Car comprising cd28 zeta and cd3 zeta

Abstract

To provide compositions and methods for targeted treatment of cancer.SOLUTION: Disclosed are compositions and methods for targeted treatment of cancer. The present disclosure provides chimeric antigen receptors and cells expressing such chimeric antigen receptors. In certain embodiments, engineered cells expressing the chimeric antigen receptors are specific for a low density cancer antigen or peptide in groove antigen.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 020,177, filed May 5, 2020, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Cancer is the second leading cause of death in the United States. Current treatments for many cancers are ineffective in certain patient populations or cause toxic side effects that greatly impact a patient's quality of life. Adoptive immunotherapy involving the transfer of ex vivo-generated antigen-specific T cells is a promising strategy for cancer treatment. The T cells used in adoptive immunotherapy can be generated, for example, by genetic modification of the T cells (e.g., by engineering them to express a chimeric antigen receptor or “CAR”). However, current approaches to adoptive immunotherapy are often ineffective when targeting low-density-expressed antigens such as peptide antigens presented by MHC class I proteins (referred to as “peptide in groove” or “PIG” antigens). Targeting of PIG antigens using adoptive immunotherapy is particularly attractive for many reasons, such as enabling the use of CAR polypeptide-expressing T cells (CAR T cells) that target intracellular cancer-related antigens. Thus, there is a need in the art for improved approaches to adoptive immunotherapy that facilitate targeting of low-density antigens, including PIG antigens, for the treatment of cancer.

Summary of the Invention

[0003] This application is based at least in part on the discovery that CAR T cells expressing a CAR comprising a CD28 / z signaling domain exhibit excellent in vitro and in vivo efficacy when targeting a grooved peptide (PIG) antigen or a low-density cancer antigen. In some aspects, provided herein are CAR polypeptides that target a PIG antigen or a low-density cancer antigen and comprise a CD28 / z signaling domain, nucleic acids encoding such CAR polypeptides, and methods of using and making such CAR polypeptides and nucleic acids.

[0004] In some aspects, provided herein are CAR polypeptides comprising at least one intracellular / costimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain. In some embodiments, the CAR polypeptide further comprises at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the CAR polypeptide further comprises an antigen-binding domain specific for a low-density cancer antigen and / or a grooved peptide antigen.

[0005] In some embodiments, the intracellular / costimulatory region of the CAR polypeptide further comprises a 4-1BB domain (e.g., in addition to a cluster of differentiation 28 zeta (CD28 / ζ) domain). The CAR polypeptide may comprise a hinge / spacer region comprising at least one cluster of differentiation 28 zeta (CD28 / ζ) domain. The CAR polypeptide may comprise a transmembrane region comprising at least one cluster of differentiation 28 zeta (CD28 / ζ) domain.

[0006] In some embodiments, the CAR polypeptides disclosed herein have antigen specificity for a cancer antigen, wherein the CAR comprises at least one differentiation antigen group 28 zeta (CD28 / ζ) amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1-3. The CD28 / ζ sequence may be within the hinge domain, transmembrane domain, or signaling domain of the CAR. Also provided herein are CAR polypeptides having antigen specificity for a cancer antigen, wherein the CAR comprises the amino acid sequences set forth in SEQ ID NOs: 6-13, 29, 30, 33, or 34.

[0007] The CAR polypeptide may comprise at least one differentiation antigen group 28 (CD28) amino acid sequence that is part of the intracellular / costimulatory region of the CAR. The CAR polypeptide may have at least one differentiation antigen group 28 (CD28) amino acid sequence that is part of the transmembrane region of the CAR. The CAR polypeptide may comprise at least one differentiation antigen group 28 (CD28) amino acid sequence that is part of the hinge / spacer region of the CAR.

[0008] As disclosed herein, the cancer antigen can be a groove peptide antigen and / or a low density cancer antigen. Less than 2,000 copies, less than 1,000 copies, or less than 500 copies of the cancer antigen may generally be found in tumors (e.g., solid tumors). The cancer antigen can be MAGEA4, tyrosinase, HPV16 E7, or an NY-ESO peptide. The cancer antigen is MAGEA4 230-239 、MAGEA4 286-294 、Tyr 369-377 、HPV16E7 11-19 、or NY-ESO-1 157-165 and may comprise at least one epitope selected from.

[0009] The CAR polypeptide may comprise a variable light chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 16-19, 31, or 37. The CAR polypeptide may comprise a variable heavy chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 20-23, 32, or 38.

[0010]

[0010] In some embodiments, provided herein are nucleic acids encoding the CAR polypeptides disclosed herein. The nucleic acids can be expression vectors (e.g., viral vectors such as lentiviral vectors). The nucleic acids encoding the CAR polypeptides can have the sequences set forth in SEQ ID NO: 14, 15, 35, or 36.

[0011]

[0011] Also provided herein are immune cells comprising the CAR polypeptides disclosed herein or nucleic acids encoding the CAR polypeptides disclosed herein. Also provided herein are compositions comprising the immune cells disclosed herein and cell banks comprising such immune cells. The cell bank can be a cell bank for adoptive immunotherapy. The immune cells can be leukocytes, lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, or eosinophils. In some embodiments, the immune cells are lymphocytes selected from αβ T cells, γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof.

[0012] In some embodiments, provided herein is a method of treating cancer (e.g., solid tumors) in a subject by administering to the subject a composition comprising cells expressing the CAR polypeptides disclosed herein (e.g., CAR polypeptides comprising at least one intracellular / costimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain). In some embodiments, the CAR polypeptide further comprises at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the CAR polypeptide further comprises an antigen-binding domain specific for a low-density cancer antigen and / or a groove peptide antigen.

[0013] In some embodiments, the methods described herein further comprise co-administering to a subject a second CAR polypeptide comprising a 4-1BB domain in the co-stimulatory region of the CAR polypeptide. In some embodiments, the second CAR comprises at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the second CAR comprises an extracellular domain specific for a low density cancer antigen and / or a groove peptide cancer antigen. The second CAR polypeptide may comprise a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region.

[0014] In some embodiments, the subject may have cancer. The cancer may be melanoma.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

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Figure 4B

Figure 5A

Figure 5B

Figure 5C

Figure 6A

Figure 6B

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0016] Overview In some aspects, provided herein are a CAR polypeptide comprising at least one intracellular / costimulatory region comprising a cluster of differentiation 28 zeta (CD28 / ζ) domain, and a polynucleotide encoding the CAR polypeptide. The CD28 / ζ domain may be a human CD28 / ζ domain. In some embodiments, the CAR polypeptide further comprises at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. The CAR polypeptide may also comprise an antigen-binding domain specific for a low-density cancer antigen and / or a groove peptide antigen. Also provided herein are immune cells expressing the CAR polypeptides disclosed herein, and cell banks comprising immune cells expressing the CAR polypeptides disclosed herein.

[0017] As disclosed herein, cancers characterized by PIG antigens or low-density cancer antigens often escape conventional cancer treatments because they are present at low target copy numbers within the tumor. In addition, solid tumors characterized by PIG or low-density cancer antigens are more resistant to CAR-T therapy and may be more difficult to treat because they are not cell surface antigens but are present in grooves within cancer-related peptides. Thus, there is a need for the improved CAR therapies provided herein that can effectively target PIG and / or low-density cancer antigens to enhance the effectiveness of CAR therapy for cancers, particularly cancers characterized by solid tumors.

[0018] The methods disclosed herein include methods of treating tumors in a subject by administering a composition comprising cells expressing a CAR polypeptide disclosed herein. In some embodiments, the CAR polypeptides disclosed herein are specific for a low density cancer antigen or a PIG antigen. Low density cancer antigens include, but are not limited to, any cancer antigen that has less than 5,000, less than 4,000, less than 3,000, less than 2,000, less than 1,500, less than 1,000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, or less than 100 copies of the cancer antigen, which are generally found in solid tumors. The cancer antigen can be MAGEA4, tyrosinase, HPV16 E7, or an NY-ESO peptide. The cancer antigen is MAGEA4 230-239 、MAGEA4 286-294 、Tyr 369-377 、HPV16E7 11-19 、or NY-ESO-1 157-165 and may include an epitope selected from. For example, the CAR can include an antigen-binding domain, or a portion thereof, using the variable light chain and / or heavy chain of an antibody specific for a low density or PIG antigen, such as an epitope sequence of MAGEA4, tyrosinase, HPV16 E7, or an NY-ESO peptide.

[0019] Definitions Here, for convenience, certain terms used in this specification, the examples, and the appended claims are listed.

[0020] The articles "a" and "an" are used herein to refer to the grammatical object of the article being one or more than one (i.e., at least one). By way of example, "an element" means one element or more than one element.

[0021] As used herein, the term "administer" means to provide a pharmaceutical agent or composition to a subject, including, but not limited to, administration by a medical professional and self-administration. Such an agent can include, for example, the CAR T cells provided herein.

[0022] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both an amino functional group and an acidic functional group and that can be included in a natural amino acid polymer. Exemplary amino acids include natural amino acids; their analogs, derivatives, and homologs; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0023] The term "bind" or "interact" refers to a stable association between two molecules, e.g., between a peptide and a binding partner or agent, e.g., a small molecule, which may be due to, for example, electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions under physiological conditions.

[0024] As used herein, the term "cancer" includes, but is not limited to, solid tumors and blood-derived tumors. The term "cancer" includes, but is not limited to, diseases of the skin, tissue, organs, bone, cartilage, blood, and blood vessels, including those of the neck, anus, vagina, vulva, penis, base of the tongue, larynx, and tonsils. The term "cancer" further encompasses primary and metastatic cancers.

[0025] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, e.g., antibody-based specificity for a desired antigen (e.g., a tumor antigen), with a T cell receptor activation intracellular domain to generate a chimeric protein that exhibits specific anti-target cell immune activity. Generally, a CAR consists of an extracellular single-chain antigen-binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain and, when expressed in T cells, has the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0026] The term "costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates, without limitation, a costimulatory response by the cell, such as proliferation. The costimulatory domain can be a human costimulatory domain. Exemplary costimulatory molecules include CD28, 4-1BB, CD27, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0027] The term "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell and thereby provides a signal that mediates, without limitation, a T cell response, including proliferative activation, differentiation, etc. Costimulatory ligands include, without limitation, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, an agonist or antibody that binds to the Toll ligand receptor, and a ligand that specifically binds to B7-H3.

[0028] The term "costimulatory signal" refers to a signal that, in combination with a primary signal, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0029] The term "epitope" means a protein determinant capable of specifically binding to an antibody or an immune cell (e.g., a T cell). An epitope usually consists of chemically active surface groups of a molecule, such as amino acids or sugar side chains. A particular epitope can be defined by a specific sequence of amino acids to which a CAR or an antibody can bind.

[0030] The term "gene construct" refers to nucleic acids such as vectors, plasmids, viral genomes, etc., which contain the "coding sequence" of a polypeptide or, if not, can be transcribed into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), can be transfected into cells, such as mammalian cells, and can cause the expression of the coding sequence in the cells transfected with the construct. A gene construct can include one or more regulatory elements operably linked to the coding sequence, as well as intervening sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0031] The terms "ligand binding domain" and "antigen binding domain" are used herein interchangeably and refer to the portion of a chimeric antigen receptor that specifically binds to a given antigen. The term "linker" is recognized in the art and refers to a molecule or group of molecules that connect two compounds, such as two polypeptides. A linker may consist of a single linking molecule or may include a spacer molecule that is intended to separate the linking molecule and the compound at a specific distance.

[0032] The term "operably linked" refers to the functional relationship between a nucleic acid and another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, the operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and the promoter such that transcription of the DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes this DNA.

[0033] As used herein, the phrase "pharmaceutically acceptable" refers to agents, compounds, materials, compositions, and / or formulations that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0034] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic and compatible substances used in pharmaceutical formulations.

[0035] The terms "polynucleotide" and "nucleic acid" are used interchangeably. These refer to natural or synthetic molecules, or some combination thereof, that include a single nucleotide, or two or more nucleotides linked by phosphate groups at the 3'-position of one nucleotide to the 5'-end of another nucleotide. The polymeric form of a nucleotide has no length limitation and may include deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function. Non-limiting examples of polynucleotides include, but are not limited to, the coding or non-coding regions of a gene or gene fragment, locus / loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may include modified nucleotides such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. A polynucleotide can be further modified, such as by conjugation to a label component. In all nucleic acid sequences provided herein, U nucleotides are replaceable with T nucleotides. A polynucleotide is not necessarily associated with a cell in which the nucleic acid occurs in nature and / or operably linked to a polynucleotide linked in nature.

[0036] The term "precancerous lesion" or "precancerous condition" refers to abnormal cells and / or tissues associated with an increased risk of cancer. The term "precancerous lesion" may refer to, for example, dysplasia, benign neoplasia, or carcinoma in situ.

[0037] As used herein, a therapeutic agent that "prevents" a disease state is a compound that, when administered to a statistical sample prior to the onset of a disorder or disease state, reduces the occurrence of the disorder or disease state in the treated sample compared to an untreated control sample, or delays or reduces the severity of the occurrence of one or more symptoms of the disorder or disease state compared to an untreated control sample.

[0038] As used herein, the "signaling domain" or "signal transduction domain" of a CAR is responsible for intracellular signal transduction after the extracellular ligand-binding domain binds to a target, thereby resulting in the activation of immune cells and immune responses. In other words, the signaling domain is responsible for activating at least one of the normal effector functions of immune cells in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity including cytokine secretion. Thus, the term "signaling domain" refers to the portion of a protein that introduces an effector function signal and induces the cell to perform a function specific to the cell. Examples of signaling domains for use in a CAR may be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction after antigen receptor ligation, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability. In some cases, the signal transduction domain includes two different classes of cytoplasmic signal transduction sequences, a class that initiates antigen-dependent primary activation, and a class that acts antigen-independently to provide secondary or co-stimulatory signals. The primary cytoplasmic signal transduction sequence may include a signal transduction motif known as an immunoreceptor tyrosine-based activation motif of an ITAM. An ITAM is a well-defined signal transduction motif found in the cytoplasmic tails of various receptors that functions as a binding site for tyrosine kinases of the syk / zap70 class. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0039] "Spacer", as used herein, refers to a peptide that links proteins (e.g., those within a fusion protein). Generally, a spacer has no special biological activity other than linking proteins together or maintaining some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer can be selected to affect some properties of the molecule, such as the folding of the molecule, the net charge, or the hydrophobicity.

[0040] As used herein, the terms "specifically binds" or "specific binding", when referring to a polypeptide (including a CAR polypeptide), refer to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous mixture of proteins and other biological agents. Thus, under specified conditions (e.g., immunoassay conditions in the case of an antibody), a particular ligand or antibody "specifically binds" to its particular "target" when it does not bind significantly to other proteins present in the sample or to other proteins that the ligand or antibody might encounter in vivo (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule binds to that second molecule with a dissociation constant greater than about 10 5 M -1 (e.g., 10 6 M -1 、10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、and 10 12 M -1It has an affinity constant (Ka) of or greater than. For example, with respect to the ability of a PIG-specific CAR to bind to a peptide presented on MHC (e.g., class I MHC or class II MHC), typically, the CAR specifically binds to the peptide / MHC with an affinity of at least about 10-4 M or less KD, and has an affinity (expressed as KD) for a given antigen / binding partner that is at least one-tenth, at least one-hundredth, or at least one-thousandth of the affinity when binding to non-specific and non-related peptide / MHC complexes (e.g., those containing BSA peptide or casein peptide).

[0041] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy. The terms "transformation", "transfection", or "transduction" mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell (e.g., a mammalian cell), including the introduction of the nucleic acid into the chromosomal DNA of the cell.

[0042] As used herein, the term "treatment" refers to a clinical intervention designed to modify the natural course of an individual being treated during the course of a clinical condition. Desirable effects of treatment include deceleration, alleviation, or reduction of the rate of progression of a pathological condition, as well as remission or improvement of the prognosis of a particular disease, disorder, or condition. For example, if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated, the individual is considered to have been "treated" normally.

[0043] The term "variant" refers to a conservative amino acid substitution, a non-conservative amino acid substitution (e.g., a denaturing variant), a substitution within the wobble position of each codon encoding an amino acid (e.g., DNA and RNA), an amino acid added to the C-terminus of a peptide, or an amino acid or peptide sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0044] The term "vector" refers to a means by which nucleic acids can be propagated and / or transferred between organisms, between cells, or between cell components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, and artificial chromosomes, etc., to which nucleic acids can be ligated and which may or may not be able to replicate autonomously, or may or may not be able to integrate into the host cell chromosome. Such vectors can include any vector (e.g., plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by a cell (e.g., linked to transcriptional control elements).

[0045] In certain embodiments, the agent may be used alone or co-administered with another type of therapeutic agent. As used herein, the phrases "co-administered" or "co-administered" refer to any form of administration of two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still efficacious in the body (e.g., including the synergistic effect of the two agents, such that the two agents are simultaneously efficacious in the subject). For example, different therapeutic agents may be administered simultaneously or sequentially, in the same formulation or in separate formulations. In certain embodiments, different therapeutic agents may be administered to each other within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week. Thus, a subject receiving such treatment can benefit from the combined effects of different therapeutic agents.

[0046] Chimeric antigen receptor (CAR) It has been successful so far to generate new specificities in T cells through genetic transfer of a transgenic T cell receptor or chimeric antigen receptor (CAR). A CAR is a receptor that contains a targeting moiety that binds to one or more signaling domains and / or co-stimulatory domains within a single fusion molecule. In certain embodiments, the binding moiety of the CAR comprises the antigen-binding domain of a single-chain antibody (scFv) comprising the variable light and heavy chain fragments of a monoclonal antibody linked by a flexible linker. In certain embodiments, the binding moiety further comprises the transmembrane and hinge domains of a monoclonal antibody.

[0047] In certain embodiments, the CAR polypeptides provided herein include at least one intracellular / costimulatory region that includes a cluster of differentiation 28 zeta (CD28 / ζ) domain. In addition, the hinge / spacer region and / or transmembrane region of the CAR, or the transmembrane region of the CAR, may include a CD28 / ζ domain. The CAR may include at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1-3. The CAR polypeptides disclosed herein have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences set forth in SEQ ID NOs: 1-3 and may include at least one cluster of differentiation 28 zeta (CD28 / ζ) amino acid sequence. The CAR polypeptide may include all three sequences set forth in SEQ ID NOs: 1-3. For example, the CAR hinge domain may include SEQ ID NO: 2 and the costimulatory domain may include SEQ ID NO: 3.

[0048]

Table 1

[0049] In some embodiments, the CAR includes an antigen-binding domain that is specific for a low-density cancer antigen and / or a groove peptide antigen. Exemplary PIG antigens and low-density cancer antigens can be identified in Table 1. In some embodiments, the CAR polypeptides disclosed herein specifically bind to the epitopes listed in Table 2. In some embodiments, the present disclosure provides a melanoma-associated antigen A4 (MAGE-A4)-specific chimeric antigen receptor (CAR), wherein the MAGE-A4-specific chimeric antigen receptor interacts with amino acids 286-294 of SEQ ID NO: 4, i.e., a portion thereof. The polypeptide sequence (286-294) of MAGE-A4 is shown in SEQ ID NO: 5.

[0050]

Table 2

[0051]

Table 3

[0052] Furthermore, exemplary PIG epitopes may be derived from melanoma-associated antigen 3 (MAGE-A3); melanoma-associated antigen 1 (MAGE-1); melanoma-associated antigen 10 (MAGEA10); melanoma antigen recognized by T cells 1 (MART-1), melanoma antigen recognized by Epstein-Barr virus (EBV) latent membrane protein (LMP2); mouse double minute 2 (MDM2); melanoma-associated antigen 1 (MAGE-A1); glycoprotein 100 (gp100); tumor protein p53 (P53); minor histocompatibility antigen (mHag); minor histocompatibility antigen HA-1 (HA-1); ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome (UTY); ribosomal protein S4, Y-linked (RPS4Y); MHC class II-restricted DEAD-box type RNA helicase Y (DBY); melanoma cytotoxic T lymphocyte (CTL) recognition antigen (CAMEL); Wilms tumor 1 (WT1); renal cell carcinoma (RCC) tumor antigen; mouse mastocytoma P815; leucine zipper protein 4 (LUZP4); cancer / testis-related SPANX antigen; ATPase family AAA domain-containing protein 2 (ATAD2); Rhox homeobox family member 2 (RHOXF2); cancer / testis antigen 136; F-Box protein 39 (FBXO39); TDRD4; WW domain-binding protein 2 N-terminal-like (WBP2NL) or carcinoembryonic antigen (CEA).

[0053] The present specification also provides a CAR polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 6-13, 29, 30, 33, or 34. The CAR polypeptides disclosed herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences set forth in SEQ ID NOs: 6-13, 29, 30, 33, or 34.

[0054]

Table 4-1

[0055]

Table 4-2

[0056]

Table 4-3

[0057]

Table 4-4

[0058]

Table 4-5

[0059]

Table 4-6

[0060]

Table 4-7

[0061]

Table 4-8

[0062]

Table 4-9

[0063]

Table 4-10

[0064]

Table 4-11

[0065]

Table 4-12

[0066]

Table 5-1

[0067]

Table 5-2

[0068]

Table 5-3

[0069]

Table 5-4

[0070]

Table 5-5

[0071]

Table 5-6

[0072]

Table 5-7

[0073]

Table 5-8

[0074]

Table 5-9

[0075]

Table 5-10

[0076] In certain embodiments, the CAR binding domains and / or extracellular domains provided herein provide a CAR that has the ability to bind to a target antigen of interest. A binding domain (e.g., a ligand binding domain or an antigen binding domain) can be any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and specifically bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). A binding domain can include any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for the biological molecule of interest. For example, as further described herein, the binding domain can be an antibody light chain and heavy chain variable regions, or the light chain and heavy chain variable regions can be single chain and in either orientation (e.g., V L -V H or V H -V L) can be linked together. Various assays are known for identifying the binding domains of the present disclosure that specifically bind to a particular target, such as Western blot, ELISA, flow cytometry, or surface plasmon resonance (e.g., using BIACORE analysis). The target can be a clinically important antigen that is desirable to induce an effector immune response that results in tumor killing. The CAR polypeptide can include a variable light chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 16-19, 31, or 37. The variable light chains disclosed herein can have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences set forth in SEQ ID NOs: 16-19, 31, or 37. The CAR polypeptide can include a variable heavy chain comprising any one of the amino acid sequences set forth in SEQ ID NOs: 20-23, 32, or 38. The variable heavy chains disclosed herein can have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences set forth in SEQ ID NOs: 20-23, 32, or 38.

[0077]

Table 6

[0078]

Table 7

[0079]

Table 8-1

[0080]

Table 8-2

[0081] In one embodiment, the binding domain of the CAR is a single-chain antibody (scFv) specific for an in-groove peptide or a low-density cancer antigen, and may be a murine, human, or humanized scFv. The single-chain antibody may be cloned from the V-region gene of a hybridoma specific for the desired target. Techniques that can be used for cloning the variable heavy chain (VH) and variable light chain (VL) regions are described, for example, in Orlandi et al., PNAS, 1989; 86:3833-3837. Thus, in certain embodiments, the binding domain comprises a binding domain derived from an antibody, but may also be a binding domain not derived from an antibody. The binding domain derived from an antibody can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, where the fragment is involved in binding to an antigen.

[0082] In certain embodiments, the CARs of the present disclosure may include linkers between various domains added for proper spacing and conformation of the molecules. For example, in one embodiment, there may be a linker between the binding domains VH or VL that can be 1 to 10 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor can be 1 to 20 or 20 amino acids in length. In this context, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In further embodiments, the linker can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges including the numbers described herein, for example, a linker 10 to 30 amino acids in length, are also included herein.

[0083] In certain embodiments, the linker suitable for the CARs described herein is a flexible linker. Suitable linkers can be readily selected and can be of any suitable different length, including from 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, 7 to 8 amino acids, from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0084] Exemplary flexible linkers include glycine polymers (G)n where n is at least one integer, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thus may be able to function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine approaches significantly more Φ-Ψ space than alanine and is much less restricted than residues with longer side chains. Those skilled in the art will understand that the design of the CAR can include a linker that is fully or partially flexible such that it can include a flexible linker as well as one or more moieties that impart a less flexible structure to provide the desired CAR structure.

[0085] Following the binding domain of the CAR, there may be a "spacer" or "hinge", which refers to a region that moves the antigen-binding domain away from the effector cell surface and enables appropriate cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of the CAR is typically between the transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified.

[0086] The "transmembrane" region or domain is the part of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell and facilitates the binding of the binding domain to the target antigen. In some embodiments, the transmembrane domain may be the CD3ζ transmembrane domain. Other transmembrane domains that can be used in some embodiments include those derived from CD8, CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is a synthetic compound, in which case the transmembrane domain contains mainly hydrophobic residues such as leucine and valine.

[0087] In certain embodiments, the CARs provided herein include an intracellular signaling domain. The intracellular signaling domain (also referred to herein as the "signaling domain") transmits the message of effective CAR binding to the target antigen into the interior of the immune effector cell to induce effector cell functions, such as the release of cytotoxic factors to CAR-bound target cells or other cellular responses induced by antigen binding to the extracellular CAR domain, for example, activation, cytokine production, proliferation, and cytotoxic activity. It includes a portion of the chimeric antigen receptor receptor protein involved in inducing cytotoxic activity.

[0088] In certain embodiments, the CARs provided herein include one or more immunoreceptor tyrosine-based activation motifs, i.e., ITAMs. Examples of ITAMs containing useful primary cytoplasmic signaling sequences include those derived from TCRζ, FcR gamma, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling domain of the CAR described herein is derived from CD3ζ.

[0089] In certain embodiments, the CARs provided herein further include a co-stimulatory domain. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors and provide the secondary signals necessary for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such co-stimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Accordingly, the present disclosure further provides exemplary co-stimulatory domains derived from CD28. By including one or more co-stimulatory signaling domains, the efficacy and proliferation of T cells expressing the CAR receptor can be enhanced. Also disclosed herein are CAR polypeptides in which the cytoplasmic / co-stimulatory region of the CAR polypeptide further includes a 4-1BB domain (e.g., in addition to the CD28 / ζ domain). The co-stimulatory region of such CAR polypeptides can include the full 4-1BB domain or a fragment thereof, and / or the full CD28 / ζ domain or a fragment thereof. The intracellular signaling and co-stimulatory signaling domains may be tandemly linked in any order at the carboxyl terminus of the transmembrane domain.

[0090] The CAR polypeptide sequences disclosed herein may include any one of the amino acid sequences set forth in SEQ ID NOs: 26 - 28. The CAR polypeptides disclosed herein may include at least an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequences set forth in SEQ ID NOs: 26 - 28.

[0091]

Table 9

[0092] Nucleic Acids and Vectors In certain embodiments, nucleic acids and polynucleotide vectors encoding the CAR polypeptides disclosed herein are also disclosed.

[0093] The nucleic acid sequences and regions encoding the disclosed CARs can be obtained using methods such as, for example, screening a library of cells expressing the gene using recombinant methods known in the art, such as standard techniques, deriving the gene from a vector known to contain it, or directly isolating it from cells and tissues containing it. Alternatively, the gene of interest can be made synthetically rather than by cloning.

[0094] Expression of the nucleic acid encoding the CAR is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0095] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. A vector is a vehicle into which a polynucleotide encoding a protein can be covalently inserted to cause expression of the protein and / or cloning of the polynucleotide. Such vectors are sometimes referred to as “expression vectors.” The isolated polynucleotide can be inserted into the vector using any suitable method known in the art. For example, without limitation, the vector may be digested with an appropriate restriction enzyme and then ligated to the isolated polynucleotide having compatible restriction ends. An expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence encoding at least a portion of a gene product that can be transcribed intracellularly. In most cases, the RNA molecule is then translated into a protein. An expression vector may contain various control sequences that refer to nucleic acid sequences necessary for transcription and optionally translation of a coding sequence operably linked within a particular host organism. Vectors and expression vectors can also contain nucleic acid sequences that perform other functions in addition to the control sequences that control transcription and translation, which will be discussed below. An expression vector can contain other elements. For example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification.

[0096] The expression vector can have the necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences like CMV, PGK, and EF1 alpha promoter, ribosome recognition and binding TATA box, and 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in its corresponding host cell. Other suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters including, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter may also be used. In certain embodiments, inducible promoters are also contemplated as part of the vector expressing the chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0097] The expression vector can have additional sequences such as 6×-histidine, c-Myc, and FLAG tags incorporated into the expressed CAR. Thus, the expression vector can be engineered to include 5' and 3' untranslated regulatory sequences that may sometimes function as enhancer sequences, a promoter region and / or terminator sequence that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest contained in the expression vector. The expression vector can also be engineered for replication and / or expression functions (e.g., transcription and translation) in a specific cell type, cell location, or tissue type. The expression vector may include a selectable marker for maintaining the vector within the host or recipient cell.

[0098] In various embodiments, the vector is a plasmid, an autonomous replication sequence, and a transposable element. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs)), bacteriophages (such as lambda phage or M13 phage), and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the Lenti-X (trademark) Bicistronic Expression System (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST (trademark), pLenti6 / V5-DEST (trademark), and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the CARs disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0099] In certain embodiments, the nucleic acid encoding the CAR is provided in a viral vector. The viral vector may be derived from, for example, a retrovirus (e.g., a foamy virus) or a lentivirus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. The viral vector can contain the coding sequences of the various chimeric proteins described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transcribing DNA, RNA, or other nucleic acids into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0100] In certain embodiments, the viral vector comprising the CAR coding sequence described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector containing structural and functional genetic elements primarily derived from retroviruses. The term "lentiviral vector" refers to a vector containing structural and functional genetic elements outside of the LTRs primarily derived from lentiviruses.

[0101] The retroviral vectors used herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). Examples of "retroviruses" further include retroviruses of the lentivirus family such as human T-cell leukemia virus, HTLV-1 and HTLV-2, as well as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0102] The lentiviral vector used in this specification refers to a vector derived from lentiviruses, which are a group (or genus) of retroviruses that cause slowly progressing diseases. Viruses included in this group are HIV (human immunodeficiency virus; HIV type 1 and HIV type 2); Visna - Maedi; Caprine arthritis encephalitis virus; Equine infectious anemia virus; Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using the methods by Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72:8463 - 8471 and Zufferey et al., J. Virol. 1998; 72:9873 - 9880).

[0103] The retroviral vectors to be used (i.e., both lentivirus and non-lentivirus) can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application No. WO89 / 07136; PCT Application No. WO89 / 02468; PCT Application No. WO89 / 05345; and PCT Application No. WO92 / 07573).

[0104] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) arrays for vector formation include, for example, genomic RNA and cDNA available from commercial suppliers such as the Type Culture Collection (ATCC), Rockville, Maryland. The sequences can also be chemically synthesized.

[0105] For the expression of the CAR, the vector can be introduced into a host cell to enable the expression of the polypeptide within the host cell. The expression vector may include various elements for controlling expression, such as, but not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selectable marker, and a signal sequence. These elements can be appropriately selected by those skilled in the art as described above. For example, the promoter sequence can be selected to promote the transcription of the polynucleotide within the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. The enhancer sequence can be selected to enhance the transcription of the polynucleotide. The selectable marker can be selected to enable the selection of host cells into which the vector has been inserted from those that have not, and for example, the selectable marker can be a gene conferring antibiotic resistance. The signal sequence can be selected to enable the transport of the expressed polypeptide outside the host cell.

[0106] Regarding the cloning of polynucleotides, the vector may be introduced into a host cell (an isolated host cell) to enable the replication of the vector itself and thereby the amplification of copies of the polynucleotide contained in the vector. Cloning vectors may generally contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription start sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication can be selected to facilitate the autonomous replication of the vector within the host cell.

[0107] In certain embodiments, the present disclosure provides an isolated host cell comprising the vectors provided herein. A host cell comprising a vector can be useful for the expression or cloning of the polynucleotide contained within the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose can include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescens, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, and Pseudomonas such as P. aeruginosa and Streptomyces.

[0108] The CAR is introduced into the host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid may be maintained transiently or introduced stably. Transfection can be performed by various methods known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistic methods. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by viral infection instead of transfection. In certain embodiments, the retroviral vector is transduced by packaging the vector into virions prior to contact with the cell. For example, the nucleic acid encoding the CAR carried by the retroviral vector can be transduced into the cell via infection and proviral integration.

[0109] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cell can include either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of the expressing cells from the population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate fragment of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be located adjacent to appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0110] Reporter genes are used to identify cells that may have been transfected and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression is revealed by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct containing the minimal 5′ flanking region that shows high-level expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate agents for their ability to regulate promoter-induced transcription.

[0111] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0112] A representative delivery vehicle in cases where a non-viral delivery system is utilized is a liposome. In another aspect, the nucleic acid may be bound to a lipid. The nucleic acid bound to a lipid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that binds to both the liposome and the oligonucleotide, trapped within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, included as a suspension in a lipid, included with or complexed with a micelle, or otherwise bound to a lipid. The lipid, lipid / DNA, or lipid / expression vector binding composition is not limited to any particular structure in solution. For example, they may exist as micelles or in a bilayer structure with a "collapsed" structure. They may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape in some cases. Lipids are lipid substances that may be natural or synthetic lipids. For example, lipids include classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acid droplets that occur naturally within the cytoplasm, as well as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids for use are available from commercial suppliers. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, Missouri; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, New York); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Alabama).

[0113] immune effector cells In certain embodiments, immune effector cells engineered to express the disclosed CAR polypeptides are also disclosed herein. In some embodiments, the cells can be harvested from the subject being treated (i.e., are autologous). However, in certain embodiments, immune effector cell lines or donor effector cells (allogeneic) are used.

[0114] Immune effector cells can be harvested from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusions, spleen tissue, and tumors. Immune effector cells can be harvested from blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be harvested by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Particular subpopulations of immune effector cells may be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies against surface markers unique to cells that have undergone positive selection, for example, by incubation with antibody-conjugated beads for a time interval sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of an immune effector cell population can be achieved by negative selection using a combination of antibodies against surface markers unique to cells that have undergone negative selection.

[0115] The present disclosure provides methods for generating immune effector cells that express the CARs described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having a tumor cell that expresses PIG and / or a low density cancer antigen, such that the immune effector cells express one or more CARs as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically engineered without further manipulation in vitro. Such cells can then be readministered directly to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically engineered to express the CAR. In this context, the immune effector cells can be cultured before and after being genetically engineered (i.e., transduced or transfected to express the CARs described herein).

[0116] Prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source can be obtained from a subject. In particular, the immune effector cells for use with the CARs described herein include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, the T cells can be obtained from a blood unit collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL separation. In one embodiment, the cells from an individual's circulating blood are collected by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and placed in a buffer or medium appropriate for subsequent processing. In one embodiment, the cells are washed with PBS. In alternative embodiments, the washing solution may lack calcium and / or may lack magnesium or may lack many, but not all, divalent cations. As will be understood by those of skill in the art, the washing step can be performed by methods known to those of skill in the art, such as using a semi-automatic flow centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions, with or without buffers. In certain embodiments, unwanted components of the apheresis sample can be removed with the medium in which the cells are directly resuspended.

[0117] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved by a combination of antibodies against surface markers specific to cells that have undergone negative selection. One method used herein is negative magnetic immunoadhesion or flow cytometry cell sorting and / or selection using a cocktail of monoclonal antibodies against cell surface markers present on cells that have undergone negative selection. For example, to enrich CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate the desired cell population.

[0118] PBMCs may be used directly for CAR-mediated genetic modification using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated and, in certain embodiments, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subsets, either before or after gene modification and / or expansion. CD8+ cells can be harvested by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each type of CD8+ cell. In embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, the expression of phenotypic markers of central memory TCM includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127 and positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0119] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying a cell population having cell surface antigens. CD4+ lymphocytes can be harvested by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L and CD45RO negative.

[0120] Immune effector cells such as T cells can be genetically engineered after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro prior to being genetically engineered. In another embodiment, immune effector cells such as T cells are genetically engineered with a chimeric antigen receptor described herein (e.g., transduced using a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874; U.S. Patent No. 6,867,041; U.S. Patent No. 6,797,514; WO2012079000. Generally, such methods involve contacting PBMCs or isolated T cells with a stimulant and co-stimulant, such as anti-CD3 and anti-CD28 antibodies, generally attached to beads or other surfaces, in a culture medium containing an appropriate cytokine such as IL-2 (e.g., recombinant human IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads function as "surrogate" antigen-presenting cells (APCs). In other embodiments, T cells can be activated and stimulated and grown with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177; U.S. Patent No. 5,827,642; and WO2012129514.

[0121] In some embodiments, immune effector cells include any white blood cells involved in defending the body against infections and foreign substances. For example, immune effector cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells may include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0122] Helper T cells (T H cells) assist other white blood cells in immune processes including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. These cells are known as CD4 + T cells because they express the CD4 glycoprotein on their surface. Helper T cells begin to activate when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response. These cells can differentiate into one of several subtypes, including T H 1, T H 2, T H 3, T H 17, T H 9, or T FH and secrete different cytokines, promoting different types of immune responses.

[0123] Cytotoxic T cells (T C cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are known as CD8 + T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8 +The cells are inactivated into anergy state, thereby preventing autoimmune diseases.

[0124] Memory T cells are a subset of antigen-specific T cells that persist for a long time even after an infection has been cured. When re-exposed to their cognate antigen, they rapidly proliferate into large numbers of effector T cells, thereby giving the immune system "memory" of past infections. Memory cells can be either CD4 + or CD8 + Memory T cells usually express the cell surface protein CD45RO.

[0125] Regulatory T cells (T reg cells), formerly known as suppressor T cells, are very important for maintaining immune tolerance. Their main roles are to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two main classes of CD4 + T reg cells, natural T reg cells and adaptive T reg cells have been described so far.

[0126] Natural killer T (NKT) cells (so as not to be confused with natural killer (NK) cells) bridge the adaptive and innate immune systems. Different from conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0127] In some embodiments, the T cells comprise a mixture of CD4 + cells. In other embodiments, the T cells are enriched in one or more subsets based on cell surface expression. For example, in some cases, the T composition is cytotoxic CD8 + T lymphocytes.

[0128] Natural killer (NK) cells are CD56 + + CD3 - - large granular lymphocytes (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8 + + T lymphocytes, NK cells can initiate cytotoxic effects against tumor cells without the need for prior sensitization and can eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and off-target off-tumor effects.

[0129] Binding characteristics of chimeric antigen receptors As used herein, the term "binding" in the context of chimeric antigen receptor binding is to a given antigen, such as a cell surface protein or a fragment thereof (or to an antigen that binds to a cell surface protein such as an HLA molecule). Binding generally refers to an interaction or association between at least two independent entities or molecular structures, such as an antigen-binding domain:antigen interaction. For example, binding affinity is typically about 10 -8 -8 mol / L (10 -8 -8 M) or less, such as about 10 -9 -9 mol / L (10 -9 -9 M) or less, such as about 10 -7 -7 mol / L (10 -7 -7 M) or less of the K D Dcorresponds to a value. Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data strongly correlate with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0130] Accordingly, in some embodiments, the chimeric antigen receptor of the present disclosure has a K D value that is at least 10-fold lower than the affinity when binding to a non-specific antigen (e.g., BSA, casein) and binds to a predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to the value. As described herein, the chimeric antigen receptor of the present disclosure can bind to the HLA-presented antigen described herein. According to the present disclosure, in some embodiments, the K D value of the chimeric antigen receptor that is 10-fold or less lower than that for a non-specific antigen may be considered undetectable binding.

[0131] The term "K D " (M) refers to the dissociation equilibrium constant of a particular antigen-binding domain:antigen interaction. Since K D is inversely related to the binding affinity, the smaller the K D value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" mean a higher ability to form an interaction and thus a smaller K D value, and conversely, the terms "lower affinity" or "weaker affinity" mean a lower ability to form an interaction and thus a larger K D value. Depending on the situation, compared to the binding affinity of a molecule (e.g., a chimeric antigen receptor) for another interacting partner molecule (e.g., antigen Y), a higher binding affinity (or K D ) of that molecule (e.g., a chimeric antigen receptor) for its interacting partner molecule (e.g., antigen X) means a larger K DA value (lower, i.e., weaker affinity) is represented as a binding ratio determined by dividing by a smaller K D and can be expressed, for example, in some cases, as a binding affinity 5 or 10 times greater.

[0132] The term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a specific antigen-binding domain:antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. The above value is also called the k off value.

[0133] The term "k a " (M-1×sec-1 or 1 / M) refers to the association rate constant of a specific antigen-binding domain:antigen interaction, or the association rate constant of a chimeric antigen receptor. The term "K A " (M-1 or 1 / M) refers to the association equilibrium constant of a specific antigen-binding domain:antigen interaction, or the association equilibrium constant of a chimeric antigen receptor. The association equilibrium constant is obtained by dividing k a by k d .

[0134] The term "EC50" or "EC 50 " refers to the maximum half-maximal effective concentration, which includes the concentration of a chimeric antigen receptor that induces a response midway between the baseline and the maximum value after a given exposure time. EC 50 essentially represents the concentration of a chimeric antigen receptor at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 value is equal to, for example, the concentration of a chimeric antigen receptor of the present disclosure that gives half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen) when measured by a FACS binding assay. Thus, as the EC 50 , i.e., the maximum half-maximal effective concentration value, increases, a decrease or weakening of binding is observed.

[0135] In one embodiment, the decrease in binding is the chimeric antigen receptor concentration that can bind to the maximum half-maximal amount of target cells, EC50 It can be defined as an increase. The present disclosure provides a chimeric antigen receptor comprising an antigen-binding domain derived from an antibody that binds to a human antigen with high affinity (e.g., a K value in the nanomolar or sub-nanomolar range). D Value) and binds to a human antigen.

[0136] According to certain embodiments, the present disclosure provides a chimeric antigen receptor comprising an antigen-binding domain derived from a corresponding antibody that binds to a human antigen (e.g., at 25°C) with a K of less than about 5 nM. D In certain embodiments, the corresponding antibody binds to the antigen protein with a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM when measured by surface plasmon resonance. D And binds to the antigen protein.

[0137] The present disclosure also provides a chimeric antigen receptor comprising an antigen-binding domain derived from a corresponding antibody that binds to an antigen protein with a dissociation half-life (t1 / 2) of greater than about 10 minutes or greater than about 125 minutes when measured by surface plasmon resonance at 25°C. In certain embodiments, the corresponding antibody binds to the antigen protein with a t1 / 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes when measured by surface plasmon resonance at 25°C.

[0138] The present disclosure also provides a chimeric antigen receptor comprising an antigen-binding domain derived from a corresponding antibody that specifically binds to a human cell line expressing endogenous MAGE-A4 when measured by a FACS binding assay.

[0139] Therapeutic method The immune effector cells expressing the CAR disclosed herein induce a therapeutically beneficial immune response against cancer cells expressing PIG or low-density antigens. For example, the anti-tumor immune response induced by the disclosed CAR-modified immune effector cells can be an active or passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which the CAR-modified immune effector cells induce an immune response specific for PIG or low-density cancer antigens.

[0140] The immune effector cells expressing the CAR prepared as described herein can be used in methods and compositions of adoptive immunotherapy according to known techniques or modifications thereof that will be apparent to those skilled in the art based on the present disclosure. For example, see U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; see also U.S. Patent No. 4,690,915 to Rosenberg.

[0141] In some embodiments, provided herein is a method of treating cancer (e.g., solid tumors) in a subject by administering to the subject a composition comprising cells expressing the CAR polypeptides disclosed herein. In some embodiments, the methods described herein further comprise co-administering to the subject a composition comprising cells expressing a second CAR polypeptide comprising a 4-1BB domain in the co-stimulatory region of the CAR polypeptide. In some embodiments, the second CAR comprises at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain. In some embodiments, the second CAR comprises an extracellular domain specific for a low density cancer antigen and / or a groove peptide cancer antigen. The second CAR polypeptide may comprise a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region. Without being bound by theory, immune cells expressing a first CAR (i.e., a CAR polypeptide comprising a CD28 / ζ domain in the co-stimulatory domain of the CAR) provide an initial burst that rapidly promotes the killing of cancer cells, while administration of immune cells by 4-1BB, although at a lower level of killing compared to administration of immune cells expressing the first CAR, provides sustained cancer cell killing. The 4-1BB / CD3z CAR is associated with the persistence of the patient's CAR T cells, thereby providing sustained cancer cell killing.

[0142] In some embodiments, the cells are formulated by first harvesting them from the culture medium and then washing the cells and concentrating them in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media may be any isotonic media formulation, typically standard saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's solution may also be used. The infusion media may be supplemented with human serum albumin.

[0143] The therapeutically effective amount of cells in the composition is at least two cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset), or typically contains more than 102, up to 106, and sometimes 108 or 109 cells, and may be more than 1010 cells. The number of cells varies depending on the intended final use of the composition, as does the type of cells included in the composition.

[0144] The cells can be autologous or heterologous to the patient being treated. The cells can be allogeneic. Optionally, the treatment can also include administration of mitogens (e.g., PHA), or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0145] The population of immune effector cells expressing a CAR can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions disclosed herein can include a population of immune effector cells expressing a CAR, such as T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions disclosed herein can be formulated for intravenous administration.

[0146] By administering T cells expressing the CARs described herein using the methods described herein or other methods known in the art, the anti-tumor immune responses induced in a subject may include cytotoxic T cells capable of killing infected cells, cell-mediated immune responses mediated by regulatory T cells, and helper T cell responses. Humoral immune responses mainly mediated by helper T cells that can activate B cells and thereby result in antibody production may also be induced. A variety of techniques can be used to analyze the types of immune responses induced by the compositions disclosed herein, which are well described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, N.Y., N.Y.

[0147] Accordingly, provided herein is a method of treating an individual diagnosed or suspected of having or being at risk of developing a malignant tumor, the method comprising administering to the individual an effective amount of immune effector cells expressing a CAR as described herein for such treatment.

[0148] Administration of the disclosed compositions can be performed in any convenient manner including injection, infusion, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions can also be injected directly into a tumor, lymph node, or site of infection.

[0149] Provided herein is a method of co-administering a second CAR polypeptide comprising a 4-1BB domain in the co-stimulatory region of the CAR polypeptide. The second CAR polypeptide may further comprise at least one intracellular signaling region comprising a differentiation antigen group 3 zeta (CD3 / ζ) domain and / or an extracellular domain specific for a low density cancer antigen and / or a grooved peptide cancer antigen. The second CAR polypeptide may comprise a differentiation antigen group 8 alpha (CD8 / α) peptide in the hinge / transmembrane region.

[0150] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of related therapies including, but not limited to, additional cancer therapies. In some embodiments, the CAR-modified immune effector cells can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immune-depleting agents such as CAM PATH, anti-CD3 antibodies or other antibody therapeutic agents, cytokines, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, T cell depletion therapy using any chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In other embodiments, the cell composition is administered after a B cell depletion therapy with an agent that reacts with CD20, such as Rituxan. For example, in some embodiments, a subject can receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells. In another embodiment, the expanded cells are administered before or after surgery to treat the subject's cancer or pre-cancerous lesions.

[0151] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of engineered cells may be administered to a subject over a defined period of time. The method according to this aspect includes continuously administering multiple doses of cells to a subject. As used herein, "administering continuously" means that each dose is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., time, day, week, or month). The present disclosure provides a method that includes continuously administering to a patient a single initial dose, followed by one or more secondary doses, and optionally one or more subsequent tertiary doses.

[0152] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the chronological order of administration of the engineered cells of the present disclosure. Thus, an "initial dose" is the dose administered at the start of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is the dose administered after the initial dose; and a "tertiary dose" is the dose administered after the secondary dose. All of the initial, secondary, and tertiary doses may contain the same amount of engineered cells, but generally vary from each other depending on the dosing frequency. However, in certain embodiments, the amount of engineered cells included in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., is appropriately adjusted up or down). In certain embodiments, two or more doses (e.g., two, three, four, or five doses) are administered at the start of a treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") being administered at a lower frequency.

[0153] In one embodiment of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or more) after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means, in a series of multiple administrations, the dose administered to a patient prior to the administration of the next dose in a series without intervening doses.

[0154] The method according to an aspect of the present disclosure may include administering any number of secondary and / or tertiary doses to a patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0155] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The dosing frequency can also be adjusted by a physician during treatment according to the needs of an individual patient after clinical examination.

[0156] Index In certain aspects, provided herein are methods of treating cancer using the CAR T cells provided herein. In certain embodiments, the cancer being treated expresses a PIG antigen and / or a low density antigen to which the CAR expressed on the T cells specifically binds.

[0157] In some embodiments, cancers that can be treated by the methods and compositions provided herein include, but are not limited to, cancers of the cervix, anus, vagina, vulva, penis, base of the tongue, larynx, tonsils, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, non - melanoma skin cancer (NMSC), cutaneous squamous cell carcinoma (SCC), stomach, testis, tongue or uterus. Additionally, cancers specifically include the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; trichilemmal carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combined hepatocellular and cholangiocarcinomas; trabecular adenomas; adenoid cystic carcinomas; adenocarcinomas of adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid carcinomas; malignant carcinoid tumors; bronchiolo - alveolar adenocarcinomas; papillary adenocarcinomas; chromophobic carcinomas; eosinophilic carcinomas; eosinophilic adenocarcinomas; basophilic carcinomas; clear cell adenocarcinomas; granular cell carcinomas; follicular adenocarcinomas; papillary and follicular adenocarcinomas; unencapsulated sclerosing carcinomas; adrenocortical carcinomas; endometroid carcinomas; carcinomas of skin appendages; apocrine adenocarcinomas; sebaceous gland carcinomas; ceruminous gland carcinomas; mucoepidermoid carcinomas; cystadenocarcinomas; papillary cystadenocarcinomas; papillary serous cystadenocarcinomas; mucinous cystadenocarcinomas; mucinous adenocarcinomas; signet ring cell carcinomas; invasive ductal carcinomas; medullary carcinomas; lobular carcinomas; inflammatory carcinomas; Paget's disease of the breast; acinar cell carcinomas; adenosquamous carcinomas; adenocarcinomas associated with squamous metaplasia; malignant thymomas; malignant ovarian stromal tumors; malignant cystomas; malignant granular cell tumors; malignant male hormone - producing cell tumors; sertoli cell tumors; malignant leydig cell tumors; malignant lipoid cell tumors; malignant paragangliomas; malignant extra - mammary paragangliomas; pheochromocytomas; glomus angiosarcomas; malignant melanomas; amelanotic melanomas; superficially spreading melanomas; malignant melanomas of giant pigmented nevi; epithelioid melanomas; malignant blue nevi; sarcomas; fibrosarcomas; malignant fibrous histiocytomas; myxosarcomas; liposarcomas; leiomyosarcomas; rhabdomyosarcomas; fetal rhabdomyosarcomas; alveolar rhabdomyosarcomas; stromal sarcomas; malignant mixed tumors; müllerian duct mixed tumors; nephroblastomas; hepatoblastomas; carcinosarcomas; malignant mesenchymal tumors; malignant Brenner tumors; malignant phyllodes tumors; synovial sarcomas; malignant mesotheliomas; undifferentiated embryonal cell tumors; fetal carcinomas; malignant teratomas; malignant struma ovarii; choriocarcinomas; malignant mesonephromas; angiosarcomas; malignant vascular endothelial tumors; Kaposi sarcomas; malignant hemangiopericytomas; lymphangiosarcomas; osteosarcomas; parosteal osteosarcomas; chondrosarcomas; malignant chondroblastomas; mesenchymal chondrosarcomas; giant cell tumors of bone;Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontogenic sarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligoastrocytoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; lateral granuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; fungating polypoid tumor; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myelogenous leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and hairy cell leukemia, but not limited to these.;

[0158] In certain embodiments, the disclosed CAR-T cells can be used in combination with any compound, moiety, or group having a cytotoxic or cytostatic effect. The drug moiety includes chemotherapeutic agents, which may function as microtubule inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators, and are particularly used in cancer treatment. Exemplary anti-cancer compounds include alemtuzumab (Campath®), alitretinoin (Panretin®), anastrozole (Arimidex®), bevacizumab (Avastin®), bexarotene (Targretin®), bortezomib (Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), cabozantinib (Cometriq™), carfilzomib (Kyprolis™), cetuximab (Erbitux®), crizotinib (Xalkori®), dasatinib (Sprycel®), denileukin diftitox (Ontak™), erlotinib hydrochloride (Tarceva®), everolimus (Afinitor®), exemestane (Aromasin®), fulvestrant (Faslodex®), gefitinib (Iressa®), ibritumomab tiuxetan (Zevalin®), imatinib mesylate (Gleevec™), ipilimumab (Yervoy™), lapatinib ditosylate (Tykerb®), letrozole (Femara®), nilotinib (Tasigna®), ofatumumab (Arzerra®), panitumumab (Vectibix®), pazopanib hydrochloride (Votrient®), pertuzumab (Perjeta™), pralatrexate (Folotyn®), regorafenib (Stivarga®), rituximab (Rituxan®), romidepsin (Istodax®), sorafenib tosylate (Nexavar®),Sunitinib malate (Sutent®), tamoxifen, temsirolimus (Torisel®), toremifene (Fareston®), tositumomab and 131I tositumomab (Bexxar™), trastuzumab (Herceptin®), tretinoin (Vesanoid®), vandetanib (Caprelsa®), vemurafenib (Zelboraf®), vorinostat (Zolinza®), and aflibercept (Zaltrap®) are mentioned, but not limited thereto. Examples of additional chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobelensin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin,Particularly calicheamicin γ1 and calicheamicin Ω1; dynemicins including dynemicin A; bisphosphonates such as clodronate; esperamicin; similarly neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, cardinophilin, chromomycins, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, promycin, quelamycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; aminoglutethimide, mitotane,Adrenal suppressants such as trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; erformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids and ansamycins such as maytansine; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; antibiotics such as PSK polysaccharide complex); razoxane; lysocine; sizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2´,2´´-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and angidicin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included, but are not limited to, examples of chemotherapeutic agents.

[0159] In some embodiments, the subject is also administered an additional immunotherapeutic agent. Immunotherapy refers to a treatment that uses the subject's immune system to treat cancer, such as the use of cancer vaccines, cytokines, cancer-specific antibodies, T cell therapy, and dendritic cell therapy.

[0160] In some embodiments, the subject is also administered an immunomodulatory protein. Examples of immunomodulatory proteins include B lymphocyte chemoattractant ("BLC"), C-C motif chemokine 11 ("Eotaxin-1"), eosinophil chemotactic protein 2 ("Eotaxin-2"), granulocyte colony-stimulating factor ("G-CSF"), granulocyte macrophage colony-stimulating factor ("GM-CSF"), 1-309, intercellular adhesion molecule 1 ("ICAM-1"), interferon gamma ("IFN-γ"), interleukin-1α ("IL-1α"), interleukin-1β ("IL-1β"), interleukin 1 receptor antagonist ("IL-1ra"), interleukin-2 ("IL-2"), interleukin-4 ("IL-4"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-6 soluble receptor ("IL-6sR"), interleukin-7 ("IL-7"), interleukin-8 ("IL-8"), interleukin-10 ("IL-10"), interleukin-11 ("IL-11"), subunit β of interleukin-12 ("IL-12p40" or "IL-12p70"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 ("IL-17"), chemokine (C-C motif) ligand 2 ("MCP-1"), macrophage colony-stimulating factor ("M-CSF"), monokine induced by gamma interferon ("MIG"), chemokine (C-C motif) ligand 2 ("MIP-1α"), chemokine (C-C motif) ligand 4 ("MIP-1β"), macrophage inflammatory protein-1-δ ("MIP-1δ"), platelet-derived growth factor subunit B ("PDGF-BB"), chemokine (C-C motif) ligand 5, regulated on activation, normal T cell expressed and secreted ("RANTES"), TIMP metallopeptidase inhibitor 1 ("TIMP-1"), TIMP metallopeptidase inhibitor 2 ("TIMP-2"), tumor necrosis factor, lymphotoxin α ("TNFα"), tumor necrosis factor, lymphotoxin β ("TNFβ"), type 1 soluble TNF receptor ("sTNFRI"),sTNFRIIAR, brain-derived neurotrophic factor ("BDNF"), basic fibroblast growth factor ("bFGF"), bone morphogenetic protein 4 ("BMP-4"), bone morphogenetic protein 5 ("BMP-5"), bone morphogenetic protein 7 ("BMP-7"), nerve growth factor ("b-NGF"), epidermal growth factor ("EGF"), epidermal growth factor receptor ("EGFR"), endocrine gland-derived vascular endothelial growth factor ("EG-VEGF"), fibroblast growth factor 4 ("FGF-4"), keratinocyte growth factor ("FGF-7"), growth differentiation factor 15 ("GDF-15"), glial cell-derived neurotrophic factor ("GDNF"), growth hormone, heparin-binding EGF-like growth factor ("HB-EGF"), hepatocyte growth factor ("HGF"), insulin-like growth factor binding protein 1 ("IGFBP-1"), insulin-like growth factor binding protein 2 ("IGFBP-2"), insulin-like growth factor binding protein 3 ("IGFBP-3"), insulin-like growth factor binding protein 4 ("IGFBP-4"), insulin-like growth factor binding protein 6 ("IGFBP-6"), insulin-like growth factor 1 ("IGF-1"), insulin, macrophage colony-stimulating factor ("M-CSF R"), nerve growth factor receptor ("NGF R"), neurotrophin-3 ("NT-3"), neurotrophin-4 ("NT-4"), osteoclast differentiation inhibitory factor ("osteoprotegerin"), platelet-derived growth factor receptor ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp, Cullin, F-box-containing complex ("SCF"), stem cell factor receptor ("SCF R"), transforming growth factor α ("TGFα"), transforming growth factor β1 ("TGFβ1"), transforming growth factor β3 ("TGFβ3"), vascular endothelial growth factor ("VEGF"), vascular endothelial growth factor receptor 2 ("VEGFR2"), vascular endothelial growth factor receptor 3 ("VEGFR3"), VEGF-D 6Ckine, tyrosine-protein kinase receptor UFO ("Axl"), Betacellulin ("BTC"), mucosa-associated epithelial chemokine ("CCL28"), chemokine (C-C motif) ligand 27 ("CTACK"), chemokine (C-X-C motif) ligand 16 ("CXCL16")C-X-C motif chemokine 5 ("ENA-78"), chemokine (C-C motif) ligand 26 ("eotaxin-3"), granulocyte chemotactic protein 2 ("GCP-2"), GRO, chemokine (C-C motif) ligand 14 ("HCC-1"), chemokine (C-C motif) ligand 16 ("HCC-4"), interleukin-9 ("IL-9"), interleukin-17F ("IL-17F"), interleukin-18 binding protein ("IL-18 BPa"), interleukin-28A ("IL-28A"), interleukin 29 ("IL-29"), interleukin 31 ("IL-31"), C-X-C motif chemokine 10 ("IP-10"), chemokine receptor CXCR3 ("I-TAC"), leukemia inhibitory factor ("LIF"), Light, chemokine (C motif) ligand ("lymphotactin"), monocyte chemoattractant protein 2 ("MCP-2"), monocyte chemoattractant protein 3 ("MCP-3"), monocyte chemoattractant protein 4 ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibitory factor ("MIF"), chemokine (C-C motif) ligand 20 ("MIP-3α"), C-C motif chemokine 19 ("MIP-3β"), chemokine (C-C motif) ligand 23 ("MPIF-1"), macrophage stimulating protein α chain ("MSPα"), nucleosome assembly protein 1-like 4 ("NAP-2"), secreted phosphoprotein 1 ("osteopontin"), pulmonary activation-regulated chemokine ("PARC"), platelet factor 4 ("PF4"), stromal cell-derived factor 1α ("SDF-1α"), chemokine (C-C motif) ligand 17 ("TARC"), thymus-expressed chemokine ("TECK"), thymic stromal lymphopoietin ("TSLP 4-IBB"), CD166 antigen ("ALCAM"), cluster of differentiation 80 ("B7-1"), tumor necrosis factor receptor superfamily member 17 ("BCMA"), cluster of differentiation 14 ("CD14"), cluster of differentiation 30 ("CD30"), cluster of differentiation 40 ("CD40 ligand"), carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) ("CEACAM-1"), death receptor 6 ("DR6"), deoxythymidine kinase ("Dtk")Type 1 membrane glycoprotein ("endoglin"), receptor tyrosine-protein kinase erbB-3 ("ErbB3"), endothelial leukocyte adhesion molecule 1 ("E-selectin"), apoptosis antigen 1 ("Fas"), Fms-like tyrosine kinase 3 ("Flt-3L"), tumor necrosis factor receptor superfamily member 1 ("GITR"), tumor necrosis factor receptor superfamily member 14 ("HVEM"), intercellular adhesion molecule 3 ("ICAM-3"), IL-1R4, IL-1RI, IL-10Rβ, IL-17R, IL-2Rγ, IL-21R, lysosomal membrane protein 2 ("LIMPII"), neutrophil gelatinase-associated lipocalin ("lipocalin-2"), CD62L ("L-selectin"), lymphatic endothelium ("LYVE-1"), MHC class I polypeptide-related sequence A ("MICA"), MHC class I polypeptide-related sequence B ("MICB"), NRGl-βl, platelet-derived growth factor receptor beta ("PDGF Rβ"), platelet endothelial cell adhesion molecule ("PECAM-1"), RAGE, hepatitis A virus cellular receptor 1 ("TIM-1"), tumor necrosis factor receptor superfamily member IOC ("TRAIL R3"), Trappin protein transglutaminase binding domain ("Trappin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDAR, activin A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, cathepsin S, CD40, cryptic family protein IB ("Cripto-1"), DAN, Dickkopf-related protein 1 ("DKK-1"), E-cadherin, epithelial cell adhesion molecule ("EpCAM"), Fas ligand (FasL or CD95L), FcgRIIB / C, follistatin, galectin-7, intercellular adhesion molecule 2 ("ICAM-2"), IL-13Rl, IL-13R2, IL-17B, IL-2Ra, IL-2Rb, IL-23, LAP, neuronal cell adhesion molecule ("NrCAM"), plasminogen activator inhibitor 1 ("PAI-1"), platelet-derived growth factor receptor ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1β"), sgpl30, secreted frizzled-related protein 2 ("ShhN")Sialic acid-binding immunoglobulin-type lectin ("Siglec-5"), ST2, transforming growth factor β2 ("TGFβ2"), Tie-2, thrombopoietin ("TPO"), tumor necrosis factor receptor superfamily member 10D ("TRAIL R4"), triggering receptor expressed on myeloid cells 1 ("TREM-1"), vascular endothelial growth factor C ("VEGF-C"), VEGFR1, adiponectin, adiposin ("AND"), α-fetoprotein ("AFP"), angiopoietin-like 4 ("ANGPTL4"), β-2-microglobulin ("B2M"), basal cell adhesion molecule ("BCAM"), carbohydrate antigen 125 ("CA125"), cancer antigen 15-3 ("CA15-3"), carcinoembryonic antigen ("CEA"), cAMP receptor protein ("CRP"), human epidermal growth factor receptor 2 ("ErbB2"), follistatin, follicle-stimulating hormone ("FSH"), chemokine (C-X-C motif) ligand 1 ("GROα"), human chorionic gonadotropin ("βHCG"), insulin-like growth factor 1 receptor ("IGF-1sR"), IL-1sRII, IL-3, IL-18Rb, IL-21, leptin, matrix metalloproteinase-1 ("MMP-1"), matrix metalloproteinase-2 ("MMP-2"), matrix metalloproteinase-3 ("MMP-3"), matrix metalloproteinase-8 ("MMP-8"), matrix metalloproteinase-9 ("MMP-9"), matrix metalloproteinase-10 ("MMP-10"), matrix metalloproteinase-13 ("MMP-13"), neural cell adhesion molecule ("NCAM-1"), entactin ("nidogen-1"), neuron-specific enolase ("NSE"), oncostatin M ("OSM"), procalcitonin, prolactin, prostate-specific antigen ("PSA"), sialic acid-binding Ig-like lectin 9 ("Siglec-9"), ADAM17 endopeptidase ("TACE"), thyroglobulin, metalloproteinase inhibitor 4 ("TIMP-4"), TSH2B4, disintegrin and metalloprotease domain-containing protein 9 ("ADAM-9"), angiopoietin 2Tumor necrosis factor ligand superfamily member 13 / acidic leucine-rich nuclear phosphoprotein 32 family member B ("APRIL"), bone morphogenetic protein 2 ("BMP-2"), bone morphogenetic protein 9 ("BMP-9"), complement component 5a ("C5a"), cathepsin L, CD200, CD97, chemrin, tumor necrosis factor receptor superfamily member 6B ("DcR3"), fatty acid-binding protein 2 ("FABP2"), fibroblast activation protein α ("F AP"), fibroblast growth factor 19 ("FGF-19"), galectin-3, hepatocyte growth factor receptor ("HGF R"), IFN-α / βR2, insulin-like growth factor 2 ("IGF-2"), insulin-like growth factor 2 receptor ("IGF-2R"), interleukin-1 receptor 6 ("IL-1R6"), interleukin 24 ("IL-24"), interleukin 33 ("IL-33"), kallikrein 14, aspartyl endopeptidase ("legumain"), oxidized low-density lipoprotein receptor 1 ("LOX-1"), mannose-binding lectin ("MBL"), neprilysin ("NEP"), Notch homolog 1, translocation associated (Drosophila) ("Notch-1"), overexpressed in renal oncocytoma ("NOV"), osteoactivin, programmed cell death protein 1 ("PD-1"), N-acetylmuramoyl-L-alanine amidase ("PGRP-5"), serpin A4, secreted frizzled-related protein 3 ("sFRP-3"), thrombomodulin, Toll-like receptor 2 ("TLR2"), tumor necrosis factor receptor superfamily member 10A ("TRAIL Rl"), transferrin ("TRF"), WIF-lACE-2, albumin, AMICA, angiopoietin 4, B cell-activating factor ("BAFF"), carbohydrate antigen 19-9 ("CA19-9"), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif) ligand 14 ("CXCL14"), Cystatin C, Decorin ("DCN"), Dickkopf-related protein 3 ("Dkk-3"), Delta-like protein 1 ("DLL1"), Fetuin A, Heparin-binding growth factor 1 ("aFGF"), Folate receptor alpha ("FOLR1"), Furin, GPCR-associated sorting protein 1 ("GASP-1"), GPCR-associated sorting protein 2 ("GASP-2"), Granulocyte colony-stimulating factor receptor ("GCSF R"), Serine protease hepsin ("HAI-2"), Interleukin-17B receptor ("IL-17B R"), Interleukin 27 ("IL-27"), Lymphocyte activation gene 3 ("LAG-3"), Apolipoprotein A-V ("LDL R"), Pepsinogen I, Retinol-binding protein 4 ("RBP4"), SOST, Heparan sulfate proteoglycan ("Syndecan-1"), Tumor necrosis factor receptor superfamily member 13B ("TACI"), Tissue factor pathway inhibitor ("TFPI"), TSP-1, Tumor necrosis factor receptor superfamily member 10b ("TRAIL R2"), TRANCE, Troponin I, Urokinase plasminogen activator ("uPA"), Cadherin 5, also known as VE-cadherin (vascular endothelial) type 2 or CD144 ("VE-cadherin"), WNT1-inducible-signaling pathway protein 1 ("WISP-1"), and Receptor activator of nuclear factor kappa B ("RANK"), but are not limited to these.

[0161] The disclosed CARs and immune cells expressing the CARs can be used in combination with immune checkpoint inhibitors. Immune checkpoint inhibition broadly refers to inhibiting checkpoints that may be generated by cancer cells to prevent or downregulate the immune response. Two known immune checkpoint pathways are involved in signal transduction via the cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play important roles at all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the major ligand, while PD-L2 has a much more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is transmitted to the T cell, thereby reducing cytokine production and suppressing T cell proliferation. Immune checkpoint inhibitors include, but are not limited to, those that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475, AMP-514), those that block PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), those that block PD-L2 (rHIgM12B7, AMP-224), those that block CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), those that block IDO, B7-H3 (MGA271), those that block B7-H4, TIM3, LAG-3 (BMS-986016) aptamers and antibodies.The immune checkpoint inhibitors may be semiprimab (REGN2810), nivolumab (BMS-936558, MDX-1106, ONO-4538), pembrolizumab (MK-3475, SCH 900475), atezolizumab (MPDL3280A, RG7446, RO5541267), durvalumab (MEDI4736, MEDI-4736), avelumab (MSB0010718C), ipilimumab (BMS-734016, IBI310, MDX-010), SHR1210, sintilimab (IBI308), spartalizumab (PDR001), surlizumab (BGB-A317), pidilizumab, BCD-100, toripalimab (JS001), BAY 1905254, ASP 8374, PF-06801591, AMP-224, AB122, AK105, AMG 404, BCD-100, BI 754091, F520, HLX10, HX008, JTX-4014, LZM009, MEDI0680, MGA012, Sym021, TSR-042, PSB205, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, INCB086550, FS118, BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, HLX20, KN035, MDX-1105, MSB2311, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, M7824, LY3415244, INCB086550, CA-170, CX-072, ADU-1604, AGEN1181, AGEN1884, MK-1308, REGN4659, XmAb22841, ATOR-1015, PSB205, MGD019, AK104, XmAb20717, BMS-986249, tremelimumab, BMS-986258, BGB-A425, INCAGN02390, Sym023, JNJ 61610588, BI 754111, LAG525, MK-4280, REGN3767, Sym022, TSR-033, relatlimab, JTX-2011, MGD009, BMS-986207, OMP-313M32, MK-7684 or TSR-022.

[0162] Methods for treating cancer using human monoclonal antibodies against Programmed Death 1 (PD-1) and anti-PD-1 antibodies alone or in combination with other immunotherapies are described in U.S. Patent No. 8,008,449, and these antibodies are incorporated by reference. Anti-PD-L1 antibodies and their use are described in U.S. Patent No. 8,552,154, and these antibodies are incorporated by reference. Anti-cancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, and these antibodies are incorporated by reference.

[0163] In some embodiments, the PD-L1 inhibitor comprises an antibody that specifically binds to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD-1 inhibitor comprises an antibody that specifically binds to PD-1, such as pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Methods for treating cancer using human monoclonal antibodies against PD-1 and anti-PD-1 antibodies alone or in combination with other immunotherapies are described in U.S. Patent No. 8,008,449, and these antibodies are incorporated by reference. Anti-PD-L1 antibodies and their use are described in U.S. Patent No. 8,552,154, and these antibodies are incorporated by reference. Anti-cancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, and these antibodies are incorporated by reference.

[0164] Optimal "killer" CD8 T cell responses require costimulation in addition to T cell receptor activation, which can be achieved by ligation of tumor necrosis factor receptor family members including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because administration of activating (agonist) anti-OX40 mAb enhances T cell differentiation and cytolysis, leading to enhanced anti-tumor immunity against various tumors.

[0165] In some embodiments, such additional therapeutic agents can be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0166] In some embodiments, such additional therapeutic agents can be selected from alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin.

[0167] In some embodiments, such additional therapeutic agents can be selected from antimitotic agents such as taxanes, for example, docetaxel, and paclitaxel, and vinca alkaloids, for example, vindesine, vincristine, vinblastine, and vinorelbine.

[0168] In some embodiments, such additional therapeutic agents can be selected from topoisomerase inhibitors such as topotecan or irinotecan, or cell growth inhibitors such as etoposide and teniposide.

[0169] In some embodiments, such additional therapeutic agents can be selected from growth factor inhibitors such as inhibitors of ErbB1 (EGFR) (such as EGFR antibodies, for example, cetuximab, panitumumab, or nimotuzumab, or other EGFR inhibitors such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as HER2 antibodies, for example, trastuzumab, trastuzumab-DM1, or pertuzumab), or inhibitors of both EGFR and HER2 (such as lapatinib).

[0170] In some embodiments, such additional therapeutic agents can be selected from tyrosine kinase inhibitors such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0171] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanilimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0172] In some embodiments, the therapeutic agents used in combination with CAR (e.g., the CARs disclosed herein and immune cells expressing such CARs) for treating the aforementioned disorders can be anti-cancer cytokines, chemokines, or combinations thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INF a2b), IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, anestrim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1a from the human CXC and C-C chemokine families. Suitable cytokines may include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0173] In some embodiments, the therapeutic agent used in combination with the CAR to treat the aforementioned disorders may be a cell cycle control / apoptosis regulator (or "regulator"). Examples of cell cycle control / apoptosis regulators may include molecules that target and modulate (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overly stimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modifiers (such as BIBR1532, SOT-095, GRN163, and compositions described, for example, in US6,440,735 and US6,713,055). Non-limiting examples of molecules that interfere with the apoptosis pathway may include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFN, and antisense Bcl-2.

[0174] In some embodiments, the therapeutic agent used in combination with a CAR (e.g., the CARs disclosed herein and immune cells expressing such CARs) to treat the aforementioned disorders may be a hormonal regulator such as an anti-androgen agent and an agent useful for estrogen therapy. Examples of such hormonal regulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / ethinyl, anti-androgens (such as flutamide / oilixine), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone / provera, megestrol acetate / acepate / megace, etc.), adrenal corticosteroids (hydrocortisone, prednisone, etc.), luteinizing hormone releasing hormone (and its analogs, as well as other LHRH agonists such as buserelin and goserelin), aromatase inhibitors (anastrozole / arimidex, aminoglutethimide / cytraden, exemestane, etc.), or hormone inhibitors (such as octreotide / sandostatin).

[0175] As described above, the combined administration may be carried out simultaneously, separately or sequentially. In the case of simultaneous administration, the agents may be administered as one composition or as separate compositions, as required.

[0176] Examples Example 1: SK-MEL-37 Tumor and Anti-HLA-A2 / MAGEA4 286-294 Chimeric antigen receptor comprising scFv Preparation of CAR: V L -V H Anti-HLA-A2 / MAGEA4 in the V 286-294 direction. In addition to the scFv, a chimeric antigen receptor comprising either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) was used for anti-HLA-A2 / MAGEA4286-294 The V of antibody 31345 L and V H sequences were used for construction. As a non-binding control, the BB / z CAR was designed using a huCD8 hinge / transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3z signaling domain in addition to different scFvs. These CARs were cloned into a pLVX lentiviral vector containing an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See Part B of Figure 1 for a schematic of the constructs.

[0177] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors (“Donor 1” and “Donor 2”), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI = 5. The transduced cells were grown for 19 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo experiments.

[0178] Xenograft tumor transplantation and measurement HLA-A2 / MAGEA4 286-294 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5 × 10 6 individuals of HLA-A2 + MAGEA4 + SK-MEL-37 human melanoma tumor cells. It was confirmed by mass spectrometry that SK-MEL-37 melanoma cells expressed approximately 1,326 cell surface copies of the MAGEA4 286-294 peptide. On day 7 after tumor engraftment, mice (n = 5 per group) were given non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 286-294 BB / z CAR or anti-HLA-A2 / MAGEA4 from two different donors286-294 4×10 cells expressing either of 28 / z CAR (measured by the frequency of cells expressing GFP, a marker of cells transduced with CAR) were intravenously injected. For tumor growth, tumor volume was evaluated up to day 64 by measuring the tumor volume. 6 Calculation of heterologous tumor growth and inhibition

[0179] To measure the tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurement, the tumor volume was calculated by the formula: volume (mm ) = (length × width 3 ) / 2. 2 In summary, the results showed that MAGEA4

[0180] 28 / z CAR T cells from two donors exhibited superior in vivo antitumor effects and antitumor kinetics compared to MAGEA4 286-294 BB / z CAR T cells. 286-294

Table 10

[0181] Experimental procedure: Summary of results and conclusions:

[0182] Donor 1: The SK-MEL-37 tumors grew progressively in mice administered either control CAR T cells or MAGEA4 BB / z CAR T cells. In contrast, MAGEA4 286-294 28 / z inhibited the growth of established SK-MEL-37 tumors in vivo. Enhanced efficacy of MAGEA4 286-294 28 / z CAR compared to MAGEA4 286-294 BB / z CAR was confirmed, and the tumor sizes on days 31, 35, 40, 47, 55, and 62 were statistically significant with p < 0.0001 by two-way ANOVA test. See Part A of Figure 2. 286-294

[0183] ​Donor 2: The SK-MEL-37 tumor grew progressively in mice administered with control CAR T cells. MAGEA4 286-294 Administration of BB / z CAR T cells showed efficacy and retarded tumor growth until approximately 1 week. However, MAGEA4 286-294 28 / z CAR T cells strongly suppressed SK-MEL-37 tumor growth and made the tumors undetectable (non-palpable) in 5 out of 5 mice by day 20. These tumors remained undetectable until days 62 - 69 when the tumors recurred. MAGEA4 286-294 MAGEA4 compared to BB / z CAR 286-294 Enhanced antitumor activity of 28 / z CAR was confirmed, and the tumor size on day 35 was statistically significant by two-way ANOVA test (p = 0.0037), and the tumor sizes on days 40, 47, 55, and 62 were also statistically significant with p < 0.0001. See Figure 2, Part B.

[0184]

Table 11 - 1

[0185]

Table 11 - 2

[0186]

Table 11 - 3

[0187]

Table 11 - 4

[0188]

Table 11 - 5

[0189]

Table 11 - 6

[0190] [Table 11-7]

[0191] [Table 11-8]

[0192] [Table 11-9]

[0193] Example 2: A375 tumor and anti-HLA-A2 / MAGEA4 286-294 Chimeric antigen receptor containing scFv Construct and CAR T cells V L -V H Orientation anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, a chimeric antigen receptor containing either 1) a huCD8 hinge / transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3z signaling domain (BB / z CAR) or 2) a huCD28 hinge / transmembrane / co-stimulatory domain, and a CD3z signaling domain (28 / z CAR) was constructed against anti-HLA-A2 / MAGEA4 286-294 V of antibody 31345 L and V H sequences were used. As a non-binding control, BB / z CAR was designed using a huCD8 hinge / transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3z signaling domain in addition to the scFv. These CARs were cloned into a pLVX lentiviral vector containing an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses. See Part B of Figure 1 for a schematic of the constructs and Figure 3 for the results.

[0194] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors ("Donor 1" and "Donor 2"), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at MOI = 5. The transduced cells were grown for 19 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo experiments.

[0195] Xenograft tumor transplantation and measurement HLA-A2 / MAGEA4 286-294 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting HLA-A2 / MAGEA4, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5 × 10 6 individuals of HLA-A2 + MAGEA4 + A375 human melanoma tumor cells. It was confirmed using mass spectrometry that A375 melanoma cells expressed approximately 424 cell surface copies of the MAGEA4 286-294 peptide. On day 13 after tumor engraftment, mice (n = 5 per group) were intravenously injected with 4 × 10 286-294 individuals of either unbound control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 286-294 BB / z CAR or anti-HLA-A2 / MAGEA4 6 28 / z CAR (measured by the frequency of cells expressing GFP, a marker of cells transduced with CAR). For tumor growth, tumor volume was evaluated by measuring tumor volume until day 64.

[0196] Calculation of xenograft tumor growth and inhibition To measure tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and maximum transverse diameter (width in mm) were measured. Based on caliper measurements, tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2) Calculated by ( / 2).

[0197] Summary and conclusion of results: Donor 1: A375 tumors grew progressively in mice administered either control CAR T cells or MAGEA4 286-294 BB / z CAR T cells. In contrast, MAGEA4 286-294 28 / z inhibited the growth of established A375 tumors in vivo, and by day 64, 1 out of 5 mice had no tumors. MAGEA4 286-294 Enhanced efficacy of MAGEA4 286-294 28 / z CAR compared to control was confirmed, and tumor sizes on days 27, 29, 33, 36, 40, and 44 were statistically significant with p < 0.0001 by two-way ANOVA test. See Part A of Figure 3.

[0198] Donor 2: A375 tumors grew progressively in mice administered control CAR T cells. MAGEA4 286-294 Administration of both BB / z and MAGEA4 286-294 28 / z CAR T cells inhibited A375 tumor growth, but their kinetics were different. MAGEA4 286-294 28 / z CAR T cells acted with faster kinetics, and tumors disappeared in all 5 out of 5 mice by day 27. MAGEA4 286-294 BB / z CAR T cells acted with slower kinetics, and tumors disappeared in 4 out of 5 mice by day 44. MAGEA4 286-294 Enhanced kinetics of the antitumor effect of MAGEA4 286-294 28 / z CAR compared to control was confirmed, and tumor sizes on days 19 and 22 were statistically significant with p = 0.0071 and p = 0.0008, respectively, by two-way ANOVA test. See Part B of Figure 3.

[0199]

Table 12-1

[0200]

Table 12-2

[0201]

Table 12-3

[0202]

Table 12-4

[0203]

Table 12-5

[0204]

Table 12-6

[0205]

Table 12-7

[0206]

Table 12-8

[0207] In summary, according to the results of Examples 1 and 2, the MAGEA4 28 / z CAR T cells derived from two donors exhibited excellent in vivo antitumor effects and antitumor kinetics compared to the MAGEA4 BB / z CAR T cells. (Parts A and B of Figure 2, and Parts A and B of Figure 3). 286-294 28 / z CAR T cells against MAGEA4 286-294 It was revealed that the BB / z CAR T cells showed excellent in vivo antitumor effects and antitumor kinetics compared to the MAGEA4 BB / z CAR T cells. (Parts A and B of Figure 2, and Parts A and B of Figure 3).

[0208] Example 3: Chimeric antigen receptor containing anti-HLA-A2 / MAGEA4 230-239 scFv V L -V H anti-HLA-A2 / MAGEA4 in the V 230-239In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR) was constructed using the anti-HLA-A2 / MAGEA4 230-239 V of antibody 33229P L and V H sequences. As a non-binding control, the 28 / z CAR was designed using the huCD28 hinge / transmembrane / co-stimulatory domain and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See Part B of Figure 4 for the constructs and Part A of Figure 4 for the results.

[0209]

Table 13

[0210]

Table 14

[0211] Experimental procedures Generation of CAR constructs and CAR T cells V L -V H anti-HLA-A2 / MAGEA4 in the direction of 230-239 In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR) was constructed using the anti-HLA-A2 / MAGEA4 230-239 V of antibody 33229P L and V HConstructed using an array. As a non-binding control, the 28 / z CAR was designed using an irrelevant scFv, the huCD28 hinge / transmembrane / costimulatory domain, and the CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and the P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated.

[0212] CD3+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of standard donors, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI = 5. The transduced cells were grown for approximately 14 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo experiments.

[0213] Xenograft tumor transplantation and measurement Anti-HLA-A2 / MAGEA4 230-239 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting anti-HLA-A2 / MAGEA4, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5 × 10 6 individuals of HLA-A2 + MAGEA4 + A375 human melanoma tumor cells. It was confirmed by mass spectrometry that A375 melanoma cells expressed approximately 553 cell surface copies of the HLA-A2 / MAGEA4 230-239 peptide. On day 13 after tumor engraftment, mice (n = 4 or 5 per group) were administered non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 Either of the 28 / z CARs (measured by the frequency of cells expressing GFP, which is a marker for CAR-transduced cells) expressing 4 × 10 6Individual T cells were injected intravenously. For tumor growth, it was evaluated until day 28 by measuring the tumor volume.

[0214] Calculation of heterologous tumor growth and inhibition To measure the tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurement, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0215] Summary of results and conclusions: A375 tumors grew progressively in untreated mice and mice administered control CAR T cells. Mice administered anti-HLA-A2 / MAGEA4 230-239 BB / z CAR T cells showed reduced tumor growth on days 19 (p < 0.02), 23 (p < 0.02), and 26 (p < 0.0001) compared to mice administered control CAR T, indicating some tumor control (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Administration of anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T also resulted in suppression of established A375 tumor growth on days 19 (p = 0.007), 23 (p < 0.0001), and 26 (p < 0.0001) (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Enhanced efficacy of anti-HLA-A2 / MAGEA4

[0216] In summary, the results revealed that anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells exhibited superior in vivo anti-tumor effects and anti-tumor kinetics compared to anti-HLA-A2 / MAGEA4 230-239 BB / z CAR T cells.

[0217]

Table 15-1

[0218]

Table 15-2

[0219]

Table 15-3

[0220] Example 4: Anti-HLA-A2 / Tyrosinase 369-377 Chimeric antigen receptor containing scFv V L -V H Anti-HLA-A2 / Tyrosinase in the V 369-377 direction, in addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) was constructed for anti-HLA-A2 / Tyrosinase 369-377 The V L and V H sequences of antibody D11 (identified by patent WO / 2016 / 199141) were used for construction. The anti-HLA-A2 / Tyrosinase 369-377 antibody binds to both HLA-A2 / Tyrosinase 369-377 and HLA-A2 / Tyrosinase with N371 deamidated to D371, similar to 369-377(N371D) As a non-binding control, BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See part A of Figure 5 for the construct schematic.

[0221] In summary, depending on the results, anti-HLA-A2 / tyrosinase derived from two donors 369-377 28 / z CAR T cells showed superior in vivo antitumor effects and antitumor kinetics compared to anti-HLA-A2 / tyrosinase 369-377 BB / z CAR T cells, as revealed.

[0222]

Table 16

[0223]

Table 17

[0224] Experimental procedure Preparation of CAR constructs and CAR T cells V L -V H Anti-HLA-A2 / tyrosinase in the V 369-377 In addition to the scFv, a chimeric antigen receptor containing either 1) a huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) a huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR) was constructed for anti-HLA-A2 / tyrosinase 369-377 using the V L and V H sequences of antibody D11 (identified by patent WO / 2016 / 199141). As a non-binding control, BB / z CAR was designed using an irrelevant scFv in addition to a huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing an EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See Part A of Figure 5 for a schematic diagram of the constructs.

[0225] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) of two standard donors (“Donor 1” and “Donor 2”), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI = 5. The transduced cells were grown for 19 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo experiments.

[0226] Xenograft tumor transplantation and measurement Anti-HLA-A2 / tyrosinase 369-377 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting anti-HLA-A2 / tyrosinase, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 4 × 10 6 individual HLA-A2 + tyrosinase + WM266.4 human melanoma tumor cells. It was confirmed using mass spectrometry that WM266.4 melanoma cells expressed approximately 4,500 cell surface copies of the HLA-A2 / tyrosinase 369-377 peptide. Seven days after tumor engraftment, mice (n = 5 per group) were intravenously injected with 2 × 10 369-377 individual T cells expressing either unbound control BB / z CAR (control CAR T), anti-HLA-A2 / tyrosinase 369-377 BB / z CAR or anti-HLA-A2 / tyrosinase 6 28 / z CAR (measured by the frequency of cells expressing GFP, a marker of cells transduced with CAR). Tumor growth was evaluated by measuring tumor volume until day 90.

[0227] A second xenograft tumor study was performed using a different tumor cell line. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5 × 10 6 individual HLA-A2 +Tyrosinase + SK-MEL-23 human melanoma tumor cells were subcutaneously injected. The SK-MEL-23 melanoma cells expressed approximately 2,000 - 3,000 cell surface copies of HLA-A2 / tyrosinase 369-377 which was confirmed using mass spectrometry. On the 7th day after tumor establishment, mice (n = 5 per group) were intravenously injected with 4×10 369-377 individual T cells expressing either unbound control BB / z CAR (control CAR T), anti-HLA-A2 / tyrosinase 369-377 BB / z CAR or anti-HLA-A2 / tyrosinase 6 28 / z CAR (measured by the frequency of cells expressing GFP, a marker of cells transduced with CAR). For tumor growth, tumor volume was measured to evaluate up to day 90 by measuring the tumor volume

[0228] Calculation of xenograft tumor growth and inhibition To measure the tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, the tumor volume was calculated by the formula: volume (mm 3 ) = (length × width 2 ) / 2

[0229] Summary of results and conclusions: WM266.4 tumor model: Donor 1: WM266.4 tumors grew progressively in mice not administered with the therapeutic agent and in mice administered with control CAR T cells. Mice administered with anti-HLA-A2 / tyrosinase 369-377 BB / z CAR T cells showed a decrease in tumor growth on day 31 (p = 0.02), day 35 (p = 0.001), day 42 (p = 0.0004), and day 49 (p < 0.0001) compared to the control, indicating some efficacy (statistically analyzed by two-way ANOVA). In contrast, administration of anti-HLA-A2 / tyrosinase 369-377 28 / z CAR T resulted in regression of established WM266.4 tumors, and 4 out of 5 mice had no tumors by day 90. Anti-HLA-A2 / tyrosinase 369-377Anti-HLA-A2 / Tyrosinase as Compared to BB / z CAR 369-377 Enhanced efficacy of 28 / z CAR was confirmed, and tumor sizes on days 35, 42, and 49 were statistically significant by two-way ANOVA (p = 0.01, p < 0.0001, and p < 0.000, respectively). See Part B of Figure 5.

[0230] Donor 2: WM266.4 tumors grew progressively in mice not receiving the therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / Tyrosinase 369-377 Mice administered BB / z CAR T cells had reduced tumor growth on days 28 (p = 0.03), 31 (p = 0.004), 35 (p = 0.0001), 42 (p = 0.0001), and 49 (p < 0.0001) compared to the control and showed some efficacy (analyzed statistically by two-way ANOVA). In contrast, Anti-HLA-A2 / Tyrosinase 369-377 Administration of 28 / z CAR T resulted in regression of established WM266.4 tumors, and 5 out of 5 mice had no tumors by day 90. Anti-HLA-A2 / Tyrosinase 369-377 Anti-HLA-A2 / Tyrosinase as Compared to BB / z CAR 369-377 Enhanced efficacy of 28 / z CAR was confirmed, and tumor sizes on days 24, 28, 31, 35, 42, and 49 were statistically significant by two-way ANOVA (p = 0.03, p = 0.008, p = 0.0004, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). See Part B of Figure 5.

[0231] SK-MEL-23 Tumor Model: Donor 1: SK-MEL-23 tumors grew progressively in mice not receiving the therapeutic agent and in mice receiving control CAR T cells. Anti-HLA-A2 / Tyrosinase 369-377 Mice administered BB / z CAR T cells also had progressive tumor growth similar to the control. In contrast, Anti-HLA-A2 / Tyrosinase 369-377Administration of 28 / z CAR T inhibited established SK-MEL-23 tumor growth significantly at day 31 (p = 0.02), day 35 (p = 0.004), day 42 (p = 0.0003), and further at day 49, day 55, day 63, day 72, day 78, and day 90 (p < 0.0001 for these days) compared to animals administered with control CAR T cells (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / tyrosinase 369-377 Anti-HLA-A2 / tyrosinase compared to BB / z CAR 369-377 Enhanced efficacy of 28 / z CAR was confirmed, and tumor sizes at day 35, day 42, day 49, day 55, day 63, day 72, day 78, and day 90 were statistically significant by two-way ANOVA test (p = 0.04 and p = 0.004 at day 35 and day 42; p < 0.0001 for all other days). See Part C of Figure 5.

[0232] Donor 2: SK-MEL-23 tumors grew progressively in mice not administered with therapeutic agents and in mice administered with control CAR T cells. Anti-HLA-A2 / tyrosinase 369-377 Mice administered with BB / z CAR T cells also had progressive tumor growth similar to the control. In contrast, anti-HLA-A2 / tyrosinase 369-377 Administration of 28 / z CAR T resulted in a reduction in the size of established SK-MEL-23 tumors and inhibition of tumor growth at day 21 (p = 0.02), day 24 (p = 0.004), and day 28 (p = 0.0004), as well as at day 31, day 35, day 42, day 49, day 55, day 63, day 72, day 78, and day 90 (all p < 0.0001 for these days) compared to animals administered with control CAR T cells (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / tyrosinase 369-377 Anti-HLA-A2 / tyrosinase compared to BB / z CAR 369-377The enhanced efficacy of 28 / z CAR was confirmed, and the tumor sizes on the 21st, 24th, 28th, 31st, 35th, 42nd, 49th, 55th, 63rd, 72nd, 78th, and 90th days were statistically significant by two-way ANOVA test (p = 0.01 and p = 0.005 on the 21st and 24th days; p < 0.0001 for all other days).

[0233]

Table 18-1

[0234]

Table 18-2

[0235]

Table 18-3

[0236]

Table 18-4

[0237]

Table 18-5

[0238]

Table 18-6

[0239]

Table 18-7

[0240]

Table 18-8

[0241]

Table 18-9

[0242]

Table 18-10

[0243]

Table 18-11

[0244]

Table 18-12

[0245]

Table 18-13

[0246]

Table 18-14

[0247]

Table 18-15

[0248]

Table 18-16

[0249] Example 5: Anti-HLA-A2 / NY-ESO-1 157-165 Chimeric antigen receptor containing scFv V H -V L Anti-HLA-A2 / NY-ESO-1 in the V 157-165In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) was used against HLA-A2 / NY-ESO-1 157-165 V of antibody 28105P L and V H sequences were used for construction. As a non-binding control, the BB / z CAR was designed using an irrelevant scFv in addition to the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV pseudotyped lentiviruses. See Figure 6 for construct design (Part A) and results (Part B). In summary, the results showed that the anti-HLA-A2 / NY-ESO-1 157-165 28 / z CAR T cells exhibited superior in vivo anti-tumor effects and anti-tumor kinetics compared to the anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR T cells.

[0250]

Table 19

[0251]

Table 20

[0252] Experimental procedures: Generation of CAR constructs and CAR T cells V H -V L anti-HLA-A2 / NY-ESO-1 in the direction of 157-165In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR) was used against HLA-A2 / NY-ESO-1 157-165 V of antibody 28105P L and V H sequences were used for construction. As a non-binding control, the BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells) to generate VSV-pseudotyped lentiviruses.

[0253] CD3+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of standard donors, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. The transduced cells were grown for 19 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo experiments.

[0254] Xenograft tumor transplantation and measurement Against HLA-A2 / NY-ESO-1 157-165 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting HLA-A2 / NY-ESO-1, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5×10 6 individuals of HLA-A2 + NY-ESO-1 + A375 human melanoma tumor cells. Three days after tumor engraftment, mice (n = 5 per group) were given non-binding control BB / z CAR (control CAR T) from two different donors, anti-HLA-A2 / NY-ESO-1157-165 BB / z CAR or anti-HLA-A2 / NY-ESO-1 157-165 Either 20×10 6 T cells expressing either 28 / z CAR (measured by the frequency of cells expressing GFP, a marker of CAR-transduced cells) were injected intravenously. For tumor growth, tumor volume was measured to evaluate up to day 38 by measuring the tumor volume.

[0255] Calculation of xenograft tumor growth and inhibition To measure tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, the tumor volume was calculated by the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0256] Summary of results and conclusions: A375 tumors grew progressively in mice administered control CAR T cells. Mice administered anti-HLA-A2 / NY-ESO-1 157-165 BB / z CAR T cells showed reduced tumor growth at day 21 (p < 0.04), day 28 (p < 0.0001), and day 38 (p < 0.0001) compared to mice administered control CAR T and showed some tumor control (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / NY-ESO-1 157-165 Administration of 28 / z CAR T also resulted in suppression of established A375 tumor growth at day 16 (p = 0.03), day 19 (p = 0.0002), day 21 (p < 0.0001), day 28 (p < 0.0001), and day 38 (p < 0.0001) (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / NY-ESO-1 157-165 Enhanced efficacy of anti-HLA-A2 / NY-ESO-1 157-165 compared to BB / z CAR was confirmed, and the tumor size was statistically significant at day 21 (p = 0.002), day 28 (p < 0.0001), and day 38 (p < 0.0001) (p < 0.0001 on both days) by two-way ANOVA. See Part B of Figure 6.

[0257]

Table 21-1

[0258]

Table 21-2

[0259]

Table 21-3

[0260]

Table 21-4

[0261] Example 6: Two anti-HLA-A2 / MAGEA4 230-239 One of the scFvs, or a chimeric antigen receptor containing anti-HLA-A2 / MAGEA4 286-294 In vitro cytotoxic effect by T cells expressing a chimeric antigen receptor containing anti-HLA-A2 / MAGEA4 Two anti-HLA-A2 / MAGEA4 230-239 scFv or anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB co-stimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / co-stimulatory domain, and the CD3z signaling domain (28 / z CAR), two anti-HLA-A2 / MAGEA4 230-239 Antibodies (33229 or 34852), and anti-HLA-A2 / MAGEA4 286-294 Antibody (31345) V L And V HConstructed using an array. As non-binding controls, BB / z CAR was designed using a non-related scFv, in addition to the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain, and 28 / z CAR was designed using a non-related scFv, in addition to the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See Figures 8 and 11 for construct design and Figure 7 for results.

[0262]

Table 22-1

[0263]

Table 22-2

[0264] Experimental procedures Generation of CAR constructs and CAR T cells Two anti-HLA-A2 / MAGEA4 230-239 scFv or anti-HLA-A2 / MAGEA4 286-294 In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain (28 / z CAR) was used for the two anti-HLA-A2 / MAGEA4 230-239 antibodies (33229 or 34852), and anti-HLA-A2 / MAGEA4 286-294 antibody (31345) V L and V HConstructed using arrays. As non-binding controls, BB / z CAR was designed using a non-related scFv in addition to the huCD8 hinge / transmembrane domain, 4-1BB co-stimulatory domain, and CD3z signaling domain, and 28 / z CAR was designed using a non-related scFv in addition to the huCD28 hinge / transmembrane / co-stimulatory domain, and CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated.

[0265] CD3+ T cells were isolated from standard donor human peripheral blood mononuclear cells (PBMCs), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI = 5. Transduced cells were grown for 14 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads and then cryopreserved until use in in vivo cytotoxicity assays.

[0266] Calcein release cytotoxicity assay Anti-HLA-A2 / MAGEA4 containing either the BB / z or 28 / z intracellular signaling domain 230-239 and anti-HLA-A2 / MAGEA4 286-294 To measure the in vitro cytotoxic activity of chimeric antigen receptor (CAR) T cells directed against anti-HLA-A2 / MAGEA4, a 2.5-hour calcein release assay was performed. A375 or SK-MEL-37 cells (2×10 6Cells / mL) were labeled with 8 μm Calcein-AM (ThermoFisher) at 37°C for 35 minutes and washed three times with medium. Activated / proliferating T cells transduced with CAR and target cells labeled with calcein were co-cultured at various ratios in triplicate in 96-well round-bottom plates at 37°C for 2.5 hours. After incubation, the culture supernatant was transferred to a black clear-bottom 96-well microplate (Greiner Bio One), and calcein fluorescence was measured using a microplate reader. The percentage of calcein release was calculated as ((calcein signal - spontaneous calcein release) / (calcein maximum release - spontaneous calcein release)) * 100 and recorded as the percentage of cytotoxic effect. Spontaneous release was measured with target cells only, and maximum release was measured by lysing the target cells with 1% Triton X-114 detergent.

[0267] Summary and Conclusions of Results MAGEA4 + HLA-A2 + Some cytotoxic effects on A375 and SK-MEL-37 cells were seen with anti-MAGEA4 286-294 CAR (31345) containing the BB / z intracellular signaling domain, but in contrast, anti-MAGEA4 286-294 CAR (31345) containing the 28 / z intracellular signaling domain showed enhanced cytotoxic effects (Figure 7). Minimal background cytotoxic effects were induced by the CTL CAR construct (Figure 7). Therefore, anti-MAGEA4 286-294 CAR (31345) CAR induced superior cytotoxic effects compared to the same CAR utilizing the BB / z signaling domain.

[0268] MAGEA4 + HLA-A2 + Some cytotoxic effects on A375 and SK-MEL-37 cells were seen with anti-MAGEA4 230-239 CAR (33229) containing the BB / z intracellular signaling domain, but in contrast, anti-MAGEA4239-239 In CAR (33229), enhanced cytotoxicity was confirmed (Figure 7), and minimal background cytotoxicity was induced by the CTL CAR construct (Figure 7). Therefore, the anti-MAGEA4 containing the 28 / z signaling domain 230-239 The CAR (33229) CAR induced superior cytotoxicity compared to the same CAR utilizing the BB / z signaling domain.

[0269] MAGEA4 + HLA-A2 + Some cytotoxicity of A375 cells was seen with the anti-MAGEA4 containing the BB / z intracellular signaling domain 230-239 In CAR (34852), but in contrast, the anti-MAGEA4 containing the 28 / z intracellular signaling domain 239-239 In CAR (34852), enhanced cytotoxicity was confirmed (Figure 7). Minimal background cytotoxicity was induced by the CTL CAR construct (Figure 7). Therefore, the anti-MAGEA4 containing the 28 / z signaling domain 230-239 The CAR (34852) CAR induced superior cytotoxicity compared to the same CAR utilizing the BB / z signaling domain.

[0270] [Table 23-1]

[0271] [Table 23-2]

[0272] [Table 23-3]

[0273] [Table 23-4]

[0274]

Table 23-5

[0275]

Table 23-6

[0276]

Table 23-7

[0277]

Table 23-8

[0278]

Table 23-9

[0279] Example 7: Chimeric antigen receptor containing one of two anti-HLA-A2 / MAGEA4 230-239 single-chain variable fragments (scFvs), in addition to either 1) a huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) a huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR), was generated against anti-HLA-A2 / MAGEA4 V L -V H orientation anti-HLA-A2 / MAGEA4 230-239 scFv. In addition to the anti-HLA-A2 / MAGEA4 230-239 antibody 33229P V L and V HConstructed using an array. As a non-binding control, a 28 / z CAR was designed using an irrelevant scFv, along with the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and the P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. See FIGS. 9 and 10 for the results and construct design, respectively.

[0280] In summary, the results show that anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells 230-239 were shown to exhibit superior in vivo anti-tumor effects and anti-tumor kinetics compared to anti-HLA-A2 / MAGEA4

[0281] [Table 24]

[0282] Experimental procedures Generation of CAR constructs and CAR T cells V L -V H anti-HLA-A2 / MAGEA4 in the V 230-239 direction, in addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain (28 / z CAR) was 230-239 V of antibody 33229P L and V HConstructed using an array. As a non-binding control, the 28 / z CAR was designed using an irrelevant scFv, along with the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and the P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated.

[0283] CD3+ T cells were isolated from standard donor human peripheral blood mononuclear cells (PBMCs), stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI of 5. The transduced cells were grown for approximately 14 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and then cryopreserved until use in in vivo experiments.

[0284] Xenograft tumor transplantation and measurement Anti-HLA-A2 / MAGEA4 230-239 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting anti-HLA-A2 / MAGEA4, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5×10 6 individual HLA-A2 + MAGEA4 + A375 human melanoma tumor cells. It was confirmed using mass spectrometry that A375 melanoma cells expressed approximately 553 cell surface copies of the HLA-A2 / MAGEA4 230-239 peptide. On day 11 after tumor engraftment, mice (n≥5 per group) were injected with either non-binding control 28 / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 1×10 expressing any of the 28 / z CARs (measured by the frequency of cells expressing GFP, a marker of CAR-transduced cells) 6 individuals, 5×10 6cells, or 10×10 6 Individuals were intravenously injected with 6 T cells. Tumor growth was evaluated until day 28 by measuring tumor volume.

[0285] Calculation of heterologous tumor growth and inhibition To measure tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurements, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0286] Summary of results 1×10 6 The A375 tumors at a dose of 6 CAR T cells grew progressively in mice administered with control CAR T cells. Mice administered with anti-HLA-A2 / MAGEA4 230-239 BB / z CAR T cells showed no tumor control. In contrast, administration of anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T resulted in suppression of established A375 tumor growth on day 9 (p = 0.001), day 13 (p < 0.0001), and day 16 (p < 0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). The enhanced efficacy of anti-HLA-A2 / MAGEA4 230-239 28 / z CAR compared to anti-HLA-A2 / MAGEA4 230-239 BB / z CAR was confirmed, and the tumor size was statistically significant on day 9 (p = 0.0086), day 13 (p < 0.0001), and day 16 (p < 0.0001) by two-way ANOVA.

[0287] 5×10 6 The A375 tumors at a dose of 6 CAR T cells grew progressively in mice administered with control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239Administration of BB / z CAR T cells resulted in suppression of established A375 tumor growth on day 9 (p<0.0001), day 13 (p<0.0001), and day 16 (p<0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Administration of 28 / z CAR T cells also resulted in suppression of established A375 tumor growth on day 6 (p<0.0001), day 9 (p<0.0001), day 13 (p<0.0001), and day 16 (p<0.0001) (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Anti-HLA-A2 / MAGEA4 compared to BB / z CAR 230-239 Enhanced efficacy of 28 / z CAR was confirmed, and tumor size was statistically significant by two-way ANOVA on day 6 (p = 0.0049), day 9 (p<0.0001), day 13 (p = 0.0003), and day 16 (p = 0.0453).

[0288] 10×10 6 The 10×10 dose of CAR T:A375 tumors grew progressively in mice administered control CAR T cells. Anti-HLA-A2 / MAGEA4 230-239 Administration of BB / z CAR T cells resulted in suppression of established A375 tumor growth on day 6 (p = 0.0214), day 9 (p<0.0001), day 13 (p<0.0001), and day 16 (p<0.0001) compared to control CAR T (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Administration of 28 / z CAR T cells also resulted in suppression of established A375 tumor growth on day 6 (p<0.0001), day 9 (p<0.0001), day 13 (p<0.0001), and day 16 (p<0.0001) (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Anti-HLA-A2 / MAGEA4 compared to BB / z CAR 230-239Enhanced efficacy of 28 / z CAR was confirmed, and tumor size was statistically significant on day 6 (p = 0.0282) and day 9 (p = 0.0212) by two-way ANOVA.

[0289]

Table 25-1

[0290]

Table 25-2

[0291]

Table 25-3

[0292]

Table 25-4

[0293]

Table 25-5

[0294]

Table 25-6

[0295] Example 8: Anti-HLA-A2 / MAGEA4 230-239 Chimeric antigen receptor containing scFv V L -V H Anti-HLA-A2 / MAGEA4 in the V direction 230-239 In addition to the scFv, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain, and CD3z signaling domain (28 / z CAR) was used for anti-HLA-A2 / MAGEA4 230-239 V of antibody 34852L and V H were constructed using the arrays. As non-binding controls, 28 / z CARs were designed using the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain, in addition to an irrelevant scFv. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and the P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated. For construct design and results, see Figures 11 and 12, respectively.

[0296] Collectively, the results showed that the anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T cells exhibited superior in vivo anti-tumor effects and anti-tumor kinetics compared to the anti-HLA-A2 / MAGEA4 230-239 BB / z CAR T cells.

[0297] [Table 26]

[0298] Experimental procedures Generation of CAR constructs and CAR T cells V L -V H anti-HLA-A2 / MAGEA4 in the V 230-239 direction. In addition to the anti-HLA-A2 / MAGEA4 230-239 scFv of antibody 34852, a chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, the 4-1BB costimulatory domain, and the CD3z signaling domain (BB / z CAR) or 2) the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain (28 / z CAR) was used. L and V HConstructed using an array. As a non-binding control, the 28 / z CAR was designed using an irrelevant scFv, along with the huCD28 hinge / transmembrane / costimulatory domain and the CD3z signaling domain. These CARs were cloned into a pLVX lentiviral vector containing the EF1a promoter and the P2A:eGF sequence (to track CAR-transduced cells), and VSV pseudotyped lentiviruses were generated.

[0299] CD3+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of standard donors, stimulated with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and transduced with lentivirus at an MOI = 5. The transduced cells were grown for approximately 14 days with 100 U / ml recombinant human IL-2 in addition to CD3 / CD28 microbeads, and then cryopreserved until use in in vivo experiments.

[0300] Xenograft tumor transplantation and measurement Anti-HLA-A2 / MAGEA4 230-239 To measure the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting anti-HLA-A2 / MAGEA4, xenograft tumor studies were performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously injected with 5×10 6 individual HLA-A2 + MAGEA4 + A375 human melanoma tumor cells. It was confirmed by mass spectrometry that A375 melanoma cells expressed approximately 553 cell surface copies of the HLA-A2 / MAGEA4 230-239 peptide. On day 10 after tumor engraftment, mice (n = 5 per group) were injected with non-binding control 28 / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR or anti-HLA-A2 / MAGEA4 230-239 28 / z CAR (measured by the frequency of cells expressing any GFP, which is a marker of CAR-transduced cells) from two different donors, each expressing 4×10 6Individual T cells were injected intravenously. For tumor growth, tumor volume was measured to evaluate up to day 28 by measuring the tumor volume.

[0301] Calculation of heterologous tumor growth and inhibition To measure the tumor volume with an external caliper, the maximum longitudinal diameter (length in mm) and the maximum transverse diameter (width in mm) were measured. Based on the caliper measurement, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0302] Summary and conclusion of the results: The A375 tumor grew progressively in mice administered with control CAR T cells. Mice administered with anti-HLA-A2 / MAGEA4 230-239 BB / z CAR T cells showed no tumor control at all. In contrast, administration of anti-HLA-A2 / MAGEA4 230-239 28 / z CAR T resulted in suppression of established A375 tumor growth on day 17 (p < 0.0001 compared to control CAR T) and day 20 (p < 0.0001 compared to control CAR T) (statistically analyzed by two-way ANOVA). Anti-HLA-A2 / MAGEA4 230-239 Enhanced efficacy of 28 / z CAR compared to control anti-HLA-A2 / MAGEA4 230-239 CAR was confirmed, and the tumor size was statistically significantly different on day 13 (p = 0.0256), day 17 (p = 0.0002), and day 21 (p < 0.0001) by two-way ANOVA.

[0303]

Table 27-1

[0304]

Table 27-2

[0305]

Table 27-3

[0306] Incorporation by reference All publications, patents, patent applications, and sequence reference numbers mentioned in this specification are specifically and individually indicated as being incorporated by reference, and the entire contents thereof are hereby incorporated by reference into this specification. In case of any conflict, this application shall prevail, including any definitions in this specification.

[0307] Equivalents Numerous embodiments of the present invention have been described. It will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains.

[0308] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A CAR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, 9, 11, 13, 30, or 34.

2. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

7.

3. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

9.

4. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

11.

5. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

13.

6. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

30.

7. The CAR polypeptide according to claim 1, wherein the CAR polypeptide comprises SEQ ID NO:

34.

8. An immune cell expressing the CAR polypeptide according to claim 1.

9. A composition comprising an immune cell expressing the CAR according to claim 1.

10. A nucleic acid encoding the CAR polypeptide according to claim 1.

11. An immune cell comprising the nucleic acid according to claim 10.

12. The immune cell according to claim 11, wherein the immune cell is a leukocyte, lymphocyte, mononuclear cell, macrophage, dendritic cell, mast cell, neutrophil, basophil, eosinophil, αβ T cell, γδ T cell, natural killer (NK) cell, natural killer T (NKT) cell, innate lymphoid cell (ILC), cytokine-induced killer (CIK) cell, cytotoxic T lymphocyte (CTL), lymphokine-activated killer (LAK) cell, or regulatory T cell.

13. A composition comprising a cell expressing the CAR polypeptide according to claim 1 for use in a method of treating a tumor in a subject, the method comprising administering the composition.

14. The method comprises a) a 4-1BB domain in the co-stimulatory region of a second CAR polypeptide, b) at least one intracellular signaling region comprising a cluster of differentiation 3 zeta (CD3 / ζ) domain, and c) an extracellular domain specific for a groove peptide cancer antigen, The composition according to claim 13, further comprising co-administering the second CAR polypeptide to the subject.

15. The composition according to claim 14, wherein the second CAR polypeptide comprises a cluster of differentiation 8 alpha (CD8 / α) peptide in the hinge / transmembrane region.

16. A cell bank containing cells for adoptive immunotherapy, wherein the cells express the CAR according to claim 1, said cell bank.

17. A nucleic acid encoding a CAR polypeptide, wherein the nucleic acid has the sequence set forth in SEQ ID NO: 14, 15, 35, or 36, said nucleic acid.

18. The nucleic acid encoding a CAR polypeptide according to claim 17, wherein the nucleic acid contains SEQ ID NO:

14.

19. The nucleic acid encoding a CAR polypeptide according to claim 17, wherein the nucleic acid contains SEQ ID NO:

15.

20. The nucleic acid encoding a CAR polypeptide according to claim 17, wherein the nucleic acid contains SEQ ID NO:

35.

21. The nucleic acid encoding a CAR polypeptide according to claim 17, wherein the nucleic acid contains SEQ ID NO: 36.

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