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

Abstract

The present disclosure relates to cell-based assays for determining in vitro tumor killing activity of immune cells expressing chimeric antigens. The present disclosure provides an in vitro method for determining the potency (e.g., cytotoxicity) of an immune cell expressing a chimeric antigen receptor (CAR) molecule. In a test sample, immune cells that express the CAR are incubated with target cells that express antigens that interact with the CAR. In a control sample, an immune cell expressing the CAR is incubated with a target cell and an inhibitory molecule that prevents interaction between the CAR and the target cell. The amount of target cell death in both the test sample and the control sample is determined and compared.

Description

[0001] This application was filed on June 7, 2021, with application number 202180049139.3. (PCT / IB2021 / 054996), a divisional application of the invention patent entitled "Cell-based assay for determining the in vitro tumor-killing activity of immune cells expressing chimeric antigens".

[0002] Cross-reference to related applications This application claims the benefits of U.S. Provisional Application Serial No. 63 / 036,249, filed June 8, 2020, and U.S. Provisional Application Serial No. 63 / 125,173, filed December 14, 2020. The entire contents of the above applications are incorporated herein by reference. Technical Field

[0003] This invention provides improved assays for determining the potency (e.g., cytotoxicity) of immune cells expressing chimeric antigen receptors. These improved assays allow for the avoidance of using simulated transfected immune cells as assay controls, 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 measuring the specific in vitro cytotoxicity of T cells expressing chimeric antigen receptors (CAR-T cells) involve using autologous, untransduced expanded T cells (mimic cells) as a baseline control. These controls are used to calculate the specific cytotoxicity of transduced CAR-T cells. However, generating untransduced expanded autologous or allogeneic control T cells (mimic cells) is both expensive and time-consuming, especially since these cells are usually generated from the patient's own T cells. Furthermore, the production of these mimic cells is prone to falling short of the required yield, which may delay treatment or hinder the appropriate administration of CAR-T cells during immunotherapy.

[0005] Methods for measuring the in vitro cytotoxicity of CAR-T cells involve using autologous, untransduced expanded T cells (mimic cells) as a baseline control. The baseline control is used to calculate the percentage increase in CAR-T cell-specific cytotoxicity (CAR-T killing %). If autologous mimic cells are unavailable, qualified batches of allergen mimic cells are used instead. However, using these qualified batches results in potency relative to allogeneic mimic cells and may therefore fail to reflect the true potency of CAR-T cells. Alternatively, this baseline control is omitted, and total cytotoxic activity is used. However, total cytotoxic activity does not indicate whether the cytotoxic activity of the immune cell / target cell interaction has been enhanced due to CAR-T cells. Total cytotoxic activity cannot distinguish between the contribution of the drug product and the spontaneous death of the target cells themselves. Alternative assays (such as cytokine ELISA) have been used as alternatives to functional assays for measuring activity, but these methods do not directly measure cytotoxicity.

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

[0007] In one aspect, an in vitro method is provided for determining the potency of immune cells expressing chimeric antigen receptor (CAR) molecules, the method comprising: a) In the test sample, 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, CAR-expressing immune cells are contacted with target cells, wherein (i) the contact is performed in the presence of an inhibitory molecule, or (ii) prior to the contact, the CAR-expressing immune cells and / or target cells have been pre-incubated with an inhibitory molecule, wherein the inhibitory molecule inhibits the interaction between the CAR and the target cells. c) Measure the amount of target cell death in the test sample. d) Determine the amount of target cell death in the first control sample, and e) Determine the potency of CAR-expressing immune cells based on a comparison of the amount of target cell death measured in steps (c) and (d). In the test sample and the first control sample, the contact time, the amount of CAR-expressing immune cells, and the amount of target cells were basically the same.

[0008] In some implementations, contact steps (a) and (b) are performed simultaneously. In some implementations, measurement steps (c) and (d) are performed simultaneously.

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

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

[0011] In some embodiments, the method further includes comparing the amount of target cell death measured in step (c) with the amount of target cell death measured in a third control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that induces target cell death. In some embodiments, the detergent is Triton X-100.

[0012] In various embodiments, the target cells generate a detectable report signal upon death, and step (c) includes measuring the report signal in the test sample, step (d) includes measuring the report signal in a first control sample, and step (e) includes comparing the report signals measured 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 generates 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 variants or derivatives thereof. In some embodiments, the inhibitory molecule specifically binds to the antigen on the target cell that interacts 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 a target cell. In some embodiments, the inhibitory molecule is an antibody or antibody fragment. In some 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 the antigen within the scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the scFv domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the VH or VL domain of the CAR. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the VH or VL domain of the CAR. In some implementations, the inhibitory molecule is a soluble form of an antigen that interacts with the CAR and is expressed on the target cell, 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 cell contains 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 some embodiments, the multiple myeloma cells are MM-1R cells.

[0016] In some embodiments, the CAR interacts with G protein-coupled receptor class C5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the CAR. In some embodiments, the target cell is a multiple myeloma cell. In some embodiments, the multiple myeloma cell is an MM-1R cell.

[0017] In some embodiments, the CAR interacts with kallikerin 2 (KLK2), the target cells contain KLK2, and the inhibitory molecule is a 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 implementations, this method is performed in a high-throughput manner. Attached Figure Description

[0019] Figure 1A Flow cytometry results are presented, demonstrating BCMA-specific competition of labeled BCMA protein on the surface of LCAR-B38M CAR-T cells. Sample 1 was labeled with FITC-BCMA only.

[0020] Figure 1B Flow cytometry results are presented, demonstrating that labeled BCMA protein specifically competes with BCMA on the surface of LCAR-B38M CAR-T cells. Sample 6S competes with unlabeled BCMA for FITC-BCMA. Detailed Implementation

[0021] The methods disclosed herein can be more readily understood by referring to the following detailed descriptions in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the methods disclosed herein are not limited to the specific methods described and / or shown herein, and the terminology used herein is for illustrative purposes only and is not intended to limit the methods protected by the claims.

[0022] All patents, published patent applications and publications cited in this article are incorporated herein by reference as if fully described herein.

[0023] When a list is provided, unless otherwise indicated, it should be understood that each individual element in the list and each combination of the list is a separate implementation. For example, a list of implementations presented as “A, B or C” will be understood to include implementations “A”, “B”, “C”, “A or B”, “A or C”, “B or C”, or “A, B or C”.

[0024] definition As used herein, “an” or “a” may refer to one or more. As used herein in the claims, the word “an” or “a” when used with the word “comprising” may mean one or more, or one or more kinds.

[0025] The term “or” as used in the claims is used to mean “and / or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although this disclosure supports referring only to alternatives and the definition of “and / or”. As used herein, “another” may mean at least a second or more.

[0026] "About" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement method, result, or implementation, unless otherwise expressly stated in the embodiments or specification, "about" means a range of 10% below to 10% above the value, for example, in the range of 90 to 110 if the value is 100.

[0027] As used herein, the term “encoding” in relation to nucleic acids is used to make the invention readily understood by those skilled in the art; however, these terms may be used interchangeably with “comprising” or “including”, respectively.

[0028] "Antigen" refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, parts thereof, or combinations thereof) capable of binding by an antigen-binding domain or a T-cell receptor capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens can be genetically expressed, synthetic, or purified from biological samples such as tissue samples, tumor samples, cells or fluids containing other biological components, organisms, protein / antigen subunits, cytotoxic or inactivated whole cells, or lysate products.

[0029] "Antibody" broadly refers to and includes immunoglobulin molecules, specifically including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), dimer, tetramer, or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of immunoglobulin molecules containing an antigen-binding site with desired specificity. A "full-length antibody" comprises two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, as well as their polymers (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (composed 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 subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be designated into five major classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domain, antibody light chains of any vertebrate species can be designated into two completely different types, κ and λ.

[0030] The term "antibody fragment" refers to at least a portion of a complete antibody or a recombinant variant thereof that retains the antigen-binding properties of the full-length parent antibody. It refers, for example, an antigen-binding domain, such as the antigen-determining variable region of a complete antibody, sufficient to confer recognition and binding, for example, the specific binding of an 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 such as sdAb (VL or VH), the Camelidae VHH domain, and multispecific antibodies formed from antibody fragments.

[0031] The term "subject" is intended to include any living organism (e.g., a mammal, such as a human) in which an immune response can be elicited. 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, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0032] As used in this article, a "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, which, in their natural state, do not coexist on a single protein. This includes receptors in which the extracellular and intracellular signaling domains do not naturally coexist on a single receptor protein. CARs are primarily intended for use on lymphocytes, such as T cells and natural killer (NK) cells.

[0033] The complementarity-determining region (CDR) is the antibody region that binds to the antigen. There are three CDRs (HCDR1, HCDR2, HCDR3) in VH and three CDRs (LCDR1, LCDR2, LCDR3) in VL. CDRs can be defined using various descriptions, such as Kabat (Wu et al., (1970) J Exp Med 132: 211-50; Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27: 55-77), and AbM (Martin and Thornton, J Bmol Biol 263: 800-15, 1996). The correspondence between various depictions and variable region numbers 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; International Immunogenetics (IMGT) Database; Web resource, http: / / www_imgt_org). Available programs (such as abYsis for UCL Business PLC) can be used to depict CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” include CDRs as defined by any of the above methods (Kabat, Chothia, IMGT, or AbM).

[0034] The terms “reduction” and “decrease” are used interchangeably herein and generally refer to a weakened response (i.e., a downstream effect) mediated by the test molecule compared to a response mediated by a control or mediator. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of proteins to their antigens or receptors, enhanced binding to Fcγ, or enhanced Fc effector function, such as enhanced ADCC, CDC, and / or ADCP. A decrease can be a statistically significant difference in the response measured between the test molecule and the control (or mediator), or a decrease in the measured response, such as 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, or such as 500, 600, 700, 800, 900, or 1000 times or more (including all integers and decimals between these values ​​and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0035] The terms “enhancement,” “promotion,” “increase,” “amplification,” or “improvement” generally refer to the ability of a test molecule to mediate a stronger response (i.e., a downstream effect) compared to a response mediated by a control or mediator. Exemplary responses include T cell amplification, T cell activation, or T cell-mediated tumor cell killing, or the binding of proteins to their antigens or receptors, enhanced binding to Fcγ, or enhanced Fc effector function, such as enhanced ADCC, CDC, and / or ADCP. Enhancement can be a statistically significant difference in the response measured between the test molecule and a control (or mediator), or an increase in the measured response, such as an increase of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, or such as 500, 600, 700, 800, 900, or 1000 times or more (including all integers and decimals between these values ​​and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0036] “dAb” or “dAb fragment” refers to an antibody fragment composed of VH domains (Ward et al., Nature 341:544546 (1989)).

[0037] "Fab" or "Fab fragment" refers to an antibody fragment composed of the VH domain, CH1 domain, VL domain, and CL domain.

[0038] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.

[0039] "Fd" or "Fd fragment" refers to an antibody fragment composed of a VH domain and a CH1 domain.

[0040] "Fv" or "Fv fragment" refers to an antibody fragment consisting of the VH and VL domains of a single arm of the antibody.

[0041] A full-length antibody consists of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds and their polymers (e.g., IgM). Each heavy chain comprises a variable domain (VH) and a constant domain, the constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain comprises a variable domain (VL) and a constant domain (CL). VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and framework regions (FRs) interspersed therebetween. Each VH and VL consists of three CDRs and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0042] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one frame is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the frame, such that the frame is not an exact copy of the expressed human immunoglobulin or the germline gene sequence of a human immunoglobulin.

[0043] "Intracellular signal transduction domain" or "cytoplasmic signal transduction domain" refers to the intracellular portion of a molecule. This functional portion of a protein functions by transmitting information within the cell to regulate cellular activity via defined signal transduction pathways by generating second messengers, or to act as an effector in response to such messengers. Intracellular signal transduction domains generate signals that promote the function of immune effectors in CAR-containing cells (e.g., CAR-T cells).

[0044] "Isolated" refers to a homogeneous group of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially isolated and / or purified from other components of the system that produces the molecules (such as recombinant cells), as well as proteins that have undergone at least one purification or isolation step. "Isolated" also refers to molecules that are substantially free of other cellular material and / or chemicals, and encompasses molecules isolated to higher purities (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity).

[0045] "Monoclonal antibody" refers to antibodies obtained from a substantially homogeneous population of antibody molecules; that is, the individual antibodies constituting the population are identical except for possibly well-known alterations (such as removal of a C-terminal lysine from the antibody heavy chain) or post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation). Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific or multispecific, such as bispecific, monovalent, divalent, or multivalent.

[0046] The terms "natural killer cells" and "NK cells" are used interchangeably and synonymously in this article. NK cells refer to cells with CD16+. + CD56 + and / or CD57 + TCR - Differentiated lymphocytes are phenotypes. NK cells are characterized by their ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific lysinases, to kill tumor cells or other diseased cells that express ligands of NK activation receptors, and to release protein molecules called cytokines that stimulate or inhibit immune responses.

[0047] The terms "protein" and "peptide" are used interchangeably herein and refer to a molecule comprising one or more polypeptides, each of which contains at least two amino acid residues linked by peptide bonds. A protein can be a monomer or a protein complex of two or more subunits, which may be identical or different. Small polypeptides of fewer than 50 amino acids may be referred to as "peptides." Proteins can be heterologous fusion proteins, glycoproteins, or proteins modified through post-translational modifications such as phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, PEGylation, or biotinylation. Proteins may be recombinantly expressed.

[0048] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, created, or isolated through recombinant means. The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by phage or yeast expression systems. This term should also be understood to mean antibodies produced by synthesizing a DNA molecule encoding and expressing the antibody protein, or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing technologies available and known in the art.

[0049] "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 peptide linker and are expressible as a single-chain polypeptide. Unless otherwise specified, as used herein, scFv may have VL and VH variable regions in any order; for example, the scFv may comprise VL-linker-VH or VH-linker-VL relative to the N-terminus and C-terminus of the polypeptide.

[0050] "Specific binding" or "binding" refers to the binding of protein molecules to antigens or epitopes within antigens with a greater affinity than to other antigens. Typically, the binding of a protein molecule to an antigen or epitope within an antigen occurs at an equilibrium dissociation constant (K). D (Approximately 1×10) -7 M or lower, for example, about 5×10 -8 M or lower, approximately 1×10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, approximately 1×10 -11 M or lower or about 1×10 -12 M or lower, usually K D Compared to its binding to non-specific antigens (such as BSA, casein), K D At least a hundred times lower.

[0051] “T cells” and “T lymphocytes” are interchangeable and are used in this document with the same meaning. “T cells” include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+). + T cells, CD4 + T cells, cytotoxic T cells (CTLs; CD8) + T cells), tumor-infiltrating cytotoxic T cells (TILs; CD8) + T cells, CD4 + CD8 + T cells, or any other subset of T cells. This also includes "NKT cells," a specialized population of T cells that express the semi-invariant αβ T cell receptor but also express multiple molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1. + Cells and NK1.1 - Cells, and CD4 + Cells, CD4- Cells, CD8 + Cells and CD8 - The unique feature of the TCR on NKT cells is that it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Because NKT cells can produce cytokines that promote inflammation or immune tolerance, they can have protective or destructive effects. Also included are "gamma-delta T cells (γδ T cells)," a specialized subset of T cells with unique TCRs on their surface. Unlike most T cells, where the TCR is composed of two glycoprotein chains named α-TCR and β-TCR, the TCR in γδ T cells consists of both γ- and δ-chains. γδ T cells can play a role in immune surveillance and immune regulation and have been found to be an important source of IL-17 and induce potent CD8+. + Cytotoxic T cell responses. This also includes "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 transcription factor-positive cells. + T cells, and may also include Foxp3-negative regulatory T cells, which are CD4+ cells that produce IL-10. + T cells.

[0052] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells, whether in vivo, in vitro, or in tissue culture, exhibiting spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. However, transformation can occur due to infection with transforming viruses and the binding of new genomic nucleic acids, the uptake of exogenous nucleic acids, or it can occur spontaneously or after exposure to carcinogens, resulting in mutations in endogenous genes. Examples of transformation / cancer include morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, regulation of tumor-specific marker levels, invasion, and tumor growth in suitable animal hosts (such as nude mice), both in vitro and in vivo.

[0053] "Variant", "mutant" or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide due to one or more modifications (e.g., one or more substitutions, insertions or deletions).

[0054] As mentioned herein, the “potency” of a cell (e.g., CAR-T cells) is an indicator or measure of its ability or potential to achieve a desired function. In the case of CAR-T cells, the desired function may be targeting or killing another cell, such as a target cell (e.g., a tumor cell). Potency can be directly assessed by measuring the cell’s effect on its target (e.g., the effect of CAR-T cells on tumor cells in vitro or in vivo). Alternatively, as in the various methods of the present invention, potency can be measured indirectly. In particular, the potency of CAR-T cells can be assessed by measuring the cell’s antigen-specific cytotoxicity level in vitro (relative to the cytotoxicity of, for example, unstimulated CAR-T cells as described herein) in an assay (e.g., an assay as described herein). This measure of potency can then be correlated with the cell’s in vivo properties, and thus can be considered a predictor of the cell’s in vivo properties, such as PK / PD parameters (e.g., CMAX, TAX, and AUC) as described herein, which can be correlated with the cell’s effectiveness in killing its target. As further described herein, potency can be expressed as a cytotoxicity index, which can be normalized based on the number of cells expressing the relevant CAR.

[0055] As used herein, "reference" or "control" describes a standard or reference substance to which comparison is made. For example, in some embodiments, a reagent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control reagent, animal, individual, population, sample, sequence, or value. In some embodiments, the test and / or determination of the reference or control is performed substantially simultaneously with the test or determination of interest. Typically, as those skilled in the art will understand, the reference or control is determined or characterized under conditions or circumstances equivalent to the evaluation conditions or situation. Those skilled in the art will know when sufficient similarity exists to justify reliance on and / or comparison with a particular possible reference or control.

[0056] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that regulates immune cell activation in a stimulatory manner targeting at least some aspects of immune cell signaling pathways. In one aspect, this signal is a primary signal triggered by, for example, the binding of a TCR / CD3 complex to a peptide-carrying MHC molecule, leading to the mediation of T cell responses, including but not limited to proliferation, activation, and differentiation. The stimulatory primary cytoplasmic signaling sequence (also called a "primary signaling domain") may contain a signaling motif referred to as an immune receptor tyrosine-based activation motif, or ITAM. Examples of cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from: CD3ζ, ordinary FcRγ (FCER1 G), FcγR1a, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In CAR, intracellular signal transduction domains may include intracellular signal transduction sequences, such as the primary signal transduction sequence CD3-ζ.

[0057] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments point to preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0058] Chimeric antigen receptor Immune cells (e.g., T cells) can be genetically modified to stably express desired chimeric antigen receptors. Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain of an antibody (scFv) linked to a signaling domain of an immune cell (e.g., T cell). CAR properties can include their ability to specifically and reactivity redirect T cells to selected targets in a non-MHC-restricted manner, thereby utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0059] The CAR described herein provides a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain"), the intracellular signaling domain including functional signaling domains derived from stimulatory molecules as defined below. T cells expressing CAR are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cell can be genetically modified to stably express an antibody-binding domain on its surface, thereby conferring novel MHC-independent antigen specificity.

[0060] 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 into a single chimeric protein. In one embodiment, the stimulating molecule is a ζ chain associated with a T cell receptor complex.

[0061] As used herein, "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. This functional portion of a protein functions by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers, or by acting as an effector in response to such messengers. Intracellular signaling domains generate signals that promote the function of immune effectors in CAR-containing cells (e.g., CAR-T cells). Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and cofactor activities, including the secretion of cytokines.

[0062] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may contain a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may contain a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0063] Primary intracellular signal transduction domains may contain signal transduction motifs, referred to as immune receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signal transduction sequences containing ITAMs include, but are not limited to, those derived from CD3-ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d, DAP10, and DAP12.

[0064] The term “ζ” or alternatively “ζ chain,” “CD3-ζ,” or “TCR-ζ” is defined as a protein provided with GenBank accession number BAG36664.1, or an equivalent residue from a non-human species (e.g., mouse, rabbit, primate, rodent, monkey, ape, etc.), and “ζ-stimulatory domain” or alternatively “CD3-ζ-stimulatory domain” or “TCR-ζ-stimulatory domain” is defined as an amino acid residue from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal necessary for T cell activation. In one aspect, the cytoplasmic domain of the ζ chain contains residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.) of its functional ortholog. In one aspect, the “ζ-stimulatory domain” or “CD3-ζ-stimulatory domain” is a sequence provided as SEQ ID NO: 10, 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 SEQ ID NO: 10.

[0065] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class 1 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 intracellular signal transduction domain of a costimulatory molecule can be the intracellular portion of that molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte-activating molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0067] Intracellular signal transduction domains can contain the entire intracellular portion of their source molecule or the entire natural intracellular signal transduction domain, or a functional fragment thereof.

[0068] The term “4-1BB” or alternatively “CD137” refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and the “4-1BB co-stimulatory domain” is defined as amino acid residues 214 to 255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). In one aspect, the “4-1BB co-stimulatory domain” or the “CD137 co-stimulatory domain” is a sequence provided as SEQ ID NO: 11, or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), 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 SEQ ID NO: 11.

[0069] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 11) In one embodiment, a transmembrane domain that naturally associates with a domain in the CAR is used. In another embodiment, the transmembrane domain may be selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In an example embodiment, the transmembrane domain includes a CD8α hinge domain.

[0070] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined herein. In one embodiment, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-ζ, and / or CD28. CD28 is a T cell marker important in T cell co-stimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and acts as a co-stimulatory immune checkpoint molecule. 4-1BB delivers a potent co-stimulatory signal to T cells, thereby promoting T lymphocyte differentiation and enhancing long-term T lymphocyte survival. CD3-ζ associates with the TCR to generate a signal and contains an immune receptor tyrosine-based activation motif (ITAM). In another embodiment, the co-stimulatory molecule is MyD88 or CD40.

[0071] In one embodiment, the CAR comprises an intracellular hinge domain and an intracellular T-cell receptor signaling domain, wherein the intracellular hinge domain comprises CD8, and the intracellular T-cell receptor signaling domain comprises CD28, 4-1BB, and CD3-ζ. In another embodiment, the CAR comprises an intracellular hinge domain and an intracellular T-cell receptor signaling domain, the intracellular T-cell receptor signaling domain comprising 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; and all or part of the FcyRIIIa receptor, IgG hinge, IgM hinge, IgA hinge, IgD hinge, IgE hinge, or Ig hinge. The IgG hinge may be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0072] The CARs described herein provide recombinant polypeptide constructs that include at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain (also referred to herein as a "cytoplasmic signal transduction domain"), which includes, for example, functional signal transduction domains derived from stimulatory molecules as defined below.

[0073] In one embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes a functional signal transduction domain derived from a stimulating molecule. In another embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes a functional signal transduction domain derived from a co-stimulating molecule and a functional signal transduction domain derived from a stimulating molecule. In yet another embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more co-stimulating molecules and a functional signal transduction domain derived from a stimulating molecule.

[0074] The CAR can be designed to contain a CD28 signaling domain and / or a 4-1BB signaling domain, or in combination with any other desired cytoplasmic domains that can be used in the environment of the CAR described herein. In one embodiment, the cytoplasmic domain of the CAR may also contain the CD3-ζ signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, CD3-ζ, 4-1BB, and CD28 signaling modules, and combinations thereof.

[0075] In some embodiments, the CAR described herein includes an extracellular antigen-binding domain that specifically binds to tumor antigens. Non-limiting examples of tumor antigens that can be recognized by the CAR described herein include BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0076] This disclosure also provides variants of the CARs, nucleic acids, peptides, and proteins described herein, such as functional variants. A “variant” is a peptide or polynucleotide that differs from a reference peptide or reference polynucleotide due to one or more modifications (e.g., substitution, insertion, or deletion). As used herein, the term “functional variant” refers to a CAR, peptide, or protein that has substantial or significant sequence identity or similarity to a parental CAR, peptide, or protein, and that retains the biological activity of the CAR, peptide, or protein that is a variant of it. Functional variants encompass those variants of, for example, the CARs, peptides, or proteins (parental CARs, peptides, or proteins) described herein, that retain the ability to recognize target cells (e.g., tumor cells) to a degree, the same degree, or greater than that of the parental CAR, peptide, or protein. Regarding the parental CAR, polypeptide, or protein, the functional variant may, for example, have 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 higher identity with the amino acid sequence of the parental CAR, polypeptide, or protein.

[0077] The functional variant may, for example, comprise the amino acid sequence of a parent CAR, peptide, or protein having at least one conserved amino acid substitution. In another embodiment, the functional variant may comprise the amino acid sequence of a parent CAR, peptide, or protein having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution may not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR, peptide, or protein.

[0078] The amino acid substitutions in the CAR of the present invention can be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include substitutions in which an amino acid having specific physical and / or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), the substitution of an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), the substitution of a basic amino acid with another basic amino acid (Lys, Arg, etc.), the substitution of an amino acid with a polar side chain with another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), and so on.

[0079] The CAR, polypeptide, or protein may consist substantially of one or more specified amino acid sequences as described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0080] The CARs, peptides, and proteins (including functional moieties and functional variants) of the embodiments disclosed herein can have any length, i.e., can contain any number of amino acids, provided that the CAR, peptide, or protein (or its functional moieties or functional variants) retains its biological activity, such as the ability to specifically bind antigens, detect diseased cells (e.g., cancer cells) in the host, or treat or prevent diseases in the host. For example, the length of the peptide can be from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 47... 5, 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 amino acids. The polypeptides described herein also include oligopeptides.

[0081] The CARs, peptides, and proteins (including functional moieties and functional variants of CARs) used in various aspects and embodiments herein may comprise synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenyl Serine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, dihydroindole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, and α-tert-butylglycine.

[0082] The CARs, peptides, and proteins (including functional moieties and functional variants) used in various aspects and embodiments herein can undergo post-translational modifications. They can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, via, for example, disulfide cyclization, or converted into acid addition salts. In some embodiments, they are dimerized or polymerized, or conjugated.

[0083] The CARs, peptides, and / or proteins (including their functional moieties and functional variants) used in various aspects and embodiments herein can be obtained by methods known in the art. Suitable methods for de novo synthesis of peptides and proteins are described in references such as Chan et al. Fmoc Solid Phase Peptide Synthesis ,Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis Reid, R. (ed.), Marcel Dekker, Inc., 2000; and Epitope Mapping Edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Alternatively, standard recombination methods can be used to recombine the nucleic acids described herein to generate peptides and proteins. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual 3rd edition, Cold Spring HarborPress, Cold Spring Harbor, NY 2001; and Ausubel et al. Current Protocols in Molecular Biology Greene Publishing Associates and John Wiley & Sons, NY, 1994. Additionally, some of the CARs, peptides, and proteins described herein (including their functional moieties and functional variants) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, etc. Methods for isolation and purification are known in the art. Alternatively, the CARs, peptides, and / or proteins described herein (including their functional moieties and functional variants) can be commercially synthesized. In this respect, the CARs, peptides, and proteins can be synthetic, recombinant, isolated, and / or purified.

[0084] Examples of modified nucleotides that can be used to generate recombinant nucleic acids for producing the polypeptides described herein include, but are not limited to: 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-mannosylquinoline, 5″-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, piracetamidine, β-D-galactosylpiracetamidine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl 5-oxyuracil uracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0085] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding a CAR, polypeptide, or protein, or any functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a combination of nucleotide sequences degenerate from or derived from any of the aforementioned sequences. These nucleic acids may be incorporated into recombinant expression vectors. Recombinant expression vectors comprising one or more of these nucleic acids may be used. As used herein, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that, when comprising a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, allows the host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with the host cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors described herein are not naturally occurring as a whole; however, portions of these vectors may be naturally occurring. The recombinant expression vector may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may comprise natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring nucleotide bonds, or both. Non-naturally occurring or altered nucleotides or nucleotide bonds do not impede the transcription or replication of the vector.

[0086] The recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for proliferation and amplification or for expression, or both, such as plasmids and viruses. Vectors can be selected from the group consisting of: pUC series (Fermentas Life Sciences, GlenBurnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λEMBL4, λNM1149, and 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 can be viral vectors, such as retroviral vectors, like gamma retroviral vectors.

[0087] These recombinant expression vectors are prepared using standard recombinant DNA techniques, as described in, for example, Sambrook et al. (ibid.) and Ausubel et al. (ibid.). Circular or linear expression vector constructs can be prepared to be contained within a functional replication system in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, SV40, 2µ plasmid, λ, bovine papillomavirus, etc.

[0088] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the host type (e.g., bacteria, plants, fungi, or animals) in which the vector will be appropriately introduced, and take into account whether the vector is DNA-based or RNA-based.

[0089] Recombinant expression vectors may contain one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.), prototrophic complementation in auxotrophic hosts, and so on. Suitable marker genes for said expression vectors include, for example, neomycin / G418 resistance genes, histidine x resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0090] The recombinant expression vector may contain a nucleotide sequence operably linked to encoding the CAR, polypeptide, or protein (including its functional portion and functional variants), or a natural or standard promoter operably linked to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR, polypeptide, or protein. The selection of the promoter (e.g., a strong promoter, a weak promoter, a tissue-specific promoter, an inducible promoter, and a development-specific promoter) is within the skill of a person skilled in the art. Similarly, the combination of the nucleotide sequence with the promoter is also within the skill of a person skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter, or a promoter found in the long terminal repeat sequence of a mouse stem cell virus.

[0091] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, they can be prepared for constitutive or inducible expression.

[0092] Additionally, recombinant expression vectors can be prepared to include suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cell death in cells expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent, such as a drug, to cells expressing the gene and causes cell death upon contact with or exposure to the reagent. 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.

[0093] Inhibitory molecules can be antibodies (e.g., monoclonal antibodies), or their antigen-binding moieties; or soluble antigens, or their functional moieties or functional variants, which bind to epitopes of the CAR on immune cells, for example, specifically. The antibody can be any type of immunoglobulin known in the art. Immunoglobulins can be classified into five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. In vertebrate species, antibody light chains can be designated as one of two types, κ and λ, based on the amino acid sequence of their constant domains. Antibodies can be of any type or isotype.

[0094] The antibodies used in the methods described herein can include immunoglobulin molecules, specifically including monoclonal antibodies (including mouse monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), polyclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, monomeric antibodies, dimeric antibodies, tetrameric antibodies, or polymeric antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding site having the desired specificity. Antibodies can be naturally occurring antibodies, such as antibodies isolated and / or purified from mammals (e.g., mice, primates, rats, rabbits, goats, horses, chickens, hamsters, humans, etc.). Alternatively, antibodies can be engineered (e.g., genetically engineered) antibodies.

[0095] Humanized antibodies possess antigen-binding sites derived from non-human species, and their variable region framework is derived from human immunoglobulin sequences. Human antibodies have heavy chain variable regions and light chain variable regions, where both the framework and antigen-binding sites are derived from human-derived sequences.

[0096] Furthermore, the antibody can possess any level of affinity or affinity for the functional portion of the CAR. In some embodiments, the antibody can have a range of affinities (K... D The antibody binds to the hK2 antigen. In various embodiments, the antibody binds to the hK2 antigen with high affinity, for example, as determined by surface plasmon resonance or the Kinexa method, with a KD equal to or less than about 10. −7 M, such as, but not limited to, 1 to 9.9 (or any range or value therein, 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 M, or any range or value thereof, as practiced by those skilled in the art. An example affinity is equal to or less than 1 × 10⁻⁶. −8 M. Another example has an affinity equal to or less than 1 × 10⁻⁶. − 9 M.

[0097] Methods for testing the ability of an antibody to bind to any functional part 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.

[0098] Suitable methods for preparing antibodies are known in the art. For example, the standard hybridoma method is described in the literature by Köhler and Milstein, for instance. Eur.J. Immunol. , 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual , CSH Press (1988), and CA Janeway et al. (editors). Immunobiology , 5th edition, Garland Publishing, New York, NY (2001). Alternatively, other methods include 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 phage vector expression systems (see, for example, Huse et al., , Science Methods for generating antibodies in non-human animals are known in the art, such as U.S. Patent Nos. 5,545,806, 5,569,825 and 5,714,352 and U.S. Patent Application Publication No. 2002 / 0197266 A1.

[0099] Phage display can also be used to generate antibodies used in any of the methods described herein. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology techniques and recombinant DNA techniques (see, for example, Sambrook et al., ibid. and Ausubel et al., ibid.)). A phage encoding a variable region with desired specificity is selected for specific binding to a desired antigen (i.e., hK2), and an antibody containing the selected variable domain, either fully or partially, is reconstructed. The nucleic acid sequence encoding the reconstructed antibody is introduced into a suitable cell line (such as myeloma cells used to generate hybridomas) so that an antibody with monoclonal antibody properties is secreted by that cell (see, for example, Janeway et al., ibid., Huse et al., and U.S. Patent No. 6,265,150).

[0100] Antibodies can be produced by transgenic mice that are transgenic for specific heavy chain immunoglobulin genes and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Patents 5,545,806 and 5,569,825 and in Janeway et al. (ibid.).

[0101] Methods for generating humanized antibodies are known in the art and described, for example, in Janeway et al. (ibid.), U.S. Patents 5,225,539, 5,585,089, and 5,693,761, European Patent 0239400 B1, and British Patent 2,188,638. Humanized antibodies can also be generated using antibody surface reconstruction techniques described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

[0102] As used herein, antibodies can be multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0103] The antigen-binding portion of any of the antibodies described herein is also provided. This antigen-binding portion can be any portion having at least one antigen-binding site, such as Fab, F(ab′)2, dsFv, sFv, biantibodies, and triantibodies. In some embodiments, the antigen-binding fragment is a heavy chain complementarity-determining region (HCDR) 1, 2, and / or 3, a light chain complementarity-determining region (LCDR) 1, 2, and / or 3, a heavy chain variable region (VH) or a light chain variable region (VL), a Fab, F(ab′)2, Fd, or Fv fragment, and a domain antibody (dAb) comprising (e.g., any of the following) a VH domain or a VL domain. The VH domain and the VL domain can be linked together via a linker (e.g., a synthetic linker).

[0104] In addition, antibodies or their antigen-binding portions can be modified to include detectable tags, 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).

[0105] This disclosure also provides nucleic acids comprising nucleotide sequences encoding any of the CARs, peptides, or proteins (including their functional portions and functional variants) described herein.

[0106] The CAR containing an antibody or an antibody fragment thereof can exist in various forms, wherein the antigen-binding domain is represented as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), scFvs, and human chimeric or humanized antibodies (Harlow et al., 1999, published in: Using Antibodies: A Laboratory Manual , ColdSpring Harbor Laboratory Press, NY; Harlow et al., 1989, published in: Antibodies: A Laboratory ManualCold Spring Harbor, NY; Ashkenazi et al., 1988. Proc.Natl.Acad.Sci.USA 85:5879-5883; Bird et al., 1988, Science (242:423-426). In one aspect, the antigen-binding domain of the CAR composition includes an antibody fragment. In another aspect, the CAR comprises an antibody fragment having an scFv.

[0107] In one embodiment, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide located between the light chain variable region and the heavy chain variable region.

[0108] In recombinant expression systems, the linker is a peptide linker and can contain any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be long enough to allow VH and VL to connect in the correct conformation relative to each other, so that they retain their desired activities (such as binding to hK2).

[0109] The linker length can be approximately 5 to 50 amino acids. In some embodiments, the linker length is approximately 10 to 40 amino acids. In some embodiments, the linker length is approximately 10 to 35 amino acids. In some embodiments, the linker length is approximately 10 to 30 amino acids. In some embodiments, the linker length is approximately 10 to 25 amino acids. In some embodiments, the linker length is approximately 10 to 20 amino acids. In some embodiments, the linker length is approximately 15 to 20 amino acids. In some embodiments, the linker length is 6 amino acids. In some embodiments, the linker length is 7 amino acids. In some embodiments, the linker length is 8 amino acids. In some embodiments, the linker length is 9 amino acids. In some embodiments, the linker length is 10 amino acids. In some embodiments, the linker length is 11 amino acids. In some embodiments, the linker length is 12 amino acids. In some embodiments, the linker length is 13 amino acids. In some embodiments, the linker length is 14 amino acids. In some embodiments, the linker length is 15 amino acids. In some embodiments, the linker length is 16 amino acids. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long.Exemplary connectors that can be used include Gly-rich connectors, connectors containing Gly and Ser, connectors containing Gly and Ala, connectors containing Ala and Ser, and other flexible connectors.

[0110] In one embodiment, the extracellular antigen-binding domain comprises a signaling polypeptide. The signaling polypeptide may be localized at the N-terminus of the hK2-binding extracellular antigen-binding domain. The signaling polypeptide may optionally be cleaved from the extracellular antigen-binding domain during cell processing and CAR localization to the cell membrane. Any of a variety of signaling polypeptides known to those skilled in the art may be used as the signaling polypeptide. Non-limiting examples of peptides from which the signaling polypeptide can be derived include FcεR, the variable region of the human immunoglobulin (IgG) heavy chain (HC), CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the signaling polypeptide is compatible with the secretory pathway of T cells.

[0111] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In one embodiment, the intracellular signal transduction domain comprises a polypeptide component selected from the group consisting of: TNF receptor superfamily member 9 (CD137) component, T cell surface glycoprotein CD3ζ chain (CD3z) component, differentiation cluster (CD27) component, differentiation cluster superfamily member (such as CD28 or inducible T cell costimulators (ICOS)) component, and combinations thereof. In one embodiment, the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide. In one embodiment, the transmembrane domain comprises at least a transmembrane region of the following: the α, β, or ζ chain of a 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 comprises at least a transmembrane domain having the following: ζ, η, or FcεR1γ and -β, MB1 (Igα.), B29 or CD3-γ, ζ, or η. In yet another embodiment, the transmembrane domain is synthetic, for example, primarily comprising hydrophobic residues such as leucine and valine, a phenylalanine triplet, or tryptophan.

[0112] In one embodiment, the CAR further includes a hinge region connecting the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region.

[0113] In one aspect, this disclosure provides isolated immune-response cells comprising the CAR described herein. In some embodiments, the isolated immune-response cells are transduced with the CAR, for example, the CAR is constitutively expressed on the surface of the immune-response cells. In some embodiments, the isolated immune-response cells are further transduced with at least one co-stimulatory ligand, such that the immune-response cells express the at least one co-stimulatory ligand. In some embodiments, the at least one co-stimulatory ligand is selected from the group consisting of: 4-1BBL, CD48, CD70, CD80, CD86, OX40L, TNFRSF14, and combinations thereof. In some embodiments, the isolated immune-response cells are further transduced with at least one cytokine, such that the immune-response cells secrete the at least one cytokine. In some embodiments, the at least one 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 implementations, the isolated immune response cells are selected from the 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 precursor cells, and pluripotent stem cells from which lymphoid cells can differentiate.

[0114] In one embodiment, the CAR T-expressing immune cells of this disclosure can be generated by introducing a lentiviral vector containing the desired CAR (e.g., a CAR containing anti-hK2, a CD8α hinge and transmembrane domain, and human 4-1BB and CD3-ζ signaling domains) into cells. The CAR T-expressing immune cells of this invention are capable of replicating in vivo, thereby producing long-term persistence that can induce sustained tumor control.

[0115] Any CAR and inhibitory molecule can be expressed in a host cell 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. Host cells can be eukaryotic cells (e.g., plants, animals, or algae, fungi) or prokaryotic cells (e.g., bacteria or protozoa). Host cells can be cultured cells or primary cells (i.e., directly isolated from an organism (e.g., humans)). Host cells can be adherent cells or suspension cells, i.e., cells grown in a suspension. Suitable host cells are known in the art, including, for example, *Escherichia coli* DH5α (… E. coliHost cells include DH5α cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For amplification or replication of the recombinant expression vector, the host cell can be a prokaryotic cell, such as DH5α cells. For the production of recombinant CARs, peptides, or proteins, the host cell can be a mammalian cell. The host cell can be a human cell. Although the host cell can be any cell type, can originate from any type of tissue, and can be at any developmental stage, the host cell can be a peripheral blood lymphocyte (PBL). The host cell can be a T cell.

[0116] For the purposes of this document, T cells can be any type of T cell, such as cultured T cells (e.g., primary T cells), T cells derived from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to bone marrow, blood, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4. + / CD8 + Double-positive T cells, CD8 + T cells (e.g., cytotoxic T cells), CD4 + Helper T cells (e.g., Th1 and Th2 cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells, memory T cells, naive T cells, etc. T cells can be CD8+. + T cells or CD4 + T cells.

[0117] A cell population comprising at least one host cell described herein is also provided. The cell population can be a heterogeneous population comprising host cells having any of the recombinant expression vectors, and additionally comprising at least one other cell type, such as host cells not having any of the recombinant expression vectors (e.g., T cells), or cells other than T cells, such as B cells, macrophages, erythrocytes, neutrophils, hepatocytes, endothelial cells, epithelial cells, myocytes, brain cells, etc. Alternatively, the cell population can be a substantially homogeneous population, wherein the population primarily comprises (e.g., is substantially composed of) host cells having the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell containing the recombinant expression vector, such that all cells in the population contain the recombinant expression vector. In one embodiment, the cell population is a clonal population comprising host cells having the recombinant expression vector as described herein.

[0118] Inhibitors that bind to CAR In some embodiments, the inhibitory molecule used in the methods described herein can be a monoclonal antibody that specifically binds to a CAR peptide. For example, the monoclonal antibody can specifically bind to the constant domain of a CAR peptide described herein (such as a CAR peptide expressed on CAR-T cells). Alternatively, the antibody can bind to the antigen recognition domain of a CAR peptide (e.g., a CAR peptide that binds to CD-19). This antibody can impair the ability of CAR-T cells to bind to target cells (e.g., tumor cells). Without being bound by theory, the antibody can prevent CAR-T cells from binding to target cells (e.g., tumor cells).

[0119] In various embodiments, a monoclonal antibody that specifically binds to a CAR peptide targeting BCMA is used. In some aspects, this monoclonal antibody binds to a BCMA-specific CAR peptide and competes with that peptide for 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 capable of binding 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 variable domain of the target antibody in a manner that inhibits, disrupts, or neutralizes the activity of the target antibody (i.e., its ability to bind to the antigen).

[0120] The inhibitory molecule may be an anti-idiotypic peptide. In some embodiments, the anti-idiotypic peptide binds to the antigen-binding receptor of one or more additional cell therapeutic agents (e.g., scFv of CAR-T cells). In some embodiments, the anti-idiotypic peptide binds to the antigen-binding receptor of one or more CDRs of the antigen-binding receptor (e.g., scFv of CAR-T cells). In various embodiments, the anti-idiotypic antibody or peptide (e.g., scFv) binds to the antigen-binding portion of a B cell-specific marker of CAR-T cells (e.g., CARs that bind to CD19, CD20, CD21, CD22, CD24, CD79a, CD79b, ROR1, or BCMA). Furthermore, for example, in some embodiments, the anti-idiotypic antibody or fragment (e.g., scFv) binds to an anti-CD19 antibody or fragment (e.g., an anti-CD19 antibody expressed by CAR-T cells (e.g., anti-CD19 scFv)).

[0121] Inhibitory molecules containing all or part of the heavy chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting BCMA are also provided. Such inhibitory molecules can also specifically bind to CAR peptides targeting BCMA. Inhibitory molecules containing all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting BCMA are also provided. Such inhibitory molecules can also specifically bind to CAR peptides targeting BCMA. Inhibitory molecules containing one, two, three, four, five, or six complementarity-determining regions (CDRs) from the light chain variable region and / or heavy chain variable region of a monoclonal antibody that binds to a BCMA-specific CAR peptide are also provided.

[0122] In some embodiments, a monoclonal antibody that specifically binds to a CAR peptide targeting GPRC5D is used. In some aspects, this monoclonal antibody binds to a GPRC5D-specific CAR peptide and competes with the peptide for binding to 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 CAR peptide targeting GPRC5D are also provided. Such inhibitory molecules may also specifically bind to CAR peptides targeting GPRC5D. Inhibitory molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting GPRC5D are also provided. Such inhibitory molecules may also specifically bind to CAR peptides targeting GPRC5D. Inhibitory molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) from the light chain variable region and / or heavy chain variable region of a monoclonal antibody that binds to a GPRC5D-specific CAR peptide are also provided.

[0123] In some embodiments, a monoclonal antibody that specifically binds to a CAR peptide targeting CD79 is used. In some aspects, the monoclonal antibody binds to the CD79-specific CAR peptide and competes with the peptide for binding to multiple myeloma target cells or any other cells expressing CD79. 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 CAR peptide targeting CD79 are also provided. Such inhibitory molecules may also specifically bind to the CAR peptide targeting CD79. Inhibitory molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting CD79 are also provided. Such inhibitory molecules may also specifically bind to the CAR peptide targeting CD79. Inhibitory molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) from the light chain variable region and / or heavy chain variable region of a monoclonal antibody that binds to a CD79-specific CAR peptide are also provided.

[0124] In some embodiments, a monoclonal antibody that specifically binds to a CAR peptide targeting KLK2 is used. In some aspects, the monoclonal antibody binds to a KLK2-specific CAR peptide and competes with the peptide for binding to multiple myeloma target cells or any other cells expressing KLK2. 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 CAR peptide targeting KLK2 are also provided. Such inhibitory molecules may also specifically bind to a CAR peptide targeting KLK2. Inhibitory molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting KLK2 are also provided. Such inhibitory molecules may also specifically bind to a CAR peptide targeting KLK2. Inhibitory molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) from the light chain variable region and / or heavy chain variable region of a monoclonal antibody that binds to a KLK2-specific CAR peptide are also provided. In some embodiments, a monoclonal antibody that specifically binds to a CAR peptide targeting CD19 is used. In some respects, the monoclonal antibody binds to a CD19-specific CAR peptide and competes with that peptide for binding to multiple myeloma target cells or any other cells expressing CD19. 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 CAR peptide targeting CD19 are also provided. Such inhibitory molecules may also specifically bind to CAR peptides targeting CD19. Inhibitory molecules comprising all or part of the light chain variable region of a monoclonal antibody that specifically binds to a CAR peptide targeting CD19 are also provided. Such inhibitory molecules may also specifically bind to CAR peptides targeting CD19. Inhibitory molecules comprising one, two, three, four, five, or six complementarity-determining regions (CDRs) from the light chain variable region and / or heavy chain variable region of a monoclonal antibody that binds to a CD19-specific CAR peptide are also provided.

[0125] In various embodiments, inhibitory molecules (e.g., monoclonal antibodies, fragments of monoclonal antibodies, or derivatives of monoclonal antibodies) are capable of transgene-specific amplification. Anti-idiotype antibodies (including their antigen-binding fragments) specifically recognize, specifically target, and / or specifically bind to idiotype sites of the antibody or its antigen-binding fragments, such as the antigen-binding domain of a recombinant receptor (e.g., a chimeric antigen receptor (CAR)). An idiotype site is any single antigenic determinant or epitope within the variable portion of an antibody. These anti-idiotype antibodies or their antigen-binding fragments can be agonists and / or exhibit specific activity in stimulating cells expressing a particular antibody (including conjugates) or a recombinant receptor containing that particular antibody or its antigen-binding fragment (see, for example, U.S. Patent Publications US 2016 / 0096902, US 2016 / 0068601, US 2014 / 0322183, US 2015 / 0175711, US 2015 / 283178; U.S. Patent No. 9,102,760; Jena et al., PloSone (2013) 8(3):e57838; Long et al., Nature Medicine (2015) 21(6):581-590; Lee et al., The Lancet (2015) 385(9967):517-528; Zhao et al., PloS One (2014)). 9(5):e96697; Leung et al., MAbs.(2015) 7(1):66-76).

[0126] In some implementations, the inhibitory molecule is a soluble form of an antigen that interacts with the CAR and is expressed on the target cell, or a functional fragment or derivative thereof. For example, the soluble antigen may be a soluble form of BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3, or a functional fragment or derivative thereof.

[0127] Contact CAR-T cells with inhibitors The various methods disclosed herein include blocking or altering the ability of specific CAR-T cells (e.g., T cells expressing CARs that bind to BCMA) by contacting the cells with monoclonal antibodies or soluble antigens that bind to the antigen recognition domain of the CAR.

[0128] In one aspect, an in vitro method is provided for determining the cytotoxicity of immune cells expressing chimeric antigen receptor (CAR) molecules, the method comprising: a) In the test sample, CAR-expressing immune cells are incubated together with target cells (e.g., tumor cells), wherein the target cells express antigens that interact with the CAR. b) In a first control sample, CAR-expressing immune cells are incubated together with target cells, wherein the incubation is performed in the presence of an inhibitory molecule that mitigates, inhibits, blocks, and / or prevents the interaction between CAR and target cells. c) Measure the amount of target cell death in the test sample. d) Determine the amount of target cell death in the first control sample, and e) Determine the cytotoxicity of CAR-expressing immune cells based on a comparison of the amount of target cell death measured in steps (c) and (d). In the test sample and the first control sample, the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells were basically the same.

[0129] In some embodiments, the incubation time of the test sample is 85% to 115%, 90% to 110%, or 95% to 105% of the incubation time of the first control sample. In some embodiments, the amount of CAR-expressing immune cells is 85% to 115%, 90% to 110%, or 95% to 105% of the amount of target cells.

[0130] 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, a difference of 1 hour, 30 minutes, or 15 minutes. Simultaneous execution of steps (a) and (b) provides conditions in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same. In some embodiments, assay steps (c) and (d) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, a difference of 1 hour, 30 minutes, or 15 minutes. Simultaneous execution of steps (c) and (d) provides conditions in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same.

[0131] In some implementations, the CAR-expressing immune cells and / or target cells are pre-incubated with the inhibitory molecule prior to the contact step.

[0132] In some embodiments, the method further includes comparing the amount of target cell (e.g., tumor cell) death measured in step (c) with the amount of target cell death measured in a second control sample, wherein the target cells were incubated in the absence of CAR-expressing immune cells.

[0133] In some embodiments, the method further includes comparing the amount of target cell death measured in step (c) with the amount of target cell death measured in a third control sample, wherein the target cells are incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that induces target cell death. In some embodiments, the detergent is Triton X-100.

[0134] In various embodiments, target cells (e.g., tumor cells) generate a detectable reporter signal upon target cell death, and step (c) includes measuring the reporter signal in a test sample, step (d) includes measuring the reporter signal in a first control sample, and step (e) includes comparing the reporter signals measured in steps (c) and (d). In some embodiments, the target cells express a reporter protein that generates a signal upon the target cells undergoing cell death. Exemplary reporter proteins suitable for the methods of this 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 variants or derivatives thereof.

[0135] In some implementations, the reporter signal is luminescence. A protein capable of generating a reporter signal (e.g., luminescence) can be expressed in a cell compartment. Upon cell death, this protein is released from the cell compartment into the culture medium, where it can generate a luminescent signal, such as by acting on a reagent via an enzymatic method to produce luminescence. Exemplary cells capable of being used in this manner include KILR. ® Target cells. Target cells can be engineered to express antigens that interact with chimeric antigen receptors. In some implementations, KILR is used. ® MM-1R multiple myeloma target cells. These target cells can also stably express proteins labeled with tags or enzymes. When the target cell line is used in a cytotoxicity assay, and its membrane is damaged due to cell death, the target cell line can release the labeled protein into the culture medium. The labeled protein can be detected by adding a reagent to the culture medium, where the reagent is the substrate of the enzyme tag on the protein. For example, β-galactosidase can hydrolyze the substrate to produce a chemiluminescent output. The luminescence can be quantified on a plate reader capable of measuring chemiluminescence. Alternatively, the labeled protein can be detected via an assay for detecting the tag.

[0136] In some implementations, the reporter signal is fluorescence. A protein capable of generating a reporter signal (e.g., fluorescence) can be expressed in a cell compartment. Upon cell death, this protein is released from the cell compartment into the culture medium, where it can generate a fluorescent signal.

[0137] In some implementations, the inhibitory molecule specifically binds to the antigen on the target cells that interacts with the CAR. Not wanting to be bound by theory, using an inhibitory molecule that binds to the antigen can provide a suitable control in cytotoxicity assays or other relevant CAR potency assays, eliminating the need to use untransfected or simulated transfected immune cells as controls. This eliminates the need to generate simulated CAR-T cells parallel to the drug product. Generating simulated CAR-T cells can affect the production of the drug product in several ways. Eliminating the use of simulated cell controls simplifies the manufacturing process, ensures patient dosing by reducing the need to collect any autologous CAR-T cells from patients, and further reduces the cost of CAR-T cell therapy. The methods described herein can also reduce or eliminate testing delays due to insufficient simulated cell production. The method can also reduce testing errors by providing a more simplified format. Reduced testing delays and errors can also avoid production delays and / or patient dosing delays.

[0138] In some implementations, the inhibitory molecule specifically binds to the CAR. In some implementations, the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on the target cell that interacts with the CAR. By binding to the CAR, particularly to a region within the CAR that specifically binds to the antigen (e.g., an epitope containing one or more CDR sequences or portions thereof), the inhibitory molecule can block the interaction between CAR-T cells and target cells. This blockade prevents CAR-T cells from killing target cells. Therefore, instead of using mimicked transfected immune cells, the patient's own CAR-T cells can be used with an inhibitory molecule added as an appropriate alternative control.

[0139] In some embodiments, the inhibitory molecule is an antibody. In some embodiments, the antibody is an anti-idiotype antibody. Anti-idiotype antibodies can competitively bind to chimeric antigen receptors on host cells. Anti-idiotype antibodies can share some structural features with the antigen.

[0140] In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the scFv domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the Fab domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the VH or VL domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the VH or VL domain.

[0141] 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 cell contains 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 some embodiments, the multiple myeloma cells are MM-1R cells.

[0142] In various other implementations, CAR interacts with tumor and disease antigens, including but not limited to BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0143] In some embodiments, the CAR interacts with the tumor antigen GPRC5D. In some embodiments, the GPRC5D receptor is expressed by target cells. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the CAR. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the GPRC5D receptor. As a non-limiting example, the target cell is multiple myeloma cells. A non-limiting example of multiple myeloma cells is MM-1R cells.

[0144] In some embodiments, the 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 a soluble KLK2 protein. As a non-limiting example, the target cell is a prostate cancer cell. A non-limiting example of a prostate cancer cell is an LNCaP cell.

[0145] In various implementations, this method is performed in a high-throughput manner.

[0146] host cells The inhibitory molecules described herein can be expressed in cells containing the nucleic acid molecules, CAR peptide molecules, or vectors described herein, such as immune effector cells (e.g., cell populations, such as immune effector cell populations). Immune effector cells can 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 prior to use in any of the assays described herein.

[0147] The CAR-T molecules described herein can be expressed in immune effector cells, such as T cells or NK cells. Immune effector cells can be expressed using many techniques known to those skilled in the art, such as Ficoll. ™Cells are obtained from units of blood collected from a subject. Cells from an individual's circulating blood can be obtained via apheresis. Apheresis products typically contain lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected via apheresis can be washed to remove the plasma fraction, and then these cells can be placed in an appropriate buffer or culture medium for subsequent processing steps. These cells can be washed with phosphate-buffered saline (PBS). The wash solution may be calcium-free, magnesium-free, and / or free of all divalent cations.

[0148] The methods described herein may include, for example, using negative selection techniques as described herein to select a specific subset of immune effector cells, such as T cells, wherein the specific subset is a depleted population of CD25+ cells and depleted T regulatory cells. Preferably, the depleted population of T regulatory cells comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells.

[0149] potency assay This article discloses methods for characterizing the potency of chimeric antigen receptor (CAR)-T cells (CAR-T cells). These methods include: (a) stimulating CAR-T cells in an antigen-specific manner (i.e., via CAR-T cells), and (b) measuring the antigen-specific cytotoxicity level of the stimulated cells.

[0150] In one aspect, an in vitro method is provided for determining the potency of immune cells expressing chimeric antigen receptor (CAR) molecules, the method comprising: a) In the test sample, CAR-expressing immune cells are incubated together with target cells (e.g., tumor cells), wherein the target cells express antigens that interact with the CAR. b) In a first control sample, CAR-expressing immune cells are incubated together with target cells, wherein the incubation is performed in the presence of an inhibitory molecule that mitigates, inhibits, blocks, and / or prevents the interaction between CAR and target cells. c) Measure the amount of interaction between CAR-expressing immune cells and target cells in the test sample. d) Determine the amount of interaction between CAR-expressing immune cells and target cells in the first control sample, and e) Determine the potency of CAR-expressing immune cells based on a comparison of the amounts of interaction measured in steps (c) and (d). In the test sample and the first control sample, the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells were basically the same.

[0151] The interaction between CAR-expressing immune cells and target cells can be measured directly, such as by assessing the binding of CAR-expressing immune cells to target cells. The interaction can also be measured indirectly, such as by assessing cell death, apoptosis, necrosis, cytokine release, cell morphology changes, and so on.

[0152] In some embodiments, the incubation time of the test sample is 85% to 115%, 90% to 110%, or 95% to 105% of the incubation time of the first control sample. In some embodiments, the amount of CAR-expressing immune cells is 85% to 115%, 90% to 110%, or 95% to 105% of the amount of target cells.

[0153] 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, a difference of 1 hour, 30 minutes, or 15 minutes. Simultaneous execution of steps (a) and (b) provides conditions in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same. In some embodiments, assay steps (c) and (d) are performed simultaneously. Simultaneous execution allows for some difference between the start and end times of these steps, for example, a difference of 1 hour, 30 minutes, or 15 minutes. Simultaneous execution of steps (c) and (d) provides conditions in which the incubation time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same.

[0154] In some embodiments, the method further includes comparing the amount of interaction between the CAR-expressing immune cells and the target cells as determined in step (c) with the amount of target cell death as determined in a second control sample, wherein the target cells were incubated in the absence of CAR-expressing immune cells.

[0155] In some embodiments, the method further includes comparing the amount of interaction between the CAR-expressing immune cells and the target cells, as determined in step (c), with the amount of target cell death, as determined in a third control sample, wherein the target cells were incubated in the absence of CAR-expressing immune cells but in the presence of a detergent that induces target cell death. In some embodiments, the detergent is Triton X-100.

[0156] In various embodiments, the target cells generate a detectable reporter signal upon target cell death, and step (c) includes measuring the reporter signal in a test sample, step (d) includes measuring the reporter signal in a first control sample, and step (e) includes comparing the reporter signals measured in steps (c) and (d). In some embodiments, the target cells express a reporter protein that generates a signal when the target cells interact with CAR-expressing immune cells.

[0157] In some implementations, the reporter signal is luminescence. A protein capable of generating a reporter signal (e.g., luminescence) can be expressed in a cell compartment. Upon cell death, this protein is released from the cell compartment into the culture medium, where it can generate a luminescent signal, such as by acting on a reagent via an enzymatic method to produce luminescence. Exemplary cells capable of being used in this manner include KILR. ® Target cells. Target cells can be engineered to express antigens that interact with chimeric antigen receptors. In some implementations, KILR is used. ® MM-1R multiple myeloma target cells. These target cells can also stably express proteins labeled with tags or enzymes. When the target cell line is used in a cytotoxicity assay, and its membrane is damaged due to cell death, the target cell line can release the labeled protein into the culture medium. The labeled protein can be detected by adding a reagent to the culture medium, where the reagent is the substrate of the enzyme tag on the protein. For example, β-galactosidase can hydrolyze the substrate to produce a chemiluminescent output. The luminescence can be quantified on a plate reader capable of measuring chemiluminescence. Alternatively, the labeled protein can be detected via an assay for detecting the tag.

[0158] In some implementations, the reporter signal is fluorescence. A protein capable of generating a reporter signal (e.g., fluorescence) can be expressed in a cell compartment. Upon cell death, this protein is released from the cell compartment into the culture medium, where it can generate a fluorescent signal.

[0159] In some implementations, the inhibitory molecule specifically binds to antigens on target cells (e.g., tumor cells) that interact with the CAR. Not wanting to be bound by theory, using an inhibitory molecule that binds to the antigen can provide a suitable control in CAR potency assays, eliminating the need to use untransfected or simulated transfected immune cells as controls. This eliminates the need to generate simulated CAR-T cells parallel to the drug product. Generating simulated CAR-T cells can affect the production of the drug product in several ways. Eliminating the use of simulated cell controls simplifies the manufacturing process, ensures patient dosing by reducing the need to collect any autologous CAR-T cells from patients, and further reduces the cost of CAR-T cell therapy. The methods described herein can also reduce or eliminate testing delays due to insufficient simulated cell production. The method can also reduce testing errors by providing a more simplified format. Reduced testing delays and errors can also avoid production delays and / or patient dosing delays.

[0160] In some implementations, the inhibitory molecule specifically binds to the CAR. In some implementations, the inhibitory molecule specifically binds to a region within the CAR that specifically binds to an antigen expressed on the target cell that interacts with the CAR. By binding to the CAR, particularly to a region within the CAR that specifically binds to the antigen (e.g., an epitope containing one or more CDR sequences or portions thereof), the inhibitory molecule can block the interaction between CAR-T cells and target cells. This blockade prevents CAR-T cells from interacting with target cells. Therefore, instead of using mimicked transfected immune cells, the patient's own CAR-T cells can be used with an inhibitory molecule added as an appropriate alternative control.

[0161] In some embodiments, the inhibitory molecule is an antibody. In some embodiments, the antibody is an anti-idiotype antibody. Anti-idiotype antibodies can competitively bind to chimeric antigen receptors on host cells. Anti-idiotype antibodies can share some structural features with the antigen.

[0162] In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the scFv domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the scFv domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the Fab domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the Fab domain. In some embodiments, the antibody or antibody fragment specifically binds to the antigen within the VH or VL domain of the chimeric antigen receptor. In some embodiments, the antibody or antibody fragment specifically binds to the CDR within the VH or VL domain.

[0163] In some implementations, the inhibitory molecule is a soluble form of an antigen that interacts with the CAR and is expressed on the target cell, or a functional fragment or derivative thereof.

[0164] 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 cell contains 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 some embodiments, the multiple myeloma cells are MM-1R cells.

[0165] In various other implementations, CAR interacts with tumor and disease antigens, including but not limited to BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, and FLT3.

[0166] In some embodiments, the CAR interacts with the tumor antigen GPRC5D. In some embodiments, the GPRC5D receptor is expressed by target cells. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the CAR. In some embodiments, the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the GPRC5D receptor. As a non-limiting example, the target cell is multiple myeloma cells. A non-limiting example of multiple myeloma cells is MM-1R cells.

[0167] In some embodiments, the 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 a soluble KLK2 protein. As a non-limiting example, the target cell is a prostate cancer cell. A non-limiting example of a prostate cancer cell is an LNCaP cell.

[0168] When measuring antigen-specific cytotoxicity levels, instead of using simulated transfected CAR-T cells as a control, the same CAR-T cells tested in the assay can be treated with an inhibitory molecule as described herein. Treatment with an inhibitory molecule (e.g., a monoclonal antibody or soluble antigen to which the CAR specifically binds) prevents CAR-T cells from binding to the antigen and exerting cytotoxicity. Detecting an increased level of cytotoxicity in antigen-specifically stimulated cells compared to the same CAR-T cells treated with an inhibitory molecule or non-specifically stimulated CAR-T cells (i.e., stimulated CAR-T cells not stimulated in an antigen-specific manner) can be used as an indication that stimulated CAR-T cells were used in treatment. These methods can be performed in vitro.

[0169] In various implementations, inhibitory molecules (such as tumor antigens or anti-idiotype antibodies) are ineffective in stimulating CAR-T cells that are responsive to antigens (such as tumor antigens), and the CAR on the CAR-T cells is specific to that antigen. Such implementations can provide the advantage of not activating the efficacy parameters resulting from the activation of CAR-T cells.

[0170] Methods for measuring the potency and cytotoxicity of CAR-T cells are also provided. Generally, these methods involve antigen-specific stimulation of the CAR on the CAR-T cells, followed by quantification of the cytotoxicity of the antigen-specific CAR-T cells. Measurements of CAR-T cell potency can be used as an in vitro indicator of the expected in vivo pharmacokinetics of CAR-T cell therapy products. CAR-T potency assays can also be used to determine the suitability of CAR-T cell products for clinical use, assess the potential efficacy of CAR-T cell products, determine the dosage of CAR-T cells administered, and / or characterize novel manufacturing methods for CAR-T cell therapy products.

[0171] The efficacy of a CAR-T cell therapy product can be expressed based on its antigen-specific cytotoxicity level, which reflects the product's efficacy. For example, this cytotoxicity level can be compared to the cytotoxicity level of a control sample of the CAR-T cell therapy product exposed to both antigen-specific stimulation and the inhibitory molecule described herein. Furthermore, the calculation can be standardized based on, for example, the number of cells in the test sample expressing CAR. Based on this information, the cytotoxicity index (CI), expressed according to the following formula, can be used as a measure of the efficacy of the CAR-T cell therapy product: CI = [(Cytotoxicity in the stimulated group) - (Cytotoxicity in the control group)] / % CAR-expressing cells Methods known in the art can be used to determine the percentage of cells expressing CAR (e.g., transduction levels) to support this standardization. For example, in a flow cytometry assay, an antibody against CAR can be included in the assay and used to quantify the level of CAR-expressing cells relative to the total number of T cells.

[0172] The antigen-specific in vitro cytotoxicity level of CAR-T cell therapy products can be correlated with the in vivo pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of the CAR-T products. PK / PD characteristics of CAR-T cell preparations that can 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. The relationship between the in vitro cytotoxicity of a CAR-T cell therapy product (reflected by, for example, the cytotoxicity index described above) and the in vivo PK / PD characteristics of the product can be shown using standard methods capable of assessing linear associations between these characteristics, such as the Spearman correlation coefficient method. The various methods described herein can provide a basis for predicting PK / PD parameters based on the shown antigen-specific in vitro cytotoxicity (e.g., by measuring CI).

[0173] Reagent test kit Any of the compositions described herein may be included in a kit. In some embodiments, a CAR-binding antibody is provided in the kit, which may also include reagents suitable for amplifying cells, such as culture medium, APC, growth factors, antigens, other antibodies (e.g., for sorting or characterizing CAR T cells), and / or plasmids encoding CAR or transposases.

[0174] In one non-limiting example, the CAR-binding antibody, a chimeric receptor expression construct (or reagents for generating a chimeric receptor expression construct), reagents for transfecting the expression construct, and / or one or more tools (such tools may be syringes, pipettes, forceps, and / or any such medically approved instruments) for transfecting the expression construct with allogeneic cells are provided in the kit. In some aspects, the kit includes reagents or devices for cell electroporation.

[0175] The kit may include target cells expressing an antigen that specifically interacts with a CAR-binding antibody, reagents for transfecting an expression construct encoding the antigen, and / or one or more tools (such tools may be syringes, pipettes, forceps, and / or any such medically approved instruments) provided in the kit for obtaining allogeneic cells for transfecting the expression construct. In some aspects, the kit includes reagents or devices for cell electroporation.

[0176] The kit may include one or more suitably aliquoted compositions of the invention or reagents for generating compositions of the invention. The components of the kit may be packaged in an aqueous medium or in lyophilized form. The container device of the kit may include at least one vial, test tube, flask, bottle, syringe, or other container device capable of containing, and preferably suitably aliquoting, the components. Where more than one component is present in the kit, the kit will typically also contain a second, third, or other additional container to which additional components can be contained separately. However, various combinations of components may be contained in the vial. The kit of the invention will also typically include a device for containing chimeric receptor constructs and any other tightly sealed reagent container for commercial sale. For example, such containers may include injection or blow-molded plastic containers in which the desired vials are held.

[0177] Implementation Plan 1. An in vitro method for determining the potency of immune cells expressing chimeric antigen receptor (CAR) molecules, the method comprising: a) In the test sample, 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, CAR-expressing immune cells are contacted with target cells, wherein (i) the contact is performed in the presence of an inhibitory molecule, or (ii) prior to the contact, the CAR-expressing immune cells and / or target cells have been pre-incubated with an inhibitory molecule, wherein the inhibitory molecule inhibits the interaction between the CAR and the target cells. c) Measure the amount of target cell death in the test sample. d) Determine the amount of target cell death in the first control sample, and e) Determine the potency of CAR-expressing immune cells based on a comparison of the amount of target cell death measured in steps (c) and (d). In the test sample and the first control sample, the contact time, the amount of CAR-expressing immune cells, and the amount of target cells were basically the same.

[0178] 2. The method according to implementation scheme 1, wherein contact steps (a) and (b) are performed simultaneously.

[0179] 3. The method according to implementation scheme 1 or implementation scheme 2, wherein the determination steps (c) and (d) are performed simultaneously.

[0180] 4. The method according to any one of embodiments 1 to 3, wherein in step (b)(i), the CAR-expressing immune cells and / or target cells have been pre-incubated with the inhibitory molecule prior to the contact step.

[0181] 5. The method according to any one of embodiments 1 to 4, wherein 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 immune cells expressing CAR.

[0182] 6. The method according to any one of embodiments 1 to 5, wherein 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 detergent that causes target cell death.

[0183] 7. The method according to embodiment 6, wherein the detergent is Triton X-100.

[0184] 8. The method according to any one of embodiments 1 to 7, wherein the target cells generate a detectable report signal when the target cells die, and step (c) includes measuring the report signal in the test sample, step (d) includes measuring the report signal in a first control sample, and step (e) includes comparing the report signals measured in steps (c) and (d).

[0185] 9. The method according to embodiment 8, wherein the reporting signal is light emission.

[0186] 10. The method according to embodiment 8, wherein the reporting signal is fluorescence.

[0187] 11. The method according to any one of embodiments 8 to 10, wherein the target cell expresses a reporter protein that generates a signal when the target cell undergoes cell death.

[0188] 12. The method according to embodiment 11, wherein the reporter protein is β-galactosidase, luciferase, green fluorescent protein (GFP), or variants or derivatives thereof.

[0189] 13. The method according to any one of embodiments 1 to 12, wherein the inhibitory molecule specifically binds to the antigen on the target cell that interacts with the CAR.

[0190] 14. The method according to any one of embodiments 1 to 13, wherein the inhibitory molecule specifically binds to the CAR.

[0191] 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 a target cell.

[0192] 16. The method according to embodiments 13, 14 or 15, wherein the inhibitory molecule is an antibody or an antibody fragment.

[0193] 17. The method according to embodiment 16, wherein the antibody is an anti-idiotype antibody.

[0194] 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.

[0195] 19. The method according to embodiments 16, 17 or 18, wherein the antibody or antibody fragment specifically binds to the antigen within the scFv domain of the CAR.

[0196] 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.

[0197] 21. The method according to embodiments 16, 17 or 18, wherein the antibody or antibody fragment specifically binds to the antigen within the VH domain or VL domain of the CAR.

[0198] 22. The method according to embodiment 21, wherein the antibody or antibody fragment specifically binds to the CDR within the VH domain or VL domain of the CAR.

[0199] 23. The method according to embodiment 14 or 15, wherein the inhibitory molecule is a soluble form of an antigen that interacts with CAR expressed on target cells, or a functional fragment or derivative thereof.

[0200] 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.

[0201] 25. The method according to any one of embodiments 1 to 24, wherein the CAR interacts with a B cell maturation antigen (BCMA) receptor, the target cell contains a BCMA receptor, and the inhibitory molecule is a soluble cytoplasmic domain of BCMA.

[0202] 26. The method according to implementation scheme 25, wherein the target cells are multiple myeloma cells.

[0203] 27. The method according to embodiment 26, wherein the multiple myeloma cells are MM-1R cells.

[0204] 28. The method according to any one of embodiments 1 to 24, wherein the CAR interacts with a G protein-coupled receptor class C5 member D (GPRC5D), the target cell contains a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the CAR.

[0205] 29. The method according to any one of embodiments 1 to 24, wherein the CAR interacts with a G protein-coupled receptor class C5 member D (GPRC5D), the target cell contains a GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the GPRC5D receptor.

[0206] 30. The method according to implementation scheme 28 or 29, wherein the target cells are multiple myeloma cells.

[0207] 31. The method according to embodiment 30, wherein the multiple myeloma cells are MM-1R cells.

[0208] 32. The method according to any one of embodiments 1 to 24, wherein the CAR interacts with kallikerin 2 (KLK2), the target cell contains KLK2, and the inhibitory molecule is a soluble KLK2 protein.

[0209] 33. The method according to implementation scheme 32, wherein the target cells are prostate cancer cells.

[0210] 34. The method according to implementation scheme 33, wherein the prostate cancer cells are LNCaP cells.

[0211] 35. The method according to any one of embodiments 1 to 34, wherein the method is performed in a high-throughput manner. Example

[0212] The present invention is also described and illustrated by the following embodiments. However, the use of these and other embodiments anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the invention or any exemplary terminology. Similarly, the invention is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the invention. Therefore, the invention is limited only by the terms of the appended claims and the full scope of their equivalents.

[0213] Example 1 The following materials were used in this embodiment. CAR-T DP samples were used as test materials, 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 the housekeeping gene marked with an enhanced Prolabel (ePL) tag. Once the cells have lysed, the ePL-tagged protein is released into the culture medium. Adding an enzyme receptor induces complementation between the β-galactosidase fragment EA and ePL. The resulting functional enzyme hydrolyzes its substrate to generate a chemiluminescent signal.

[0214] Make KILR MM-1R ® Cells were grown in RPMI 1640 (ATCC formulation) (Gibco catalog 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). Assays were performed in assay medium consisting of RPMI 1640 (Corning catalog number 10-041-CV, ThermoFisher, Waltham, MA) containing L-glutamine and 25 mM HEPES and 10% HI-FBS (6140-071, Life Technologies, Carlsbad, CA).

[0215] Soluble human BCMA protein (sBCMA) (BCA-H522y, AcroBiosystems, Newark DE), the blocking protein, was prepared in the assay medium. A 10% Triton X-100 solution (catalog number 93443-100ML, SigmaAldrich Co., St. Louis, Mo) was used as a control for the total mortality count. KILR was used. ® Detection ™ The kit (catalog number 97-001, Eurofins Discoverx Corp., Fremont, CA) is used to detect cytotoxicity.

[0216] In this embodiment, the ability of a CAR-T cell drug product (DP) to kill multiple myeloma target cells expressing relevant antigens was measured. Target cells expressing reporter genes were used, which produce measurable signals (e.g., luminescence) due to cell death when bound by effector CAR-T cells. The results are presented as activity measurements used to determine whether the DP exhibited an appropriate level of release activity.

[0217] The assay consisted of four components used in a total of 16 wells. These four components were: (i) CAR-T drug product (DP) with target cells (total activity); (ii) CAR-T DP cells blocked by the blocking agent with target cells (baseline control); (iii) target cells with culture medium (cell death-free control); and (iv) target cells with 0.1% TitonX-100 (total cell death control). All four assay components were run as four separate replicates, as shown in the assay plate layout in Table 1A below. Detailed descriptions of the samples in the 96-well plate are provided in Table 1B. Six assays were performed using a single 96-well plate, one of which was a QC CAR-T cell assay for system suitability and chemotactic method performance. The QC CAR-T cells had previously been identified as activity-qualified.

[0218] Table 1A. Layout of 96-well measuring plate Table 1B. Description of samples in the 96-well plate 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 RPMI 1640 and 10% HI-FBS. KILR cells were cultured according to the manufacturer's instructions. ® MM-1R cells. A baseline control was generated by blocking LCAR-B38M DP cells with the soluble cytoplasmic domain of B cell maturation antigen (sBCMA). KILR ® The assay kit (Eurofins Discoverx Corp., Fremont CA) is used to measure the assay reagents. It is used for effector cells (DPs) and target cells (e.g., KILRs). ®The E:T ratio (E:T) and the amount of blocking reagent required to completely block the interaction between CAR-T cells (DPs) and their target cells were optimized. The E:T ratio and blocking reagent concentration were optimized for each drug product and its corresponding target cell line. Once determined, the E:T ratio was a fixed value in the assay and remained constant for all drug product tests. The blocking reagent was qualified for each batch and used at this level for drug product testing. Furthermore, the assay conditions could be optimized based on the reporting system used.

[0219] The LCAR-B38M CAR-T drug product was assayed in 96-well white opaque, TC-treated assay plates as described below. Each assay plate held a maximum of 6 assays, as described in Table 1A above. For each assay, 25 µL of blocking reagent was added to the CAR-T cell blocking well (baseline control) of the assay plate, followed by 25 µL of assay culture medium to the CAR-T test well. Then, 25 µL of CAR-T DP cells were introduced at 8 × 10⁻⁶ cells / well. 5 viable cells / mL (total 2×10⁻⁶) 4 (1 live cells / 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, 5% CO2, and humidified for 10 min (± 5 min).

[0220] After incubation, 50 µL of target cells were added at 8 × 10⁻⁶. 4 1 live cells / mL were added to all wells (final 4×10⁶ cells / mL). 3 (1 live cells / well), resulting in a final E:T ratio of 5:1. In each assay, 0.1% Triton X-100 (50 µL / well) was added last to the total cell death control wells. The well contents were incubated at 37°C, 5% CO2, and humidified for 22 h (± 1 h). After co-incubation, the assay plate was removed from the incubator and allowed to equilibrate at room temperature for 30 min (+ / - 5 min). The assay reagents were thawed while maintaining equilibration to room temperature for 30 min. Once equilibrated, 100 µL of KILR was added to each well. ® The test reagents were incubated in the dark for 50 minutes (+ / - 5 minutes). After shaking for 10 seconds, the plate was read on a Molecular Devices Paradigm plate reader designed for chemiluminescence.

[0221] The data are shown in Tables 2 and 3 below. Table 2 shows the batch titration of sBCMA protein, demonstrating that BCMA blocks the killing of KILR MM-1R multiple myeloma target cells by LCAR-B38M in the range of 50 μg / mL to 400 μg / mL. Table 3 shows that sBCMA has blocking specificity against multiple myeloma target cells (RPMI8226_Luc) compared to non-B cell target cells (K562_luc cells).

[0222] Table 2: BCMA titration demonstrates the activity of protein-blocking CAR-T cells against target cells. .

[0223] Table 3: Demonstrating specificity for multiple myeloma and non-B cell lineages .

[0224] Example 2 GPRC5D CAR-T cells were tested using an anti-ID antibody against the CAR-T cells. The assay conditions for GPRC5D CAR-T cells were similar to those used for LCAR-B38M in Example 1 above. For GPRC5D CAR-T, another multiple myeloma target cell, the same KILR MM-1R target cells were used. The assay medium and reagents were the same as those used in Example 1. The 5:1 E:T ratio and seeding density were the same as in Example 1. The blocking reagent was a GCPR5D anti-idiotyping antibody against GPRC5D CAR. Examples of GCPR5D anti-idiotyping antibodies used for this assay include GP5B337, GP5B332, GP5B324, and GP5B206. The heavy and light chain sequences of these anti-idiotyping antibodies are provided in Table 5. The incubation time was consistent with that in Example 1 above. All other reagents were the same as those used in Example 1. The initial titration study shown in Table 4 below indicates the use of anti-ID antibody (GP5B337) to block GPRC5DCAR-T cells.

[0225] Table 4: GPRC5D CAR-T Titration Data .

[0226] Table 5. Exemplary heavy and light chain sequences of GCPR5D anti-idiotype antibodies Example 3 GPRC5D CAR-T cells were tested using an anti-ID antibody Fab fragment targeting CAR-T cells. The assay conditions for GPRC5D CAR-T cells were similar to those used for LCAR-B38M in Example 1 above. For GPRC5D CAR-T, another multiple myeloma target cell, the same KILR MM-1R target cells were used. The assay medium and reagents were the same as those used in Examples 1 and 2. The 5:1 E:T ratio and seeding density were the same as in Examples 1 and 2. The blocking reagent was a GCPR5D anti-idiotypic Fab antibody (GP5B337) fragment targeting GPRC5D CAR. The incubation time was consistent with that in Examples 1 and 2 above. All other reagents were the same as those used in Examples 1 and 2. The initial titration studies shown in Table 6 below indicate the blocking of GPRC5D CAR-T cells with the anti-ID Fab antibody (GP5B337) fragment.

[0227] Table 6. Titration data of GPRC5D CAR-T anti-ID Fab fragment The GPRC5D anti-idiotype Fab fragment was generated from a full-length anti-idiotype antibody against GPRC5DCAR using the Pierce Fab Preparation Kit (ThermoFisher, catalog number: VF299292), with slight modifications to the kit's instructions. In short, BupH phosphate-buffered saline (PBS) and digestion buffer were prepared. IgG was removed from the refrigerator, diluted with the prepared PBS, and passed through a desalting column. The eluent was collected and transferred to a prepared papain digestion column for digestion at 37°C. After digestion and rotation at 37°C for approximately 5 hours, the tubes were removed from the incubator and centrifuged to collect the sample. The Protein A column from the kit was prepared, the sample was added, and the column was rotated overnight at 2°C to 8°C for approximately 20 hours. The Protein A column was centrifuged to collect the sample in fractional form. The first and second fractions were collected, combined, and stored. Previous work such as A280 and 1D silver staining has confirmed that these fractions contain the most desired fragment. Prepare a fresh, unused protein A column, add the sample again, and perform a second rotation at 2°C to 8°C for approximately 1 hour. Perform a second incubation on protein A to further purify and remove any potential Fc region contaminants considered to affect bioassays. Elute the fragment, concentrate it in 10K Amicon tubes (catalog number UFC501008), and store at 2°C to 8°C.

[0228] Example 4 GPRC5D CAR-T cells were tested using an anti-GPRC5D antibody or its Fab fragment against an antigen (GPRC5D receptor) targeting CAR-T cells. The assay conditions for GPRC5D CAR-T cells were similar to those used for GPRC5D CAR-T cells in Example 2 above. For GPRC5D CAR-T, another multiple myeloma target cell, the same KILR MM-1R target cells were used. The assay medium and reagents were the same as those used in Examples 2 and 3. The 5:1 E:T ratio and seeding density were the same as in Examples 2 and 3. The blocking reagents were anti-GPRC5D antibodies or their Fab antibody fragments. 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 their target. The anti-GPRC5D antibody Fab was produced from the full-length anti-GPRC5D antibody using the established method detailed above. The incubation time was consistent with that in Examples 2 and 3. All other reagents were the same as those used in Examples 2 and 3. The initial titration studies shown in Table 7 below indicate the use of anti-GPRC5D antibody or Fab fragment to block KILR MM-1R cells.

[0229] Table 7. Titration protocol for GPRC5D CAR-T anti-ID Fab fragment Example 5 KLK2 CAR-T cells were tested using a soluble human KLK2 protein targeting CAR-T cells. The assay conditions for KLK2 CAR-T cells were similar to those used for LCAR-B38M in Example 1 above. KLK2 CAR-T targets the kallikerin 2 (KLK2) molecule expressed on malignant luminal prostate cells. KLK2-overexpressing reporter cell lines were generated using LNcaP cells (ATCC, CRL1740) and DiscoverX Killer Immunoreplication (KILR) reporter cells. The killing mechanism was the same as described in Example 1. These cells expressed an enhanced Prolabel (ePL)-tagged housekeeping gene, and upon cell lysis, the tagged reporter protein was released into the culture medium. The addition of an enzyme receptor caused the β-galactosidase fragment EA to be complementary to ePL. The resulting functional enzyme hydrolyzed its substrate to generate a chemiluminescent signal. The assay medium, assay reagents, and target seeding density were the same as those used in Example 1, but the E:T ratio was 10:1. The blocking agent is a soluble protein of the 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): MWDLVLSIALSVGCTGA VPLIEGRIVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWVLTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHDLMLLRLSEPAKITDVVKVLGLPTQ EPALGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKPAVYTKVVHYRKWIKDTIAANPHHHHHH (SEQ ID NO: 9) KILR LNcaP-KLK2 cells were grown in RPMI 1640 (Gibco catalog 11875-093, ThermoFisher, Waltham, MA) containing 10% FBS (97068-085, VWR, Radnor, PA) and 250 µg / mL G418 sulfate (Corning catalog 30-234-CR, Corning, Tewksbury, MA). Assays were performed in assay medium consisting of RPMI 1640 (Corning catalog 10-040-CV, Corning, Tewksbury, MA) containing L-glutamine and 10% FBS (97068-085, VWR, Radnor, PA).

[0230] Although the target cell line in this embodiment is different from that in Embodiments 1 and 2, the strategy and setup are similar to those in Embodiments 1 and 2.

[0231] The following assay conditions were used for KLK2 CAR-T DP target cells and KILR LNcaP-KLK2 target cells. The assay medium was RPMI 1640 and 10% HI-FBS. KILR LNcaP-KLK2 cells were grown in RPMI 1640 and 10% FBS, 1x non-essential amino acids, 2.5 ug / mL puromycin, and 500 ug / mL G418 sulfate. Baseline controls were obtained and the assay was optimized according to Example 1. Baseline controls were generated by blocking KLK2 CAR-T DP cells with soluble human KLK2 protein (sKLK2). KILR ®The assay kit (Eurofins Discoverx Corp., Fremont CA) was used as the assay reagent. 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 were optimized. The E:T ratio and blocking reagent concentration were optimized for each drug product and its corresponding target cell line. Once determined, the E:T ratio was a fixed value in the assay and remained constant for all drug product tests. The blocking reagent was qualified for each batch and used at this level for drug product testing. Furthermore, the assay conditions were optimized based on the reporting system used.

[0232] The KLK2 CAR-T drug product was assayed in a 96-well white opaque, TC-treated assay plate as described in Example 1. The assay procedure is as follows, with each assay plate accommodating a maximum of 6 assays, as described in Table 1A of Example 1. For each assay, 25 µL of blocking reagent was added to the CAR-T cell blocking well (baseline control) of the assay plate, followed by 25 µL of assay culture medium to the CAR-T test well. Then, 25 µL of CAR-T DP cells were introduced at a concentration of 1.6 × 10⁻⁶. 6 viable cells / mL (total 4×10⁴) 4 (Number of live cells / 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, 5% CO2, and humidified for 15 min (± 5 min).

[0233] After incubation, 50 µL of target cells were added at 8 × 10⁻⁶. 4 1 live cells / mL were added to all wells (final 4×10⁶ cells / mL). 3 (1 live cells / well), resulting in a final E:T ratio of 10:1. The contents of the wells were incubated at 37°C, 5% CO2, and humidified for 20 h (± 2 h). After co-incubation, the assay plate was removed from the incubator and allowed to equilibrate at room temperature for 30 min (+ / - 5 min). The assay reagents were thawed and equilibrated to room temperature for 30 min. In each assay, once equilibration was complete, 0.1% Triton X-100 (50 µL / well) was added to the total cell death control wells, followed by the addition of 100 µL KILR to each well. ® The test reagent was incubated for 50 minutes (+ / - 5 minutes) in the dark. After shaking for 10 seconds, the plate was read on a Molecular Devices Paradigm plate reader designed for chemiluminescence.

[0234] The data are shown in Table 8 below. The data demonstrate that the fractional titration of sKLK2 protein showed that KLK2 blockade of KLK2 CAR-T DP killed KILR LNcaP-KLK2 target cells in the range of 10 ug / mL to 500 ug / mL.

[0235] The incubation time was consistent with that in Example 1 above. All other reagents were the same as those used in Example 1. The initial titration studies shown in Table 8 below indicate the blocking of KLK2 CAR-T cells with KLK2 soluble protein (internal, KL2W12.009).

[0236] Table 8. KLK2 CAR-T titration data This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. It should also be understood that all values ​​are approximate and are provided for illustrative purposes.

[0237] This application incorporates throughout patents, patent applications, publications, product descriptions, and solutions, the full contents of which are incorporated herein by reference for all purposes.

Claims

1. An in vitro method for determining the potency of immune cells expressing chimeric antigen receptor (CAR) molecules, the method comprising: a) In a test sample, the CAR-expressing immune cells are brought into contact with target cells, wherein the target cells express an antigen that interacts with the CAR. b) In a first control sample, the CAR-expressing immune cells are contacted with the target cells, wherein (i) the contact is made in the presence of an inhibitory molecule, or (ii) prior to the contact, the CAR-expressing immune cells and / or the target cells have been pre-incubated with the inhibitory molecule, wherein the inhibitory molecule inhibits the interaction between the CAR and the target cells. c) Determine the amount of target cell death in the test sample. d) Determine the amount of target cell death in the first control sample, and e) Determine the potency of CAR-expressing immune cells based on a comparison of the amount of target cell death measured in steps (c) and (d). In both the test sample and the first control sample, the contact time, the amount of CAR-expressing immune cells, and the amount of target cells are substantially the same.

2. The method according to claim 1, wherein the contacting steps (a) and (b) are performed simultaneously.

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

4. The method according to claim 1, wherein in step (b)(i), the CAR-expressing immune cells and / or the target cells have been pre-incubated with the inhibitory molecule prior to the contact step.

5. The method of claim 1, wherein 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 were incubated in the absence of the CAR-expressing immune cells.

6. The method of claim 1, wherein 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 were incubated in the absence of the CAR-expressing immune cells but in the presence of a detergent that causes the target cell death.

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

8. The method of claim 1, wherein the target cell generates a detectable report signal upon target cell death, and step (c) includes measuring the report signal in the test sample, step (d) includes measuring the report signal in the first control sample, and step (e) includes comparing the report signals measured in steps (c) and (d).

9. The method of claim 8, wherein the reporting signal is light emission.

10. The method of claim 8, wherein the reporting signal is fluorescence.

11. The method of claim 8, wherein the target cell expresses a reporter protein that generates a signal when the target cell undergoes cell death.

12. The method of claim 11, wherein the reporter protein is β-galactosidase, luciferase, green fluorescent protein (GFP), or variants or derivatives thereof.

13. The method of claim 1, wherein the inhibitory molecule specifically binds to the antigen on the target cell that interacts with the CAR.

14. The method of claim 1, wherein the inhibitory molecule specifically binds to the CAR.

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

17. The method of claim 16, wherein the antibody is an anti-idiotype antibody.

18. The method of claim 16, 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.

19. The method of claim 16, wherein the antibody or antibody fragment specifically binds to the antigen within the scFv domain of the CAR.

20. The method of claim 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 of claim 16, wherein the antibody or antibody fragment specifically binds to the antigen within the VH domain or VL domain of the CAR.

22. The method of claim 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 of claim 14, wherein the inhibitory molecule is a soluble form of the antigen expressed on the target cell that interacts with the CAR, or a functional fragment or derivative thereof.

24. The method of claim 1, wherein the immune cells are selected from T cells, induced pluripotent stem cells (iPSCs), and natural killer (NK) cells.

25. The method of 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 a soluble cytoplasmic domain of BCMA.

26. The method of claim 25, wherein the target cell is a multiple myeloma cell.

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

28. The method of claim 1, wherein the CAR interacts with a G protein-coupled receptor class C5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the CAR.

29. The method of claim 1, wherein the CAR interacts with a G protein-coupled receptor class C5 member D (GPRC5D), the target cell contains the GPRC5D receptor, and the inhibitory molecule is an anti-idiotypic antibody or antibody fragment of the GPRC5D receptor.

30. The method of claim 28, wherein the target cell is a multiple myeloma cell.

31. The method of claim 30, wherein the multiple myeloma cells are MM-1R cells.

32. The method of claim 1, wherein the CAR interacts with kallikerin 2 (KLK2), the target cell contains the KLK2, and the inhibitory molecule is a soluble KLK2 protein.

33. The method of claim 32, wherein the target cell is a prostate cancer cell.

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

35. The method of claim 1, wherein the method is performed in a high-throughput manner.

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