015) 7(1):66-76).

[0127] In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interact with CAR, or a functional fragment or derivative thereof. For example, the soluble antigens may be soluble forms of BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3, or functional fragments or derivatives thereof.

[0128] Contact between CAR-T cells and inhibitors Various methods are disclosed herein, including preventing or modifying the ability of specific CAR-T cells (e.g., T cells expressing BCMA-binding CARs) by contacting cells with a monoclonal antibody or soluble antigen that binds to the antigen-recognition domain of a CAR.

[0129] In one embodiment, an in vitro method for determining the cytotoxicity of immune cells expressing a chimeric antigen receptor (CAR) molecule, wherein the method is a) Incubating CAR-expressing immune cells with target cells (e.g., tumor cells) in a test sample, wherein the target cells express antigens that interact with CAR, b) Incubating CAR-expressing immune cells with target cells in a first control sample, wherein the incubation is carried out in the presence of an inhibitory molecule, the inhibitory molecule reduces, inhibits, blocks, and / or prevents the interaction between the CAR and the target cells. c) To determine the amount of target cell death in the test sample, and d) To determine the amount of target cell death in the first control sample, e) Determining the cytotoxicity of CAR-expressing immune cells based on a comparison of the amount of target cell death determined in steps (c) and (d), The present invention provides a method in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same in the test sample and the first control sample.

[0130] 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 amount of CAR-expressing immune cells is 85–115%, 90–110%, or 95–105% of the amount of target cells.

[0131] In some embodiments, incubation steps (a) and (b) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, 1 hour, 30 minutes, or 15 minutes. Performing steps (a) and (b) simultaneously can provide conditions under which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same. In some embodiments, determining steps (c) and (d) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, 1 hour, 30 minutes, or 15 minutes. Performing steps (c) and (d) simultaneously can provide conditions under which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same.

[0132] In some embodiments, CAR-expressing immune cells and / or target cells are pre-incubated with inhibitory molecules prior to the contact step.

[0133] In some embodiments, the method further comprises comparing the amount of target cell death (e.g., tumor cells) determined in step (c) with the amount of target cell death determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

[0134] In some embodiments, the method further comprises comparing the amount of target cell death determined in step (c) with the amount of target cell death determined in a third control sample, wherein the target cells are incubated in the presence of a surfactant that induces target cell death but is absent from CAR-expressing immune cells. In certain embodiments, the surfactant is TritonX-100.

[0135] In various embodiments, target cells (e.g., tumor cells) produce a detectable reporter signal upon death of the target cells, and step (c) comprises determining the reporter signal in a test sample, step (d) comprising determining the reporter signal in a first control sample, and step (e) comprising comparing the reporter signals determined in steps (c) and (d). In some embodiments, target cells express a reporter protein that produces a signal when the target cells undergo cell death. Exemplary reporter proteins suitable for use in the methods of the present disclosure include, but are not limited to, β-galactosidase, luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), and their variants or derivatives.

[0136] In some embodiments, the reporter signal is luminescence. A protein capable of generating a reporter signal (e.g., luminescence) may be expressed in a cell compartment. Upon cell death, the protein is released from the cell compartment into the culture medium, and the protein may generate a luminescence signal by enzymatically acting on, for example, a drug to generate luminescence. An exemplary cell that may be used in this manner is KILR® target cells. Target cells can be engineered to express antigens that interact with chimeric antigen receptors. In certain embodiments, KILR® MM-1R multiple myeloma target cells are used. Target cells can also stably express labeled or enzyme-tagged proteins. When a target cell line is used in a cytotoxic assay and its membrane is damaged due to cell death, the target cell line may release tagged proteins into the culture medium. Tagged proteins can be detected by adding a reagent to the culture medium that is a substrate for the enzyme tag on the protein. For example, the β-galactosidase enzyme can hydrolyze the substrate to give a chemiluminescent output. The luminescence can be quantified on a plate reader capable of measuring chemiluminescence. Alternatively, tagged proteins can be detected via assays to detect the label.

[0137] In some embodiments, the reporter signal is fluorescence. A protein capable of generating a reporter signal (e.g., fluorescence) may be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into the culture medium, where it can generate a fluorescent signal.

[0138] In some embodiments, the inhibitory molecule specifically binds to an antigen on a target cell that interacts with the CAR. While we do not wish to be bound by theory, the use of an antigen-binding inhibitory molecule can provide a suitable control in cytotoxicity assays or other relevant CAR efficacy assays, eliminating the need to use untransfected or mock-transfected immune cells as controls. This eliminates the need to produce mock CAR-T cells in parallel with the formulation. The production of mock CAR-T cells can affect the production of the formulation in several ways. Eliminating the use of mock cell controls can simplify the manufacturing process and ensure that patient dosing can be achieved by reducing the sampling of any autologous CAR-T cells from patients, otherwise reducing the cost in CAR-T cell therapy. The method described herein can also reduce or eliminate test delays due to mock cell failure. The method can also reduce test errors by providing a simpler format. The reduction of test delays and errors can also provide the avoidance of production delays and / or patient dosing delays.

[0139] In some embodiments, the inhibitory molecule specifically binds to CARs. In some embodiments, the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on a target cell interacting with the CAR. By binding to the CAR, particularly to a region within the CAR that specifically binds to an antigen (e.g., an epitope containing one or more CDR sequences or a portion thereof), the inhibitory molecule can block CAR-T cells from interacting with target cells. Such blockage can prevent CAR-T cells from killing target cells. Therefore, instead of using mock-transfected immune cells, the patient's own CAR-T cells can be used, instead with the inhibitory molecule added as a suitable control.

[0140] In some embodiments, the inhibitory molecule is an antibody. In certain embodiments, the antibody is an anti-idiotype antibody. The anti-idiotype antibody can compete with the antigen on the host cell for binding to the chimeric antigen receptor. The anti-idiotype antibody may share some structural features with the antigen.

[0141] In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the scFv domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the Fab domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the VH or VL domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the VH or VL domain.

[0142] 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 the B cell maturation antigen (BCMA) receptor, the target cells contain the BCMA receptor, and the inhibitory molecule is the soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In one embodiment, the multiple myeloma cells are MM-1R cells.

[0143] In various other embodiments, CAR interacts with tumor and disease antigens including, but not limited to, BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0144] In some embodiments, 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-idiotype antibody or antibody fragment against CAR. In some embodiments, the inhibitory molecule is an anti-idiotype antibody or antibody fragment against the GPRC5D receptor. In a non-limiting example, the target cells are multiple myeloma cells. A non-limiting example of multiple myeloma cells is MM-1R cells.

[0145] In some embodiments, CAR interacts with the tumor antigen KLK2. In some embodiments, the KLK2 antigen is expressed by target cells. In some embodiments, the inhibitory molecule is the soluble KLK2 protein. In a non-limiting example, the target cells are prostate cancer cells. A non-limiting example of prostate cells is LNCaP cells.

[0146] In various embodiments, this method is performed in a high-throughput format.

[0147] host cell The inhibitory molecules described herein may be expressed in cells containing nucleic acid molecules, CAR polypeptide molecules, or vectors as described herein, for example, in immune effector cells (e.g., cell populations, e.g., immune effector cell populations). Immune effector cells may be, for example, T cells or NK cells. The inhibitory molecules can be expressed in various mammalian cell types (e.g., Chinese hamster ovary cells) and then purified before use in any of the assays described herein.

[0148] The CAR-T molecules described herein can be expressed in immunoeffector cells, such as T cells or NK cells. Immunoeffector cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll® isolation. Cells from the circulating blood of an individual can be obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction, and then the cells may be placed in a suitable buffer or medium for subsequent processing steps. Cells may be washed with phosphate-buffered saline (PBS). The washing solution may be calcium-deficient, magnesium-deficient, and / or all divalent cations-deficient.

[0149] The methods described herein may, for example, include the selection of a regulatory T cell depletion population, a CD25+ depletion population, which is a specific subpopulation of immune effector cells, such as T cells, using, for example, the negative selection technique described herein. Preferably, the regulatory T cell depletion population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells.

[0150] Efficacy assay This specification discloses a method for characterizing the efficacy of chimeric antigen receptor (CAR)-T cells (CAR-T cells). The method comprises (a) stimulating CAR-T cells in an antigen-specific manner (i.e., via the CAR of the CAR-T cell) and (b) determining the level of antigen-specific cytotoxicity of the stimulated cells.

[0151] In one embodiment, an in vitro method for determining the efficacy of immune cells expressing a chimeric antigen receptor (CAR) molecule, wherein the method is a) Incubating CAR-expressing immune cells with target cells (e.g., tumor cells) in a test sample, wherein the target cells express antigens that interact with CAR, b) Incubating CAR-expressing immune cells with target cells in a first control sample, wherein the incubation is carried out in the presence of an inhibitory molecule, the inhibitory molecule reduces, inhibits, blocks, and / or prevents the interaction between the CAR and the target cells. c) To determine the amount of interaction between CAR-expressing immune cells and target cells in the test sample, d) To determine the amount of interaction between CAR-expressing immune cells and target cells in the first control sample, e) Determining the potency of CAR-expressing immune cells based on comparing the amounts of interaction determined in steps (c) and (d), The present invention provides a method in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same in the test sample and the first control sample.

[0152] The interaction between CAR-expressing immune cells and target cells can be measured directly, for example, by evaluating the binding between CAR-expressing immune cells and target cells. Alternatively, the interaction can be measured indirectly, for example, by evaluating cell death, apoptosis, necrosis, cytokine release, and changes in cell morphology.

[0153] 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 amount of CAR-expressing immune cells is 85–115%, 90–110%, or 95–105% of the amount of target cells.

[0154] In some embodiments, incubation steps (a) and (b) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, 1 hour, 30 minutes, or 15 minutes. Performing steps (a) and (b) simultaneously can provide conditions under which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same. In some embodiments, determining steps (c) and (d) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, 1 hour, 30 minutes, or 15 minutes. Performing steps (c) and (d) simultaneously can provide conditions under which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same.

[0155] 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 target cell death determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

[0156] In some embodiments, the method further includes comparing the amount of interaction between CAR-expressing immune cells and target cells in step (c) with the amount of target cell death 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 surfactant that induces target cell death. In certain embodiments, the surfactant is TritonX-100.

[0157] In various embodiments, the target cells produce a detectable reporter signal upon death of the target cells, and step (c) includes determining the reporter signal in a test sample, step (d) includes determining the reporter signal in a first control sample, and step (e) includes comparing the reporter signals determined in steps (c) and (d). In some embodiments, the target cells express a reporter protein that produces a signal when the target cells interact with CAR-expressing immune cells.

[0158] In some embodiments, the reporter signal is luminescence. A protein capable of generating a reporter signal (e.g., luminescence) may be expressed in a cell compartment. Upon cell death, the protein is released from the cell compartment into the culture medium, and the protein may generate a luminescence signal by enzymatically acting on, for example, a drug to generate luminescence. An exemplary cell that may be used in this manner is KILR® target cells. Target cells can be engineered to express antigens that interact with chimeric antigen receptors. In certain embodiments, KILR® MM-1R multiple myeloma target cells are used. Target cells can also stably express labeled or enzyme-tagged proteins. When a target cell line is used in a cytotoxic assay and its membrane is damaged due to cell death, the target cell line may release tagged proteins into the culture medium. Tagged proteins can be detected by adding a reagent to the culture medium that is a substrate for the enzyme tag on the protein. For example, the β-galactosidase enzyme can hydrolyze the substrate to give a chemiluminescent output. The luminescence can be quantified on a plate reader capable of measuring chemiluminescence. Alternatively, tagged proteins can be detected via assays to detect the label.

[0159] In some embodiments, the reporter signal is fluorescence. A protein capable of generating a reporter signal (e.g., fluorescence) may be expressed in a cellular compartment. Upon cell death, the protein is released from the cellular compartment into the culture medium, where it can generate a fluorescent signal.

[0160] In some embodiments, the inhibitory molecule specifically binds to an antigen on a target cell (e.g., tumor cell) that interacts with the CAR. While we do not wish to be bound by theory, the use of an antigen-binding inhibitory molecule can provide a suitable control in CAR efficacy assays, eliminating the need to use untransfected or mock-transfected immune cells as controls. This eliminates the need to produce mock CAR-T cells in parallel with the formulation. The production of mock CAR-T cells can affect the production of the formulation in several ways. Eliminating the use of mock cell controls can simplify the manufacturing process and ensure that patient dosing can be achieved by reducing the sampling of any autologous CAR-T cells from patients, otherwise reducing the cost in CAR-T cell therapy. The method described herein can also reduce or eliminate test delays due to mock cell failure. The method can also reduce test errors by providing a simpler format. The reduction of test delays and errors can also provide the avoidance of production delays and / or patient dosing delays.

[0161] In some embodiments, the inhibitory molecule specifically binds to CARs. In some embodiments, the inhibitory molecule specifically binds to regions within the CAR that specifically bind to antigens expressed on target cells that interact with the CAR. By binding to the CAR, particularly to regions within the CAR that specifically bind to antigens (e.g., epitopes containing one or more CDR sequences or portions thereof), the inhibitory molecule can block CAR-T cells from interacting with target cells. Such blockage can prevent CAR-T cells from interacting with target cells. Therefore, instead of using mock-transfected immune cells, the patient's own CAR-T cells can be used, instead with the inhibitory molecule added as a suitable control.

[0162] In some embodiments, the inhibitory molecule is an antibody. In certain embodiments, the antibody is an anti-idiotype antibody. The anti-idiotype antibody can compete with the antigen on the host cell for binding to the chimeric antigen receptor. The anti-idiotype antibody may share some structural features with the antigen.

[0163] In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the scFv domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the Fab domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen in the VH or VL domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR in the VH or VL domain.

[0164] In some embodiments, the inhibitory molecule is a soluble form of an antigen expressed on target cells that interact with CAR, or a functional fragment or derivative thereof.

[0165] 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 the B cell maturation antigen (BCMA) receptor, the target cells contain the BCMA receptor, and the inhibitory molecule is the soluble cytoplasmic domain of BCMA. In some embodiments, the target cells are multiple myeloma cells. In one embodiment, the multiple myeloma cells are MM-1R cells.

[0166] In various other embodiments, CAR interacts with tumor and disease antigens including, but not limited to, BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0167] In some embodiments, 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-idiotype antibody or antibody fragment against CAR. In some embodiments, the inhibitory molecule is an anti-idiotype antibody or antibody fragment against the GPRC5D receptor. In a non-limiting example, the target cells are multiple myeloma cells. A non-limiting example of multiple myeloma cells is MM-1R cells.

[0168] In some embodiments, CAR interacts with the tumor antigen KLK2. In some embodiments, the KLK2 antigen is expressed by target cells. In some embodiments, the inhibitory molecule is the soluble KLK2 protein. In a non-limiting example, the target cells are prostate cancer cells. A non-limiting example of prostate cells is LNCaP cells.

[0169] Instead of using mock-transfected CAR-T cells as a control when determining the level of antigen-specific cytotoxicity, the same CAR-T cells tested in the assay can be treated with inhibitory molecules described herein. Treatment with inhibitory molecules (e.g., monoclonal antibodies or soluble antigens to which CARs specifically bind) can prevent CAR-T cells from binding to the antigen and exerting cytotoxicity. Stimulated CAR-T cells for therapeutic use can be indicated by detecting an increase in the level of cytotoxicity of antigen-specific stimulated cells compared to the level of cytotoxicity of the same CAR-T cells treated with the inhibitory molecule, or non-specifically stimulated CAR-T cells (i.e., stimulated CAR-T cells that are not antigen-specifically stimulated). This method can be performed in vitro.

[0170] In various embodiments, inhibitory molecules (e.g., tumor antigens or anti-idiotype antibodies) are not effective in stimulating CAR-T cells that can be stimulated by antigens (e.g., tumor antigens) that are specific to the CAR on the CAR-T cell. Such embodiments can offer the advantage of not activating the efficacy parameters resulting from the activation of CAR-T cells.

[0171] Methods for determining the potency and cytotoxic function of CAR-T cells are also provided. Generally, these methods involve antigen-specific stimulation of CARs on CAR-T cells and subsequent quantification of the cytotoxicity of antigen-specific CAR-T cells. Measures of CAR-T cell potency can be used as in vitro indicators of the expected in vivo pharmacokinetics of CAR-T cell therapy products. CAR-T potency assays can further be used to determine whether CAR-T cell products are suitable for clinical use, to assess the potential efficacy of CAR-T cell products, to determine the dosage of CAR-T cells to be administered, and / or to characterize novel manufacturing approaches for CAR-T cell therapy products.

[0172] The potency of a CAR-T cell therapy product can be expressed in terms that reflect the level of antigen-specific cytotoxicity of the product. This level of cytotoxicity can be compared, for example, to the level of cytotoxicity of a control sample of the CAR-T cell therapy product exposed to both antigen-specific stimuli and the inhibitory molecules described herein. Furthermore, the calculation can be normalized, for example, based on the number of cells in the test sample expressing CAR. Based on this information, a cytotoxicity index (CI) given by the following formula can be used as a measure of the potency of the CAR-T cell therapy product.

[0173] CI = [(Cytotoxicity in the stimulated group) - (Cytotoxicity in the control group)] / % of cells expressing CAR For normalization, the percentage of cells expressing CAR (e.g., the level of transduction) can be determined using methods known in the art. For example, in studies utilizing flow cytometry, an antibody against CAR can be included in the assay to quantify the level of CAR-expressing cells relative to the total number of T cells.

[0174] The level of antigen-specific in vitro cytotoxicity of a CAR-T cell therapy product may correlate with the in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties of the CAR-T product. PK / PD characteristics of CAR-T cell preparations that may be considered according to the present invention include, for example, Cmax, Tmax, and Area Under the Curve (AUC), which can be determined in clinical samples using standard methods in the art. For example, the relationship between the in vitro cytotoxicity of a CAR-T cell therapy product, as reflected in the above-mentioned cytotoxicity index, and the in vivo PK / PD properties of the product can be shown using, for example, standard methods such as the Spearman correlation coefficient method, which can be used to evaluate the linear relationship between these characteristics. Various methods described herein can provide criteria for predicting PK / PD parameters based on antigen-specific in vitro cytotoxicity, such as that indicated by the determination of CI.

[0175] kit Any of the compositions described herein can be incorporated into a kit. In some embodiments, a CAR-binding antibody is provided in the kit, which may also include reagents suitable for cell growth, such as culture media, APCs, growth factors, antigens, other antibodies (e.g., for sorting or characterizing CAR T cells) and / or plasmids encoding CARs or transposases.

[0176] In non-limiting examples, the kit provides a CAR-binding antibody, a chimeric receptor expression construct (or reagents for generating a chimeric receptor expression construct), reagents for transfection of the expression construct, and / or one or more instruments for obtaining allogeneic cells for transfection of the expression construct (such instruments may be syringes, pipettes, forceps, and / or any such medically approved device). In some embodiments, the kit includes reagents or equipment for electroporation of cells.

[0177] The kit may include, and may be provided in a kit, target cells expressing an antigen that specifically interacts with a CAR-binding antibody, reagents for transfection of an expression construct encoding the antigen, and / or one or more instruments for obtaining allogeneic cells for transfection of the expression construct (such instruments may be syringes, pipettes, forceps, and / or any such medically approved device). In some embodiments, the kit may include reagents or equipment for electroporation of cells.

[0178] The kit may comprise one or more preferably equally divided compositions of the present invention or reagents for producing the compositions of the present invention. The components of the kit may be packaged in either aqueous medium or lyophilized form. The container means of the kit may comprise at least one vial, test tube, flask, bottle, syringe, or other container means, which may contain the components, preferably preferably equally divided. If there are two or more components in the kit, the kit will also generally comprise a second, third, or other additional container in which additional components can be separately contained. However, various combinations of components may be contained in vials. The kit of the present invention will typically comprise means for containing the chimeric receptor construct and any other reagent containers sealed for commercial sale. Such containers may include, for example, injection-molded or blow-molded plastic containers that hold the desired vials.

[0179] Embodiment 1. An in vitro method for determining the efficacy of immune cells expressing chimeric antigen receptor (CAR) molecules, wherein the method is: a) In the test sample, CAR-expressing immune cells are brought into contact with target cells, and the target cells express antigens that interact with CARs. b) In a first control sample, CAR-expressing immune cells are brought into contact with target cells, wherein (i) the contact is made in the presence of an inhibitory molecule, or (ii) the CAR-expressing immune cells and / or target cells are pre-incubated with an inhibitory molecule before the contact, and the inhibitory molecule inhibits the interaction between the CAR and the target cells. c) To determine the amount of target cell death in the test sample, and d) To determine the amount of target cell death in the first control sample, e) Determining the potency of CAR-expressing immune cells based on a comparison of the amount of target cell death determined in steps (c) and (d), The present invention provides a method in which the contact time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same for the test sample and the first control sample.

[0180] 2. The method of Embodiment 1, wherein steps (a) and (b) of bringing into contact are performed simultaneously.

[0181] 3. The method of Embodiment 1 or Embodiment 2, wherein steps (c) and (d) for determining are performed simultaneously.

[0182] 4. The method according to any one of Embodiments 1 to 3, wherein in step (b)(i), CAR-expressing immune cells and / or target cells are pre-incubated with the inhibitory molecule before the contact step.

[0183] 5. The method according to any one of Embodiments 1 to 4, further comprising comparing the amount of target cell death determined in step (c) with the amount of target cell death determined in a second control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells.

[0184] 6. The method according to any one of Embodiments 1 to 5, further comprising comparing the amount of targeted cell death determined in step (c) with the amount of targeted cell death determined in a third control sample, wherein the target cells are incubated in the presence of a surfactant that induces targeted cell death but is in the absence of CAR-expressing immune cells.

[0185] 7. The method according to Embodiment 6, wherein the surfactant is TritonX-100.

[0186] 8. The method according to any one of Embodiments 1 to 7, wherein the target cells produce a detectable reporter signal upon the death of the target cells, step (c) comprises determining the reporter signal in a test sample, step (d) comprises determining the reporter signal in a first control sample, and step (e) comprises comparing the reporter signals determined in steps (c) and (d).

[0187] 9. The method according to Embodiment 8, wherein the reporter signal is luminescence.

[0188] 10. The method according to Embodiment 8, wherein the reporter signal is fluorescence.

[0189] 11. The method according to any one of Embodiments 8 to 10, wherein the target cell expresses a reporter protein that produces a signal when the target cell undergoes cell death.

[0190] 12. The method according to Embodiment 11, wherein the reporter protein is β-galactosidase, luciferase, green fluorescent protein (GFP), or a variant or derivative thereof.

[0191] 13. The method according to any one of Embodiments 1 to 12, wherein the inhibitory molecule specifically binds to an antigen on a target cell, and the antigen interacts with the CAR.

[0192] 14. The method according to any one of Embodiments 1 to 13, wherein the inhibitory molecule specifically binds to CAR.

[0193] 15. The method according to Embodiment 14, wherein the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on target cells.

[0194] 16. The method according to Embodiment 13, 14, or 15, wherein the inhibitory molecule is an antibody or an antibody fragment.

[0195] 17. The method according to Embodiment 16, wherein the antibody is an anti-idiotype antibody.

[0196] 18. The method according to Embodiment 16 or 17, wherein the antibody fragment is a Fab, Fab', F(ab'))2, Fv or Fd fragment, a single-chain antibody (scFv), a linear antibody, a single-domain antibody, a heavy chain variable region (VH) domain, or a light chain variable region (VL) domain.

[0197] 19. The method according to Embodiment 16, 17, or 18, wherein the antibody or antibody fragment specifically binds to an antigen within the scFv domain of a CAR.

[0198] 20. The method according to Embodiment 19, wherein the antibody or antibody fragment specifically binds to the complementarity-determining region (CDR) within the scFv domain of the CAR.

[0199] 21. The method according to Embodiment 16, 17, or 18, wherein the antibody or antibody fragment specifically binds to an antigen in the VH domain or VL domain of a CAR.

[0200] 22. The method according to Embodiment 21, wherein the antibody or antibody fragment specifically binds to a CDR within the VH domain or VL domain of the CAR.

[0201] 23. The method according to Embodiment 14 or 15, wherein the inhibitory molecule is a soluble form of an antigen expressed on target cells that interact with CAR, or a functional fragment or derivative thereof.

[0202] 24. The method according to any one of Embodiments 1 to 23, wherein the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells.

[0203] 25. The method according to any one of Embodiments 1 to 24, wherein CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cell contains a BCMA receptor, and the inhibitory molecule is the soluble cytoplasmic domain of BCMA.

[0204] 26. The method according to Embodiment 25, wherein the target cells are multiple myeloma cells.

[0205] 27. The method according to Embodiment 26, wherein the multiple myeloma cells are MM-1R cells.

[0206] 28. The method according to any one of Embodiments 1 to 24, wherein CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotype antibody or antibody fragment against CAR.

[0207] 29. The method according to any one of Embodiments 1 to 24, wherein CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotype antibody or antibody fragment against the GPRC5D receptor.

[0208] 30. The method according to Embodiment 28 or 29, wherein the target cells are multiple myeloma cells.

[0209] 31. The method according to Embodiment 30, wherein the multiple myeloma cells are human MM-1R cells.

[0210] 32. The method according to any one of Embodiments 1 to 24, wherein CAR interacts with kallikerin 2 (KLK2), the target cell contains KLK2, and the inhibitory molecule is a soluble KLK2 protein.

[0211] 33. The method according to Embodiment 32, wherein the target cells are prostate cancer cells.

[0212] 34. The method according to embodiment 33, wherein the prostate cancer cells are LNCaP cells.

[0213] 35. The method according to any one of Embodiments 1 to 34, wherein the method is performed in a high-throughput format. [Examples]

[0214] The present invention is also illustrated and demonstrated by the following embodiments. However, the use of these and other embodiments anywhere in this specification is merely illustrative and does not limit the scope and meaning of the invention, or the scope and meaning of any exemplary term. Similarly, the present invention is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the present invention will be apparent to those skilled in the art by reading this specification, and such variations may be made without departing in spirit or scope from the invention. Thus, the present invention is limited only by the appended claims and the entire scope of the equivalents to which these claims are granted.

[0215] (Example 1) The following materials were used in this example. CAR-T DP samples were used as test material, and KILR®MM1-R® reporter cells (catalog number 97-1045P052, Eurofins Discoverx Corp., Fremont, CA) were used as the target cell line. KILR®MM-1R® cells express enhanced Prolabel (ePL)-tagged housekeeping genes. When the cells are lysed, ePL-tagged proteins are released into the culture medium. Addition of an enzyme acceptor induces complementation of β-galactosidase enzyme fragments, EA, and ePL. The resulting functional enzyme hydrolyzes its substrate to generate a chemiluminescent signal.

[0216] KILR MM-1R® cells were grown in RPMI1640 (ATCC formulation) (Gibco catalog number A10491-01, ThermoFisher, Waltham, MA) containing 10% HI-FBS (6140-071, Life Technologies, Carlsbad, CA) and 250 μg / mL G418 sulfate (Corning catalog number 30-234-CR, ThermoFisher, Waltham, MA). The assay was performed in assay medium consisting of RPMI1640 containing L-glutamine and 25 mM HEPES (Corning catalog number 10-041-CV, ThermoFisher, Waltham, MA) and 10% HI-FBS (6140-071, Life Technologies, Carlsbad, CA).

[0217] A soluble human BCMA protein (sBCMA) (BCA-H522y, Acro Biosystems, Newark DE) blocking protein was incorporated into the assay medium. A total mortality control was prepared using a solution of 10% TritonX-100 (catalog number 93443-100ML, SigmaAldrich Co., St. Louis, MO). Cytotoxicity was detected using the KILR® Detection® kit (catalog number 97-001, Eurofins Discoverx Corp., Fremont, CA).

[0218] In this example, the ability of a CAR-T cell preparation (Drug Product, DP) to kill multiple myeloma target cells expressing the relevant antigen was measured. Target cells expressing a reporter gene that produces a measurable signal (e.g., luminescence) due to cell death when bound by effector CAR-T cells were used. The results were shown in activity measurements used to determine whether the DP demonstrated an appropriate level of activity for release.

[0219] The assay consisted of four components used in a total of 16 wells. The four components used were: (i) CAR-T formulation (DP) containing target cells (total activity), (ii) CAR-T DP cells blocked by a blocking reagent containing target cells (baseline control), (iii) target cells containing culture medium (non-cell death control), and (iv) target cells containing 0.1% TitonX-100 (total cell death control). All four assay components were run as four separate replicas, as shown in the plate layout in Table 1A below. A detailed description of the samples in the 96-well plate is shown in Table 1B. Six assays were performed using one 96-well plate, one of which was an assay of QC CAR-T cells used for system compatibility and trend method performance. The QC CAR-T cells were pre-qualified for activity.

[0220] [Table 1]

[0221] [Table 2]

[0222] The following assay conditions were used for LCAR-B38M CAR-T DP and KILR® MM-1R multiple myeloma target cells (Eurofins Discoverx Corp., Fremont, CA). The assay medium was RPMI1640 and 10% HI-FBS. KILR® MM-1R cells were grown according to the manufacturer's specifications. A baseline control was generated by blocking LCAR-B38M DP cells with the soluble cytoplasmic domain of B cell maturation antigen (sBCMA). The KILR® detection kit (Eurofins Discoverx Corp., Fremont CA) was used as the assay detection reagent. Optimization was performed on 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 formulation and its corresponding target cell line. Once established, the E:T ratio was fixed in the assay and remained fixed for all formulation tests. Blocking reagents were qualified lot by lot and used for formulation tests at that level. Furthermore, detection conditions could be optimized based on the reporter system used.

[0223] The LCAR-B38M CAR-T cell assay was performed in a 96-well white opaque TC-treated assay plate as follows. Each assay plate was designed to accommodate up to six assays, as shown in Table 1A above. For each assay, 25 μL of blocking reagent was added to the blocked CAR-T cell wells (baseline control) of the assay plate, followed by 25 μL of assay medium added to the CAR-T test wells. Next, 8 × 10⁶ cells were extracted. 5 25 μL of CAR-T DP cells (total 2 × 10⁶ cells) at a viable cell / mL. 4 Viable cells (for each well) were added to the CAR-T test wells and baseline control wells. 50 μL of assay medium was added to the wells containing only KILR MM-1R cells (no cell death). The contents of the wells were gently mixed and incubated at 37°C with 5% CO2 for 10 minutes (±5 minutes).

[0224] Once incubation is complete, 50 μL of 8 × 10 4 Add viable target cells / mL to all assay wells (finally 4 × 10 3 (Visible cells / well), the final E:T ratio was 5:1. In each assay, the final addition was 0.1% Triton X-100 (50 μL / well) to the total cell death control well. The well contents were incubated at 37°C with 5% CO2 for 22 hours (±1 hour). After co-culture incubation was complete, the assay plate was removed from the incubator and equilibrated at room temperature for 30 minutes (+ / - 5 minutes). The detection reagent was thawed and then simultaneously equilibrated at room temperature for 30 minutes. After equilibration, 100 μL of KILR® detection reagent was added to each well and incubated for 50 minutes (+ / - 5 minutes) while protected from light. After shaking for 10 seconds, the plate was read using a Molecular Devices Paradigm plate reader set for chemiluminescence.

[0225] The data are shown in Tables 2 and 3 below. The data in Table 2 show lot titrations of sBCMA protein demonstrating BCMA blockade of LCAR-B38M killing in KILR MM-1R multiple myeloma target cells in the range of 50–400 ug / mL. The data in Table 3 demonstrate the sBCMA blockade specificity for multiple myeloma target cells (RPMI8226_Luc) compared to non-B cell target cells (K562_luc cells).

[0226] [Table 3]

[0227] [Table 4]

[0228] (Example 2) GPRC5D CAR-T cells were tested using an anti-ID antibody against the CAR of CAR-T cells. The assay conditions for GPRC5D CAR-T cells were the same as those used for LCAR-B38M in Example 1 above. For GPRC5D CAR-T, another multiple myeloma target, the same KILR MM-1R target cells were used. The assay medium and detection reagents were the same as those used in Example 1. The E:T ratio of 5:1 and seeding density were the same as in Example 1. The blocking reagent was a GCPR5D anti-idiotype antibody against GPRC5D CAR. Examples of GCPR5D anti-idiotype antibodies for use in the assay include GP5B337, GP5B332, GP5B324, and GP5B206. The heavy and light chain sequences of these anti-idiotype antibodies are provided in Table 5. The incubation time was the same as that of Example 1 above. All other reagents were the same as those used in Example 1. The initial titration test shown in Table 4 below demonstrates the blockade of GPRC5D CAR-T cells by the anti-ID antibody (GP5B337).

[0229] [Table 5]

[0230] [Table 6]

[0231] (Example 3) GPRC5D CAR-T cells were tested using the anti-ID antibody Fab fragment against CAR in CAR-T cells. The assay conditions for GPRC5D CAR-T cells were the same as those used for LCAR-B38M in Example 1 above. For GPRC5D CAR-T, another multiple myeloma target, the same KILR MM-1R target cells were used. The assay medium and detection reagents were the same as those used in Examples 1 and 2. The E:T ratio of 5:1 and seeding density were the same as in Examples 1 and 2. The blocking reagent was the GCPR5D anti-idiotype Fab antibody (GP5B337) fragment against GPRC5D CAR. The incubation time was the same as in Examples 1 and 2 above. All other reagents were the same as those used in Examples 1 and 2. The initial titration test shown in Table 6 below demonstrates the blocking of GPRC5D CAR-T cells by the anti-ID Fab antibody (GP5B337) fragment.

[0232] [Table 7]

[0233] GPRC5D anti-idiotype Fab fragments were generated from full-length anti-idiotype antibodies against GPRC5D CAR using the Pierce Fab Preparation Kit (ThermoFisher, catalog number: VF299292) following a slightly modified kit procedure. Briefly, BupH phosphate-buffered saline (PBS) and digestion buffer were prepared. IgG was removed from the refrigerator, diluted in the prepared PBS, and passed through a desalting column. The eluate was collected and transferred to a prepared Papain Digestion Column for digestion at 37°C. After approximately 5 hours of digestion and rotation at 37°C, the tube was removed from the incubator and the sample was collected by spinning down. A Kit Protein A column was prepared, the sample was added, and the column was rotated overnight at 2–8°C for approximately 20 hours. The Protein A column was spun down and the sample in the fractions was collected. The first and second fractions were collected and stored together. Previous studies, such as A280 and 1D Silver Stains, have shown that these fractions contain the most desirable fragments. A fresh Protein A column was prepared (never previously used), and the sample was added again for a secondary rotation at 2–8°C lasting approximately 1 hour. A second Protein A incubation was added to further purify and remove any potential contamination of Fc regions that were thought to affect the bioassay. The fragments were eluted, concentrated in 10K Amicon tubes (catalog UFC501008), and stored at 2–8°C.

[0234] (Example 4) GPRC5D CAR-T cells are tested using an anti-GPRC5D antibody or its Fab fragment against the antigen (GPRC5D receptor) of CAR-T cells. The assay conditions for GPRC5D CAR-T cells are the same as those used for GPRC5D CAR-T DP in Example 2 above. For GPRC5D CAR-T, another multiple myeloma target, the same KILR MM-1R target cells are used. The assay medium and detection reagents are the same as those used in Examples 2 and 3. The E:T ratio of 5:1 and seeding density are the same as in Examples 2 and 3. The blocking reagent is an anti-GPRC5D antibody or its Fab antibody fragment. These blocking reagents bind to the GPRC5D receptor on KILR MM-1R target cells and inhibit the ability of CAR-T cells to bind to that target. The anti-GPRC 5D antibody Fab is generated from full-length anti-GPRC 5D antibody using the established method detailed above. The incubation time is consistent with that of Examples 2 and 3. All other reagents are the same as those used in Examples 2 and 3. The initial titration tests shown in Table 7 below demonstrate the blockade of KILR MM-1R cells by anti-GPRC5D antibody or Fab fragment.

[0235] [Table 8]

[0236] (Example 5) KLK2 CAR-T cells were tested against the CAR of CAR-T cells using human soluble KLK2 protein. The assay conditions for KLK2 CAR-T cells were the same as those used for LCAR-B38M in Example 1 above. KLK2 CAR-T cells target kallikerin 2 (KLK2), a molecule expressed in malignant luminal prostate gland. Overexpressed KLK2 reporter cell lines were prepared using LNcaP cells (ATCC, CRL1740) and DiscoverX's Killing Immune-Lysis Reaction (KILR) reporter. The killing principle was the same as described in Example 1. These cells expressed enhanced Prolabel (ePL) tagged housekeeping genes, and upon lysis, the tagged reporter protein was released into the culture medium. Addition of enzyme acceptors induced complementation of β-galactosidase enzyme fragments, EA, and ePL. The obtained functional enzyme hydrolyzed its substrate to generate a chemiluminescent signal. The assay medium, detection reagent, and target seeding density were all the same as those used in Example 1, but the E:T ratio was 10:1. The blocking reagent was a protein soluble to KLK2 CAR. The amino acid sequence (SEQ ID NO: 12) of this soluble KLK2 protein with a C-terminal His6 tag is provided below (the underlined sequence is the signal peptide):

[0237] JPEG0007936735000009.jpg29152

[0238] KILR LNcaP-KLK2 cells were grown in RPMI1640 (Gibco catalog number 11875-093, ThermoFisher, Waltham, MA) containing 10% FBS (97068-085, VWR, Radnor, PA) and 250 μg / mL G418 sulfate (Corning catalog number 30-234-CR, Corning, Tewksbury, MA). The assay was performed in assay medium consisting of RPMI1640 containing L-glutamine (Corning catalog number 10-040-CV, Corning, Tewksbury, MA) and 10% FBS (97068-085, VWR, Radnor, PA).

[0239] Although the target cell line in this example was different from that in Examples 1 and 2, the strategy and setup were the same as in Examples 1 and 2.

[0240] The following assay conditions were used for KLK2 CAR-T DP 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, 1× non-essential amino acids, 2.5 ug / mL puromycin, and 500 ug / mL sulfate G418. Baseline control and assay optimization followed Example 1. Baseline control was generated by blocking KLK2 CAR-T DP cells with soluble human KLK2 protein (sKLK2). The KILR® detection kit (Eurofins Discoverx Corp., Fremont CA) was used as the assay detection reagent. Optimization was performed on 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 (DP) and their target cells. The E:T ratio and blocking reagent concentration were optimized for each formulation and its corresponding target cell line. Once established, the E:T ratio was fixed in the assay and remained fixed for all formulation tests. Blocking reagents were qualified lot by lot and used at that level for formulation tests. Furthermore, detection conditions were optimized based on the reporter system used.

[0241] The KLK2 CAR-T formulation assay was performed in a 96-well white opaque TC-treated assay plate, as in Example 1. The assay steps were as follows, and each assay plate was compatible with up to six assays, as described in Example 1 in Table 1A. For each assay, 25 μL of blocking reagent was added to the blocked CAR-T cell wells (baseline control) of the assay plate, followed by 25 μL of assay medium added to the CAR-T test wells. Next, 1.6 × 10⁻⁶ 6 25 μL of CAR-T DP cells (total 4 × 10⁶ cells) at a viable cell / mL. 4Viable cells (for each well) were added to the CAR-T test wells and baseline control wells. 50 μL of assay medium was added to the wells containing only KILR LNcaP-KLK2 cells (no cell death). The contents of the wells were gently mixed and incubated at 37°C with 5% CO2 for 15 minutes (±5 minutes).

[0242] Once incubation is complete, 50 μL of 8 × 10 4 Add viable target cells / mL to all assay wells (finally 4 × 10 3 The final E:T ratio was 10:1 (viable cells / well). The contents of the wells were incubated at 37°C with 5% CO2 for 20 hours (±2 hours). After co-culture incubation was complete, the assay plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes (+ / - 5 minutes). The detection reagents were thawed and then simultaneously allowed to equilibrate at room temperature for 30 minutes. In each assay, after equilibration, 0.1% Triton X-100 (50 μL / well) was added to the total cell death control well, followed by 100 μL of KILR® detection reagent in each well, and incubated for 50 minutes (+ / - 5 minutes) while protected from light. After shaking for 10 seconds, the plates were read using a Molecular Devices Paradigm plate reader set for chemiluminescence.

[0243] The data are shown in Table 8 below. The data show lot titrations of sKLK2 protein demonstrating KLK2 blockade in KLK2 CAR-T DP killing in KILR LNcaP-KLK2 target cells in the range of 10–500 ug / mL.

[0244] The incubation time was the same as that used in Example 1 above. All other reagents were the same as those used in Example 1. The initial titration test shown in Table 8 below demonstrates the blockade of KLK2 CAR-T cells by KLK2 soluble protein (Internal, KL2W12.009).

[0245] [Table 9] * * *

[0246] The present invention is not limited to the scope defined by the specific embodiments and examples described in the present specification. In fact, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, it should be understood that all values are approximate and are provided for illustrative purposes.

[0247] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. Various embodiments of the present invention are shown below. 1. An in vitro method for determining the efficacy of immune cells expressing chimeric antigen receptor (CAR) molecules, wherein the method is: a) In a test sample, the CAR-expressing immune cells are brought into contact with target cells, wherein the target cells express antigens that interact with the CAR, b) In a first control sample, the CAR-expressing immune cells are brought into contact with the target cells, wherein (i) the contact is performed in the presence of an inhibitory molecule, or (ii) the CAR-expressing immune cells and / or the target cells are pre-incubated with the inhibitory molecule before the contact, and the inhibitory molecule inhibits the interaction between the CAR and the target cells. c) To determine the amount of target cell death in the test sample, d) To determine the amount of target cell death in the first control sample, e) Determining the potency of CAR-expressing immune cells based on a comparison of the amount of target cell death determined in steps (c) and (d), A method wherein the contact time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same for the test sample and the first control sample. 2. The method according to 1 above, wherein steps (a) and (b) of bringing the two into contact are performed simultaneously. 3. The method according to 1 above, wherein steps (c) and (d) for determining are performed simultaneously. 4. The method according to 1, wherein in step (b)(i), the CAR-expressing immune cells and / or the target cells are pre-incubated with the inhibitory molecule before the contact step. 5. The method according to 1, further comprising comparing the amount of target cell death determined in step (c) with the amount of target cell death determined in a second control sample, wherein the target cells are incubated in the absence of the CAR-expressing immune cells. 6. The method according to 1, further comprising comparing the amount of target cell death determined in step (c) with the amount of target cell death determined in a third control sample, wherein the target cells are incubated in the presence of a surfactant that induces target cell death but in the absence of CAR-expressing immune cells. 7. The method according to 6 above, wherein the surfactant is TritonX-100. 8. The method according to 1, wherein the target cells produce a reporter signal detectable upon the death of the target cells, step (c) comprises determining the reporter signal in the test sample, step (d) comprises determining the reporter signal in the first control sample, and step (e) comprises comparing the reporter signals determined in steps (c) and (d). 9. The method according to 8 above, wherein the reporter signal is light emission. 10. The method according to 8 above, wherein the reporter signal is fluorescence. 11. The method according to 8 above, wherein the target cells express a reporter protein that produces a signal when the target cells undergo cell death. 12. The method according to 11, wherein the reporter protein is β-galactosidase, luciferase, green fluorescent protein (GFP), or a variant or derivative thereof. 13. The method according to 1, wherein the inhibitory molecule specifically binds to the antigen on the target cell, and the antigen interacts with the CAR. 14. The method according to 1 above, wherein the inhibitory molecule specifically binds to the CAR. 15. The method according to 14, wherein the inhibitory molecule specifically binds to a region within the CAR that specifically binds to the antigen expressed on the target cell. 16. The method according to 13 above, wherein the inhibitory molecule is an antibody or an antibody fragment. 17. The method according to 16 above, wherein the antibody is an anti-idiotype antibody. 18. The antibody fragment is Fab, Fab', F(ab') 2 The method according to 16 above, wherein the antibody is an Fv or Fd fragment, a single-chain antibody (scFv), a linear antibody, a single-domain antibody, a heavy chain variable region (VH) domain, or a light chain variable region (VL) domain. 19. The method according to 16, wherein the antibody or antibody fragment specifically binds to the antigen in the scFv domain of the CAR. 20. The method according to 19, wherein the antibody or antibody fragment specifically binds to the complementarity-determining region (CDR) within the scFv domain of the CAR. 21. The method according to 16, wherein the antibody or antibody fragment specifically binds to an antigen in the VH domain or VL domain of the CAR. 22. The method according to 21, wherein the antibody or antibody fragment specifically binds to the CDR within the VH domain or VL domain of the CAR. 23. The method according to 14, wherein the inhibitory molecule is a soluble form of an antigen expressed on the target cell that interacts with the CAR, or a functional fragment or derivative thereof. 24. The method according to 1 above, wherein the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells. 25. The method according to claim 1, wherein the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cell contains the BCMA receptor, and the inhibitory molecule is the soluble cytoplasmic domain of BCMA. 26. The method according to 25 above, wherein the target cells are multiple myeloma cells. 27. The method according to 26 above, wherein the multiple myeloma cells are MM-1R cells. 28. The method according to claim 1, wherein the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotype antibody or antibody fragment against the CAR. 29. The method according to claim 1, wherein the CAR interacts with a G protein-coupled receptor, class C group 5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotype antibody or antibody fragment against the GPRC5D receptor. 30. The method according to 28 above, wherein the target cells are multiple myeloma cells. 31. The method according to 30 above, wherein the multiple myeloma cells are MM-1R cells. 32. The method according to claim 1, wherein the CAR interacts with kallikerin 2 (KLK2), the target cell contains KLK2, and the inhibitory molecule is a soluble KLK2 protein. 33. The method according to 32 above, wherein the target cells are prostate cancer cells. 34. The method according to 33 above, wherein the prostate cancer cells are LNCaP cells. 35. The method according to item 1 above, wherein the method is carried out in a high-throughput format.

Claims

1. An in vitro method for determining the efficacy of a chimeric antigen receptor (CAR) T cell preparation, wherein the method is a) In a test sample, the CART cell preparation is brought into contact with multiple myeloma target cells, wherein the multiple myeloma target cells express an antigen including a B cell maturation antigen (BCMA) receptor that interacts with the CART cell preparation. b) In a first control sample, the CART cell preparation is brought into contact with the multiple myeloma target cells, wherein (i) the contact is performed in the presence of an inhibitory molecule at a concentration of 50 to 400 μg / mL, or (ii) the CART cell preparation and / or the multiple myeloma target cells are pre-incubated with the inhibitory molecule at a concentration of 50 to 400 μg / mL prior to the contact, wherein the inhibitory molecule consists of a soluble domain of BCMA and inhibits the interaction between the CART cell preparation and the multiple myeloma target cells. c) To determine the amount of multiple myeloma target cell death in the test sample, d) To determine the amount of multiple myeloma target cell death in the first control sample, e) Determining the efficacy of the CART cell preparation based on a comparison of the amount of multiple myeloma target cell death determined in steps (c) and (d), A method wherein the contact time, the amount of the CART cell preparation, and the amount of the multiple myeloma target cells are substantially the same for the test sample and the first control sample.

2. The method according to claim 1, wherein steps (a) and (b) of bringing the objects into contact are performed simultaneously.

3. The method according to claim 1, wherein steps (c) and (d) of determining are performed simultaneously.

4. The method according to claim 1, wherein in step (b)(i), the CART cell preparation and / or the multiple myeloma target cells are pre-incubated with the inhibitory molecule at a concentration of 50 to 400 μg / mL prior to the contact step.

5. The method according to claim 1, further comprising comparing the amount of multiple myeloma target cell death determined in step (c) with the amount of multiple myeloma target cell death determined in a second control sample, wherein the multiple myeloma target cells are incubated in the absence of the CART cell preparation.

6. The method according to claim 1, further comprising comparing the amount of multiple myeloma target cell death determined in step (c) with the amount of multiple myeloma target cell death determined in a third control sample, wherein the multiple myeloma target cells are incubated in the absence of the CART cell preparation but in the presence of a surfactant that induces multiple myeloma target cell death.

7. The method according to claim 6, wherein the surfactant is Triton X-100.

8. The method according to claim 1, wherein the multiple myeloma target cells produce a reporter signal detectable upon death of the multiple myeloma target cells, step (c) comprises determining the reporter signal in the test sample, step (d) comprises determining the reporter signal in the first control sample, and step (e) comprises comparing the reporter signals determined in steps (c) and (d).

9. The method according to claim 8, wherein the reporter signal is light emission.

10. The method according to claim 8, wherein the reporter signal is fluorescence.

11. The method according to claim 8, wherein the multiple myeloma target cells express a reporter protein that produces a signal when the multiple myeloma target cells undergo cell death.

12. The method according to claim 11, wherein the reporter protein is β-galactosidase, luciferase, green fluorescent protein (GFP), or a variant or derivative thereof.

13. The method according to claim 1, wherein the inhibitory molecule specifically binds to the CART cell preparation.

14. The method according to claim 13, wherein the inhibitory molecule specifically binds to a region of the CART cell preparation that specifically binds to the antigen expressed on the multiple myeloma target cells.

15. The method according to claim 1, wherein the multiple myeloma target cells are MM-1R cells.

16. The method according to claim 1, wherein the method is performed in a high-throughput format.

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