Chimeric antigen receptor comprising a TMIGD2 co-stimulatory domain and method of using the same

Abstract

The present technology provides chimeric antigen receptors (CARs) containing the TMIGD2 costimulatory domain. In certain embodiments, these CARs contain an intracellular region containing the TMIGD2 costimulatory domain and an effector domain, a transmembrane domain, an extracellular region containing an antigen-binding domain, and optionally a hinge region and / or a leader sequence. In other embodiments, these CARs contain an intracellular region containing the TMIGD2 costimulatory domain and an effector domain, a transmembrane domain, an extracellular region containing an antigen-binding domain that specifically binds to the tumor-associated antigen B7-H3, and optionally a hinge region and / or a leader sequence. Also provided are nucleic acids, vectors, and cells related to these CARs, as well as methods of using the CARs, nucleic acids, vectors, and cells in the treatment of conditions associated with the expression of a specific antigen.

Description

Technical Field

[0001] Cross - reference to Related Applications and Claim of Priority This application claims the priority of U.S. Provisional Patent Application No. 63 / 368,549 (filing date: July 15, 2022), the entire content of which is incorporated herein by reference and relied upon.

[0002] Statement Regarding Federal Funding This invention was made with government support under CA175495 - 08 and CA175495 - 06 awarded by the National Institutes of Health (NIH) / National Cancer Institute (NCI). The government has certain rights in this invention.

[0003] Sequence Listing Incorporated by Reference This application includes a sequence listing compliant with ST.26, which is submitted simultaneously in xml format via the Patent Center and the entire content of which is incorporated herein by reference. The name of the xml copy created on July 14, 2023 is 1298078025WO00.xml and the size is 25KB.

Background Art

[0004] Adoptive transfer of chimeric antigen receptor - modified T cells, NK cells, macrophages or other immune cells is a powerful antigen - specific therapy for treating diseases such as human malignancies. T cells, NK cells, macrophages or other immune cells expressing CAR target tumor - associated antigens expressed on the surface of malignant cells. CAR - T, NK cell, macrophage or other immune cell - based treatment strategies target antigens already expressed on the cell surface, thus overcoming problems related to tumor - avoidance mechanisms involving the major histocompatibility complex.

[0005] Advances in the understanding of the mechanisms and molecules that regulate the immune response have provided many therapeutic targets for treating cancer. For example, co-stimulatory and co-inhibitory molecules play central roles in the regulation of T cell immune responses. However, checkpoint inhibition therapies still fail in many patients, despite the remarkable responses of patients to antibodies targeting these co-stimulatory and co-inhibitory molecules, including, for example, anti-PD-1 / PD-L1.

[0006] There is still a need for new compositions and methods that can improve the effectiveness of these agents, particularly agents that include CAR-modified T cells. SUMMARY OF THE INVENTION

[0007] In some aspects, this disclosure provides a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular region containing an antigen-binding domain; (b) a transmembrane region; and (c) an intracellular region containing an effector domain and a TMIGD2 co-stimulatory domain.

[0008] In some embodiments, the TMIGD2 co-stimulatory domain contains the intracellular region of TMIGD2. In some embodiments, the TMIGD2 co-stimulatory domain contains a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5. In some embodiments, the TMIGD2 co-stimulatory domain contains an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5.

[0009] In some embodiments, the antigen-binding domain specifically binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of HHLA2, CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3 and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; a tumor- or pathogen-associated peptide bound to HLA; an hTERT peptide bound to HLA; a tyrosinase peptide bound to HLA; a WT-1 peptide bound to HLA; LTβR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME and SSX-2 antigen.

[0010] In some embodiments, the antigen-binding domain contains an scFv. In some embodiments, the antigen-binding domain contains a linker. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker contains (Gly x Ser y ) z where x and y are each independently an integer from 0 to 10, except when both x and y are 0, and z is an integer from 1 to 10.

[0011] In some embodiments, the extracellular region further contains an N-terminal leader sequence. In some embodiments, the extracellular region further contains a hinge region. In some embodiments, the hinge region contains the amino acid sequence shown in SEQ ID NO: 2.

[0012] In some embodiments, the transmembrane region contains the transmembrane region of CD8α. In some embodiments, the transmembrane region contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0013] In some embodiments, the effector domain is the effector domain of CD3ζ. In some embodiments, the effector domain contains the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the effector domain contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 5.

[0014] In some embodiments, the CAR contains: (a) a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 1. In another embodiment, the CAR contains: (a) a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 6. In yet another embodiment, the CAR contains: (a) a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO: 5, (b) the sequence shown in SEQ ID NO: 1; and (c) the sequence shown in SEQ ID NO: 6. In some embodiments, the CAR further contains the sequence shown in SEQ ID NO: 2.

[0015] In some aspects, this disclosure provides an isolated polynucleotide encoding the CAR described herein.

[0016] In one aspect, this disclosure provides an expression vector containing the isolated polynucleotide described in this specification operably linked to an expression control sequence. In some embodiments, the expression control sequence is a promoter. In some embodiments, the expression control sequence is a promoter.

[0017] In some embodiments, the expression vector further contains an isolated polynucleotide encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is a P2A peptide. In some embodiments, the isolated polynucleotide encoding the self-cleaving peptide is 3' of the polynucleotide encoding the CAR. In some embodiments, the isolated polynucleotide encoding the self-cleaving peptide is 5' of the isolated polynucleotide encoding the marker polypeptide.

[0018] In some embodiments, the expression vector further contains an isolated polynucleotide encoding a transduction marker polypeptide. In some embodiments, the transduction marker polypeptide is a cleaved form of the epidermal growth factor receptor (EGFRt) or a portion or variant thereof, or GFP or a portion or variant thereof.

[0019] In some embodiments, the vector is a viral vector.

[0020] In one aspect, this disclosure provides a host cell expressing the CAR of this application, containing the isolated polynucleotide of this application, and / or containing the expression vector of this application.

[0021] In some embodiments, the host cell is a T cell, natural killer (NK) cell, macrophage or other immune cell. In some embodiments, the T cell is a CD4 + T cell, CD8 + T cell, CD4 - CD8 -They are double-negative T cells, NK cells, macrophages, other immune cells, or combinations thereof. In some embodiments, the T cells are naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, NK cells, macrophages, other immune cells, or combinations thereof.

[0022] In some embodiments, the host cell further expresses a transduction marker on its cell surface. In some embodiments, the transduction marker is a cleaved form of the epidermal growth factor receptor (EGFRt) or a portion or variant thereof, or GFP or a portion or variant thereof.

[0023] In some aspects, this disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the host cells of this application.

[0024] In some embodiments, the disease or condition is a malignant tumor. In some embodiments, the malignant tumor is cancer. In some embodiments, the cancer is selected from the group consisting of prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, stomach, thyroid, anus, small intestine, bone, cervical, endometrial, esophagus, eye, gallbladder, thymus, sarcoma, and osteosarcoma. In some embodiments, the cancer includes solid tumors. In some embodiments, the cancer includes hematological malignancies.

[0025] In some aspects, this disclosure provides a method of inducing an immune response against a tumor-associated antigen that specifically binds to the CAR of this application, the method comprising administering to a subject in need thereof an effective amount of the host cells of this application.

[0026] In some aspects, this disclosure provides a composition comprising the CAR of this application and a pharmaceutically acceptable excipient, carrier, or diluent.

[0027] In one aspect, this disclosure provides a composition comprising the cells of this application and a pharmaceutically acceptable excipient, carrier, or diluent.

[0028] These and other aspects of this application are disclosed in more detail hereinbelow. **Brief Description of the Drawings**

[0029]

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[0030] Detailed Description This disclosure provides CARs containing a TMIGD2 co-stimulatory domain, nucleic acid molecules and vectors encoding these CARs, cells expressing these CARs, and methods of using said CARs, nucleic acid molecules, vectors and cells.

[0031] HHLA2 (HERV-H LTR-related 2) is a functional member of the B7 family, and TMIGD2 (NCBI accession number NP_653216.2) has been identified as a co-stimulatory receptor for HHLA2 (Zhao 2013; Janakiram 2015a; Janakiram 2015b). There are at least three isoforms of TMIGD2: isoform 1 (SEQ ID NO: 3, NCBI NP_653216.2), isoform 2 (SEQ ID NO: 4, NCBI NP_001162597.1) and isoform 3 (SEQ ID NO: 5, NCBI NP_001295161.1). Representative DNA sequences encoding isoforms 1-3 are shown in SEQ ID NO: 9 (NCBI NM_144615.), SEQ ID NO: 10 (NCBI NM_001169126.1) and SEQ ID NO: 11 (NCBI NM_001308232), respectively.

[0032] Most CARs incorporate CD28 or 4-1BB as co-stimulatory molecules, but this disclosure relates to CARs using TMIGD2 as a co-stimulatory molecule. TMIGD2 is expressed in the majority of human naive T cells. After antigen stimulation, almost all T cells lose TMIGD2 expression, and this loss is associated with increased PD-1 levels, exhaustion and senescence (Zhu 2013; Crespo 2017; Janakiram 2017). Using TMIGD2 for co-stimulation in CAR-T cell therapy provides a unique opportunity to maintain TMIGD2 co-stimulatory signaling and preserve functionally active CAR-T cells.

[0033] Representative CARs of this application contain: (a) an extracellular region containing a binding domain (e.g., scFv); (b) a transmembrane region; and (c) an intracellular region containing an effector domain and a TMIGD2 costimulatory domain. The TMIGD2 costimulatory domain may contain all or part of the intracellular region of a TMIGD2 isoform or its variant, including any of isoforms 1-3.

[0034] The CARs of this application are useful in cellular immunotherapies (e.g., T cells and / or natural killer (NK) cells) for treating diseases associated with the expression of one or more antigens such as cancer. In some embodiments, when administered to a subject having malignant cells expressing one or more antigens associated with cancer, the CARs of this application reduce and / or suppress the growth, extent, volume, and / or spread of the malignant cells, eliminate (e.g., kill) the malignant cells, and / or increase the survival of the subject to a greater extent and / or for a longer period than cells not containing the CARs of this application.

[0035] The following description of this application is merely intended to illustrate various aspects of this application. Therefore, specific modifications discussed in this specification are not to be considered as limitations on the scope of this application. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of this application, and such equivalent aspects are also understood to be included in this specification.

[0036] Throughout the specification, references to "example," "embodiment," or "aspect" mean that a particular characteristic, structure, or feature described in connection with the example is included in at least one example of this application. Accordingly, the phrases "in an example," "embodiment," or "aspect" in various places throughout the specification are not necessarily all referring to the same example, embodiment, and / or aspect.

[0037] The headings used in the specification are for convenience only and are not intended to limit or interpret the scope or meaning of this application.

[0038] Definition Unless otherwise indicated, the ranges of concentrations, percentages, ratios, or integers referred to in this specification are understood to include any integers within the recited ranges and, where appropriate, fractional values thereof (such as one-tenth and one-hundredth of an integer). Also, the ranges of numbers recited in this specification are understood to include the integers included in the recited ranges unless otherwise indicated. In this specification, the term "about" means ±20% of the indicated range, value, or structure unless otherwise indicated. In this specification, it should be understood that the term "one" refers to "one or more" in the recited area. Words used in the singular or plural form respectively also include the plural or singular form. The use of the term "or" referring to a list of two or more items encompasses all of the following interpretations of the words: any of the items in the list, all of the items in the list, and combinations of the items in the list. Further, the phrase "at least one of A, B, and C, etc." is intended from the perspective that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). When a convention similar to "at least one of A, B, or C, etc." is used, generally, such a configuration is intended from the perspective that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). In this specification, the terms "comprising," "having," and "containing" are used in the same meaning, and these terms and their variants are intended to be interpreted as non-limiting.

[0039] A "nucleic acid molecule" or "polynucleotide" refers to a polymeric compound containing covalently linked nucleotides that contain natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, any of which may be single-stranded or double-stranded. Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences encoding the same amino acid sequence.

[0040] The "percent identity" or "sequence identity percent" with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as needed to achieve the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignments for the purpose of determining the percent amino acid sequence identity can be achieved by various means known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software or other software suitable for nucleic acid sequences. Appropriate parameters for sequence alignment can be determined, including the algorithms required to achieve the maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, however, the percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was made by Genentech, Inc., and the source code is deposited with the user documentation at the U.S. Copyright Office, Washington D.C., 20559 and is registered under U.S. Copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif. or can be compiled from the source code. The ALIGN-2 program should be compiled for use in a UNIX (registered trademark) operating system including Digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0041] In the situation where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having some percent amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the ALIGN-2 sequence alignment program, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. Unless specifically indicated otherwise, all percent amino acid sequence identity values used in this specification are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0042] "Conservative substitution" refers to an amino acid substitution that does not significantly affect or change the binding characteristics of a particular protein. Generally, a conservative substitution is one in which the amino acid residue being substituted is replaced with an amino acid residue having a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and, Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be classified into conservative substitution groups based on similar function, chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic or sulfur-containing). For example, the aliphatic group may include Gly, Ala, Val, Leu and Ile for the purpose of substitution. Other conservative substitution groups include: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn and Gln; small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu and Gln; polar positively charged residues: His, Arg and Lys; large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and, large aromatic residues: Phe, Tyr and Trp. Additional information can be found in Creighton (1984) Proteins , W.H. Freeman and Company. In certain embodiments, the variant proteins, peptides, polypeptides and amino acid sequences of this application may include one or more conservative substitutions compared to the reference amino acid sequence.

[0043] The term "isolated" means that a material has been removed from its original environment (e.g., its natural environment if it occurs naturally). Such a nucleic acid may be part of a vector, and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), yet still be isolated in that such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide.

[0044] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that contains only a domain, motif, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with a domain, portion, or fragment of the parent or reference compound. In certain embodiments, a functional portion refers to a "signaling portion" of an effector molecule, effector domain, co-stimulatory molecule, or co-stimulatory domain.

[0045] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or combinations thereof. The nucleic acid molecule to be expressed is typically operably linked to an expression control sequence (e.g., a promoter).

[0046] The term "operably linked" refers to the joining of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other.

[0047] As used herein, the term "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences that can effect the expression of the nucleic acid molecule in a preferred host. Such control sequences include a promoter that effects transcription, an operator sequence that controls transcription if desirable, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. When transformed into a preferred host, the vector may replicate and function independently of the host genome, or in some instances, may be integrated into the genome itself. As used herein, the terms "plasmid", "expression plasmid", "virus", and "vector" are often used interchangeably.

[0048] As used in the context of inserting a nucleic acid molecule into a cell, the term "introduced" means "transfection", "transformation", or "transduction", which includes reference to the incorporation of a nucleic acid molecule into a eukaryotic cell, where the nucleic acid molecule may be incorporated into the genome of the cell and converted into an autonomous replicon. As used herein, the terms "engineered", "recombinant", or "non-native" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic abnormality or has been modified by the introduction of an exogenous nucleic acid molecule, where such an abnormality or modification is introduced by genetic engineering. Genetic abnormalities include, for example, modifications that introduce an expressible nucleic acid molecule encoding a protein, CAR, or enzyme, or the addition, deletion, substitution, or other functional disruption of the genetic material of the cell by another nucleic acid molecule.

[0049] The term "construct" refers to a polynucleotide containing a recombinant nucleic acid molecule. The construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into the genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. The vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule that may contain chromosomal, episomal, semi-synthetic or synthetic nucleic acid molecules. Representative vectors are those capable of autonomous replication (episomal vectors), those capable of delivering a polynucleotide to a cell genome (e.g., viral vectors) or those capable of expressing a ligated nucleic acid molecule (expression vectors).

[0050] As used herein, "enrichment" or "depletion" with respect to the amount of a cell type in a mixture refers to an increase in the number of "enriched" cell types, a decrease in the number of "depleted" cell types, or both, resulting from one or more methods or processes of enrichment or depletion. In certain embodiments, CD4 + cells are enriched and CD8 + cells are depleted, or CD8 + cells are enriched and CD4 + cells are depleted, or combinations thereof, such that the amount of a particular cell type in the mixture is enriched and the amount of a different cell type is depleted.

[0051] A "chimeric antigen receptor" (CAR) refers to a CAR of this application engineered to contain two or more naturally occurring (or engineered) amino acid sequences linked in a manner not found in nature or not found in a particular cell, and which, when present on the surface of a cell, can function as a receptor. The CARs of this application include an antigen-binding domain of an antibody (e.g., one obtained or derived from an immunoglobulin, such as an scFv), an extracellular portion linked to a transmembrane region, and one or more intracellular signaling domains (which may have a co-stimulatory domain) (see, e.g., Sadelain et al., 2013; Harris & Kranz, 2016; Stone et al., 2014).

[0052] The terms "variable region" or "variable domain" refer to the heavy or light chains of an antibody that are involved in binding to an antigen. The variable domain (V H ) of the antibody heavy chain and the variable domain (V L ) of the light chain generally contain four generally conserved framework regions (FRs) and three CDRs each. The framework regions separate the CDRs, and the CDRs are positioned between the framework regions.

[0053] The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and generally confer antigen specificity and / or binding affinity and refer to the sequences of amino acids within the antibody variable region that are separated from each other in the primary structure by the framework sequences, as is known in the art. In some cases, framework amino acids may also contribute to binding. Generally, there are three CDRs in each variable region. The variable domain sequences can be aligned to numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT), and Aho) that allow annotation of the positions of equivalent residues and comparison across different molecules using the Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).

[0054] "Antigen" as used herein refers to an immunogenic molecule that induces an immune response. This immune response may include antibody production, activation of specific immunologically compatible cells, or both. Antigens may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is readily apparent that antigens may be synthesized, produced recombinantly, or derived from biological samples. Representative biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens may be produced by cells that have been modified or genetically engineered to express the antigen.

[0055] "Binding domain" (also referred to as "binding region") as used herein refers to a molecule or a portion thereof that has the ability to specifically and non-covalently bind, associate, or conjugate with a target, such as an scFv. Binding domains include naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partners of biological molecules, molecular complexes, or other intended targets. Representative binding domains include single-chain immunoglobulin variable regions, receptor ectodomains, ligands, or synthetic polypeptides selected for their specific ability to bind to biological molecules, molecular complexes, or other intended targets.

[0056] As used herein, "effector domain" is the intracellular portion or domain of a CAR or receptor that can directly or indirectly promote a biological or physiological response in a cell upon receipt of an appropriate signal. In certain embodiments, the effector domain is derived from a protein or a portion thereof or a protein complex that receives a signal when bound to a target or a cognate molecule, or triggers the signal of the effector domain when the protein or a portion thereof or a protein complex binds directly to a target or a cognate molecule.

[0057] "Transmembrane region" as used herein is a portion of a transmembrane protein that can enter or traverse the cell membrane.

[0058] "To treat", "treatment" or "alleviate" refers to the medical management of a subject's disease, disorder or condition. Generally, an appropriate dose or treatment regimen containing cells expressing the CAR of this application and, if necessary, an adjuvant is administered in an amount sufficient to induce a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefits include improvement of clinical prognosis; reduction or alleviation of symptoms associated with the disease; reduction in the occurrence of symptoms; improvement in quality of life; longer disease-free status; attenuation of the degree of the disease; stabilization of the disease state; delay in disease progression; remission; survival; extension of survival period; or a combination thereof.

[0059] As used herein, "hyperproliferative disorder" and "proliferative disorder" refer to excessive growth or proliferation compared to normal or non-diseased cells. Representative hyperproliferative disorders and proliferative disorders include tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, and pre-malignant cells.

[0060] Furthermore, "cancer" may refer to accelerated proliferation of cells, including solid tumors, ascites tumors, blood, lymph or other malignant tumors; connective tissue malignancies; metastatic diseases; minimal residual disease after organ or stem cell transplantation; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis associated with malignancies or other forms of cancer.

[0061] The "therapeutically effective amount" or "effective amount" of cells expressing the CAR of this application refers to the number of cells expressing the CAR that is sufficient to produce a therapeutic effect, in a statistically significant manner, including improvement of clinical prognosis; reduction or alleviation of symptoms associated with the disease; reduction in the occurrence of symptoms; improvement in quality of life; longer disease-free status; attenuation of the degree of the disease, stabilization of the disease state; delay in disease progression; remission; survival; or extension of survival period. When referring to an individual active ingredient administered alone or a cell expressing a single active ingredient, the therapeutically effective amount refers to the action of that ingredient or that cell alone. When referring to a combination, the therapeutically effective amount refers to the amount of the combination of active ingredients or the amount of the combination of an adjuvant active ingredient and a cell expressing an active ingredient that produces a therapeutic effect, whether administered sequentially or simultaneously.

[0062] The term "pharmaceutically acceptable" in a carrier, excipient or diluent means that the carrier, excipient or diluent is suitable for administration to human or other non-human mammalian subjects and is generally recognized as being safe or not causing serious adverse events.

[0063] The term "adoptive immunotherapy" or "adoptive immunotherapy" as used herein refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells such as T cells. Adoptive cell immunotherapy may be autologous (immune cells are derived from the recipient), allogeneic (immune cells are derived from a donor of the same species) or syngeneic (immune cells are derived from a donor genetically identical to the recipient).

[0064] "T cell" or "T lymphocyte" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR) that includes αβ T cells and γδ T cells. T cells can be naive (not exposed to antigen, increased expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA and decreased expression of CD45RO compared to...), memory T cells (experienced antigen, long-lived) and effector cells (experienced antigen, cytotoxic). T... can be further divided into subsets of central memory T cells (increased expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95 and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (decreased expression of CD62L, CCR7, CD28, CD45RA and increased expression of CD127 compared to naive T cells or T...). CM compared to increased expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA and decreased expression of CD45RO), memory T cells (T... M (experienced antigen, long-lived) and effector cells (experienced antigen, cytotoxic). T... M can be further divided into subsets of central memory T cells (T... CM , increased expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95 and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (T... EM , decreased expression of CD62L, CCR7, CD28, CD45RA and increased expression of CD127 compared to naive T cells or T... CM ).

[0065] "Natural killer cells" or "NK cells" refer to cells in this specification that are activated in response to interferons or macrophage-derived cytokines, including viral infections, but adoptive immune responses can eliminate infections and generate antigen-specific cytotoxic T cells that express CD56.

[0066] In addition, it should be understood that individual constructs or groups of constructs derived from various combinations of the structures and subunits described in this specification are disclosed by this disclosure to the same extent that each individual construct or group of constructs is separately shown. Accordingly, the selection of a particular structure or particular subunit is within the scope of this application.

[0067] The terms used in the specification are intended to be construed in their broadest reasonable manner, even if used with a detailed description of a particular embodiment.

[0068] Chimeric antigen receptor (CAR) In this specification, chimeric antigen receptors (CARs) containing the TMIGD2 costimulatory domain and compositions containing these CARs are provided. In certain embodiments, the CARs provided herein include: (a) an extracellular region containing a binding domain that specifically binds to a tumor-associated antigen expressed on the surface of cells such as malignant cells; (b) a transmembrane region; and (c) an intracellular region containing an effector domain and a TMIGD2 costimulatory domain. Also provided in this specification are cells expressing these CARs and compositions thereof, as well as unit doses of these cells and compositions. In some embodiments, these unit doses contain (i) a composition containing at least about 50% modified CD4 + T cells and (ii) a composition containing at least about 50% modified CD8 + T cells in a ratio of about 1:1, where the unit dose has a reduced number of naive T cells or is substantially free of naive T cells.

[0069] TMIGD2 co-stimulatory domain The co-stimulatory domain refers to a part of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule that is not an antigen receptor or its ligand and is required for an efficient response of lymphocytes to an antigen.

[0070] The CAR provided in this specification contains a TMIGD2 co-stimulatory domain containing all or a part of the intracellular region of TMIGD2 or its variant. In certain embodiments, the TMIGD2 intracellular region is derived from TMIGD2 isoform 1 (SEQ ID NO: 3), isoform 2 (SEQ ID NO: 4), or isoform 3 (SEQ ID NO: 5). In certain embodiments, when the TMIGD2 co-stimulatory domain contains a part of the intracellular region of TMIGD2, that part is sufficient for the transmission of signals normally associated with the binding of TMIGD2 to HHLA2.

[0071] In certain embodiments, the TMIGD2 intracellular region is derived from isoform 1 or 3 and contains, consists of, or consists essentially of the amino acid sequence shown in residues 172 - 282 of SEQ ID NO: 3 or residues 52 - 162 of SEQ ID NO: 5 or a part thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172 - 282 of SEQ ID NO: 3 or residues 52 - 162 of SEQ ID NO: 5 or a part thereof.

[0072] In certain embodiments, the TMIGD2 intracellular region is derived from isoform 2 and contains, consists of, or consists essentially of the amino acid sequence shown in residues 172 - 278 of SEQ ID NO: 4 or a part thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to amino acids 172 - 278 of SEQ ID NO: 4 or a part thereof.

[0073] In certain embodiments where the TMIGD2 co-stimulatory domain contains an amino acid sequence having less than 100% identity to the TMIGD2 intracellular region or a portion thereof, e.g., the TMIGD2 intracellular regions of isoforms 1-3, all of the substitutions that result in sequence differences are conservative substitutions. In other embodiments, the TMIGD2 co-stimulatory domain may contain one or more non-conservative substitutions relative to the TMIGD2 intracellular region. In certain embodiments, the TMIGD2 co-stimulatory domain contains 1, 2, 3, 4, or 5 or more conservative substitutions relative to the TMIGD2 intracellular region.

[0074] Intracellular effector domain In certain embodiments, the CARs provided herein contain an intracellular effector domain. In some embodiments, the intracellular effector domain is the effector domain of CD3ζ or a functional portion or variant thereof.

[0075] In some embodiments, the effector domain of CD3ζ contains, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6.

[0076] In certain embodiments where the CARs provided herein contain an intracellular effector domain, the effector domain is directly adjacent to the TMIGD2 co-stimulatory domain. In other embodiments, the effector domain and the TMIGD2 co-stimulatory domain are separated by one or more amino acids.

[0077] Transmembrane region In certain embodiments, the CARs provided herein contain a transmembrane domain that connects the extracellular and intracellular regions.

[0078] In certain embodiments, the transmembrane region is derived from CD8α. In certain of these embodiments, the transmembrane region contains, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1.

[0079] Extracellular region In certain embodiments, the CAR provided herein contains an extracellular region that includes a target-specific binding element (also known as an antigen-binding domain).

[0080] In certain embodiments, the antigen-binding domain within the extracellular region is derived from an antibody and contains the antigen-binding portion thereof. For example, in certain embodiments, the antigen-binding domain is an antibody V H chain, all or a portion of it (e.g., V H variable domain or one or more of its CDRs), an antibody V L chain, all or a portion of it (e.g., V L variable domain or one or more of its CDRs), or both. In certain of these embodiments, the antigen-binding domain is a scFv. In certain of these embodiments, the scFv linker domain contains a peptide linker between the V L component and the V H component. For example, the scFv can be designed such that the C-terminus of the V L domain is linked to the N-terminus of the V H domain by a peptide linker ((N)V L (C)-linker-(N)V H (C)) or the C-terminus of the V H domain is linked to the N-terminus of the V L domain by a peptide linker (N)V H (C)-linker-(N)V L (C). Representative linkers include Gly x Ser yThose having a glycine-serine amino acid chain containing 1 to 10 repeats of are included, where x and y are each independently an integer from 0 to 10, provided that the case where both x and y are 0 is excluded (for example, combinations such as (Gly4Ser)2; (Gly3Ser)2; Gly2Ser; or (Gly3Ser)2Gly2Ser).

[0081] Anti-B7-H3 CAR-T binding domain sequences useful in the present technology are described, for example, in International Application No. PCT / US2020 / 66002 with the title "Chimeric Antigen Receptors Targeting B7-H3 (CD276) and Associated Methods" and International Application No. PCT / US2019 / 61887 with the title "Monoclonal antibodies against IgV domain of B7-H3 and uses thereof" (these technologies and sequences are hereby incorporated by reference in their entirety).

[0082] In some embodiments, the binding domain specifically binds to one or more tumor-associated antigens selected from HHLA2; CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3 and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; tumor- or pathogen-associated peptides bound to HLA; hTERT peptides bound to HLA; tyrosinase peptides bound to HLA; WT-1 peptides bound to HLA; LTβR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME and / or SSX-2 antigen.

[0083] The source of the binding domain, including known antibodies, methods of generating antibodies, and the binding domains described in this specification, is known in the art. In some embodiments, the tumor-associated antigen is CD19. In certain embodiments, the binding domain is derived from an anti-CD19 antibody such as, for example, the FMC-63 antibody, MOR208, blinatumomab, MEDI-551, Meck's patented anti-CD19 antibody, Xmab5871 or MDX-1342.

[0084] In some embodiments, the antigen-specific receptor binding domain is derived from the FMC-63 antibody, MOR208, blinatumomab, MEDI-551, the Merck-patented anti-CD19 antibody, Xmab5871 or MDX-1342, or has a VH or (i.e., and / or) VL having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to the antibody variable region or scFv derived from the FMC-63 antibody, MOR208, blinatumomab, MEDI-551, the Merck-patented anti-CD19 antibody, Xmab5871 or MDX-1342, or has a CDR or functional CDR variant from any one of these antibodies.

[0085] In some embodiments, the antigen-binding domain is directly adjacent to the transmembrane domain. In other embodiments, there is one or more intervening residues, such as a hinge region, linking the antigen-binding domain and the transmembrane domain. In certain embodiments where the CAR contains a hinge region, the hinge region is derived from CD8α, and in certain ones of these embodiments, the hinge region contains, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2.

[0086] In some embodiments, the extracellular region further contains a leader sequence, including but not limited to a leader peptide. For example, the leader peptide may be an IgG1 V H domain or V L domain N-terminally conjugated to the V K leader domain or a portion or variant thereof.

[0087] Specific embodiments In certain embodiments, the CARs provided herein contain (a) a TMIGD2 co-stimulatory domain and (b) an effector domain of intracellular CD3ζ or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of the amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the effector domain of CD3ζ contains, consists of, or consists essentially of the amino acid sequence shown in SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6 or a portion thereof. In certain of these embodiments, the TMIGD2 co-stimulatory domain and the effector domain of intracellular CD3ζ are directly adjacent to each other. In other embodiments, one or more amino acids separating the TMIGD2 co-stimulatory domain and the effector domain of intracellular CD3ζ are present. In certain embodiments, the CARs provided herein contain the amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the CARs provided herein contain the amino acid sequence shown in residues 52-162 of SEQ ID NO: 5 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the CARs provided herein contain the amino acid sequence shown in residues 172-278 of SEQ ID NO: 4 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 6.

[0088] In certain embodiments, the CARs provided in this specification contain (a) a TMIGD2 co-stimulatory domain and (b) a transmembrane region of CD8α or a portion or variant thereof. In certain of these embodiments, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO: 1 or a portion thereof. In certain embodiments, the transmembrane region of CD8α is directly adjacent to the TMIGD2 co-stimulatory domain, and in another embodiment, there is one or more amino acids separating the transmembrane region of CD8α and the TMIGD2 co-stimulatory domain. In certain embodiments, the CARs provided in this specification contain the amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the CARs provided in this specification contain the amino acid sequence shown in residues 52-162 of SEQ ID NO: 5 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the CARs provided in this specification contain the amino acid sequence shown in residues 172-278 of SEQ ID NO: 4 or a portion thereof and the amino acid sequence shown in SEQ ID NO: 1.

[0089] In certain embodiments, the CAR provided in this specification contains (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, and (c) a transmembrane region of CD8α or a portion or variant thereof. In certain ones of these embodiments, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown by residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the effector domain of CD3ζ contains, consists of, or consists essentially of an amino acid sequence shown by SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 or a portion thereof; (c) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown by SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or a portion thereof. In certain embodiments, the TMIGD2 co-stimulatory domain is located between the transmembrane region of CD8α and the effector domain of CD3ζ. In other embodiments, the effector domain of CD3ζ is located between the transmembrane region of CD8α and the TMIGD2 co-stimulatory domain.In certain embodiments, the transmembrane region of CD8α is directly adjacent to the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2, and in other embodiments, there are one or more amino acids separating the transmembrane region of CD8α and the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2. Similarly, in certain embodiments, the co-stimulatory domain of TMIGD2 and the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there are one or more amino acids separating the co-stimulatory domain of TMIGD2 and the intracellular effector domain of CD3ζ. In certain embodiments, the CARs provided herein contain the amino acid sequences shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the CARs provided herein contain the amino acid sequences shown in residues 52-162 of SEQ ID NO: 5 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the CARs provided herein contain the amino acid sequences shown in residues 172-278 of SEQ ID NO: 4 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 1.

[0090] In certain embodiments, the CAR provided in this specification contains (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, (c) a transmembrane region of CD8α or a portion or variant thereof, and (d) a hinge region of CD8α or a portion or variant thereof. In certain ones of these embodiments, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the effector domain of CD3ζ contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6 or a portion thereof; (c) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or a portion thereof; (d) the hinge region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or a portion thereof. In certain embodiments, the TMIGD2 co-stimulatory domain is located between the transmembrane region of CD8α and the effector domain of CD3ζ.In other embodiments, the effector domain of CD3ζ is located between the transmembrane region of CD8α and the TMIGD2 costimulatory domain. In certain embodiments, the transmembrane region of CD8α is directly adjacent to the effector domain of CD3ζ or the TMIGD2 costimulatory domain, and in other embodiments, there is one or more amino acids separating the transmembrane region of CD8α and the effector domain of CD3ζ or the TMIGD2 costimulatory domain. Similarly, in certain embodiments, the TMIGD2 costimulatory domain and the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there is one or more amino acids separating the TMIGD2 costimulatory domain and the intracellular effector domain of CD3ζ. In certain embodiments, the hinge region of CD8α is directly adjacent to the transmembrane region of CD8α. In other embodiments, there is one or more amino acids separating the hinge region and the transmembrane region. In certain embodiments, the CAR provided herein contains the amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the CAR provided herein contains the amino acid sequence shown in residues 52-162 of SEQ ID NO: 5 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the CAR provided herein contains the amino acid sequence shown in residues 172-278 of SEQ ID NO: 4 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2.

[0091] In certain embodiments, the CARs provided herein contain (a) a TMIGD2 co-stimulatory domain, (b) a transmembrane region of CD8α or a portion or variant thereof, and (c) a hinge region of CD8α. In certain of these embodiments, the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or a portion thereof; (c) the hinge region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or a portion thereof. In certain embodiments, the transmembrane region of CD8α is directly adjacent to the TMIGD2 co-stimulatory domain, and in another embodiment, one or more amino acids separating the transmembrane region of CD8α and the TMIGD2 co-stimulatory domain are present. In certain embodiments, the hinge region of CD8α is directly adjacent to the transmembrane region of CD8α. In other embodiments, one or more amino acids separating the hinge region and the transmembrane region are present. In certain embodiments, the CARs provided herein contain the amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2.In certain embodiments, the CAR provided in this specification contains the amino acid sequence shown in residues 52-162 of SEQ ID NO: 5 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the CAR provided in this specification contains the amino acid sequence shown in residues 172-278 of SEQ ID NO: 4 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2.

[0092] In certain aspects, the CARs provided in this specification contain (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, (c) a transmembrane region of CD8α or a portion or variant thereof, and (d) an extracellular region containing an antigen-binding domain. In certain ones of these aspects, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the effector domain of CD3ζ contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6 or a portion thereof; (c) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or a portion thereof; (d) the antigen-binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain aspects, the TMIGD2 co-stimulatory domain is located between the transmembrane region of CD8α and the effector domain of CD3ζ. In other aspects, the effector domain of CD3ζ is located between the transmembrane region of CD8α and the TMIGD2 co-stimulatory domain.In certain embodiments, the transmembrane region of CD8α is directly adjacent to the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2, and in other embodiments, there are one or more amino acids separating the transmembrane region of CD8α and the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2. Similarly, in certain embodiments, the co-stimulatory domain of TMIGD2 and the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there are one or more amino acids separating the co-stimulatory domain of TMIGD2 and the intracellular effector domain of CD3ζ. Similarly, in certain embodiments, the antigen-binding domain is directly adjacent to the transmembrane region of CD8α, and in other embodiments, there are one or more amino acids separating the antigen-binding domain and the transmembrane region of CD8α. In certain of these embodiments, the amino acids separating the antigen-binding domain and the transmembrane region of CD8α constitute a hinge region, and in certain of these embodiments, the hinge region contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or a portion thereof, which is the hinge region of CD8α.

[0093] In certain embodiments, the CARs provided in this specification contain: (a) a TMIGD2 co-stimulatory domain; (b) a transmembrane region of CD8α or a portion or variant thereof; and (c) an extracellular region containing an antigen-binding domain. In certain of these embodiments, (a) the TMIGD2 co-stimulatory domain contains, consists of, or consists essentially of an amino acid sequence shown in residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172-282 of SEQ ID NO: 3 or a portion thereof, residues 52-162 of SEQ ID NO: 5 or a portion thereof, or residues 172-278 of SEQ ID NO: 4 or a portion thereof; (b) the transmembrane region of CD8α contains, consists of, or consists essentially of an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or a portion thereof; (c) the antigen-binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the transmembrane region of CD8α is directly adjacent to the TMIGD2 co-stimulatory domain, and in another embodiment, there are one or more amino acids separating the transmembrane region of CD8α and the TMIGD2 co-stimulatory domain. Similarly, in certain embodiments, the antigen-binding domain is directly adjacent to the transmembrane region of CD8α, and in another embodiment, there are one or more amino acids separating the antigen-binding domain and the transmembrane region of CD8α.In certain ones of these aspects, the amino acids separating the antigen-binding domain and the transmembrane region of CD8α constitute a hinge region, and in certain ones of these aspects, the hinge region contains, consists of, or consists essentially of the amino acid sequence shown in SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or a portion thereof, which is the hinge region of CD8α.

[0094] In certain aspects, the CARs provided herein contain the amino acid sequence shown in residues 172 - 282 of SEQ ID NO: 3 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and an antigen-binding domain. In certain aspects, the CARs provided herein contain the amino acid sequence shown in residues 52 - 162 of SEQ ID NO: 5 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and an antigen-binding domain. In certain aspects, the CARs provided herein contain the amino acid sequence shown in residues 172 - 278 of SEQ ID NO: 4 or a portion thereof, the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and an antigen-binding domain.

[0095]

Table 1

[0096] Nucleic acid molecules, vectors and cells In this specification, in certain aspects, nucleic acid molecules encoding one or more of the CARs provided in the specification are provided, as well as compositions and vectors containing these nucleic acid molecules. In this specification, cells containing these nucleic acid molecules or vectors and their compositions are provided, as well as unit doses of these cells and compositions. In some aspects, these unit doses are (i) at least about 50% modified CD4 +A composition containing T cells, (ii) at least about 50% of modified CD8 + A composition containing T cells in a ratio of about 1:1, wherein the unit dose has a reduced number of naive T cells or is substantially free of naive T cells.

[0097] TMIGD2 co-stimulatory domain In certain embodiments, the nucleic acid molecules provided herein encode a CAR containing a TMIGD2 co-stimulatory domain derived from TMIGD2 isoform 1 (SEQ ID NO: 9), isoform 2 (SEQ ID NO: 10), or isoform 3 (SEQ ID NO: 11).

[0098] In certain embodiments, the nucleic acid molecules provided herein encode a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to residues 172 - 282 of SEQ ID NO: 3 or a portion thereof, residues 52 - 162 of SEQ ID NO: 5 or a portion thereof, or residues 172 - 282 of SEQ ID NO: 3 or residues 52 - 162 of SEQ ID NO: 5 or a portion thereof. In certain ones of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 - 849 of SEQ ID NO: 9 or nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof.

[0099] In certain embodiments, the nucleic acid molecules provided herein encode a CAR that contains an intracellular effector domain. In some embodiments, the intracellular effector domain is the effector domain of CD3ζ or a functional portion or variant thereof.

[0100] Intracellular effector domain In certain embodiments, the nucleic acid molecules provided herein encode a CAR that contains an intracellular effector domain. In some embodiments, the intracellular effector domain is the effector domain of CD3ζ or a functional portion or variant thereof.

[0101] In certain embodiments, the nucleic acid molecules provided herein encode an amino acid sequence of SEQ ID NO: 6 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 or a portion thereof. In certain ones of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of a nucleotide sequence shown in SEQ ID NO: 12 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 12 or a portion thereof.

[0102] Transmembrane region In certain embodiments, the nucleic acid molecules provided herein encode a CAR containing a transmembrane domain. In some embodiments, the transmembrane domain is the transmembrane domain of CD8α or a variant thereof.

[0103] In certain embodiments, the nucleic acid molecules provided herein encode an amino acid sequence of SEQ ID NO: 1 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or a portion thereof. In certain ones of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7.

[0104] Extracellular region In certain aspects, the nucleic acid molecules provided in this specification contain nucleic acid sequences encoding extracellular regions. In certain ones of these aspects, the extracellular region contains an antigen-binding domain, and in certain ones of these aspects, the antigen-binding domain specifically binds to a tumor-associated antigen selected from HHLA2, CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; a tumor- or pathogen-associated peptide bound to HLA; an hTERT peptide bound to HLA; a tyrosinase peptide bound to HLA; a WT-1 peptide bound to HLA; LTβR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME and / or SSX-2 antigen.

[0105] In certain embodiments, the extracellular region further contains a hinge region that connects the antigen-binding domain to the transmembrane domain. In certain of these embodiments, the hinge region is the hinge region of CD8α. In certain embodiments, the nucleic acid molecule provided herein encodes the hinge region of CD8α of SEQ ID NO: 2 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 or a portion thereof. In certain of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8.

[0106] Marker and self-cleaving peptide In certain embodiments, the nucleic acid molecule provided herein contains a nucleotide sequence encoding a marker such as GFP and / or EGFRt. In certain of these embodiments, the nucleic acid molecule encodes the EGFRt marker of SEQ ID NO: 15 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15 or a portion thereof. In certain of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 18 or a portion thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 18.

[0107] In certain embodiments, the nucleic acid molecule provided herein contains a nucleotide sequence encoding a self-cleaving peptide such as a 2A peptide. Representative 2A peptides are P2A and F2A.

[0108] In certain of these embodiments, the nucleic acid molecule encodes a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 or a portion thereof, or to the P2A self-cleaving peptide of SEQ ID NO: 14 or a portion thereof, or to SEQ ID NO: 13 or a portion thereof, or to SEQ ID NO: 14 or a portion thereof. In certain of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 16 or a portion thereof, or in SEQ ID NO: 17 or a portion thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 16 or SEQ ID NO: 17.

[0109] In certain embodiments, the nucleic acid molecules provided herein that contain a nucleotide sequence encoding a marker such as GFP and / or EGFRt are linked to a signal leader sequence. One representative sequence is the GMCSFR alpha chain signal sequence linked to EGFRt, which induces surface expression.

[0110] Specific embodiments In certain embodiments, the nucleic acid molecules provided in this specification encode (a) a TMIGD2 co-stimulatory domain and (b) an effector domain of intracellular CD3ζ or a portion or variant thereof. In certain ones of these embodiments, the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to (a) nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof; and (b) SEQ ID NO: 12 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 12 or a portion thereof, and contains, consists of, or consists essentially of the same. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the effector domain of CD3ζ are directly adjacent to each other, and in another embodiment, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the effector domain of CD3ζ. In certain embodiments, the nucleic acid molecule further contains, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, or SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both of SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both of SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0111] In certain embodiments, the nucleic acid molecules provided in this specification encode (a) a TMIGD2 co-stimulatory domain and (b) a transmembrane region of CD8α or a portion or variant thereof. In certain of these embodiments, the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to (a) nucleotides 514 to 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 to 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 to 837 of SEQ ID NO: 10 or a portion thereof, or (b) at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 or a portion thereof, or a nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, and contains, consists of, or consists essentially of such sequences. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the transmembrane region of CD8α are directly adjacent to each other, and in another embodiment, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the transmembrane region of CD8α. In certain embodiments, the nucleic acid molecule further contains, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0112] In certain aspects, the nucleic acid molecules provided in this specification encode (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, and (c) a transmembrane region of CD8α or a portion or variant thereof. In certain ones of these aspects, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof; (b) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 or a portion thereof, or to the nucleotide sequence set forth in SEQ ID NO: 12 or a portion thereof; and (c) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 or a portion thereof, or to the nucleotide sequence set forth in SEQ ID NO: 7 or a portion thereof. In certain aspects, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the effector domain of CD3ζ. In other aspects, the nucleic acid sequence encoding the effector domain of CD3ζ is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain.In certain embodiments, the nucleic acid sequence encoding the transmembrane region of CD8α is directly adjacent to the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2. Similarly, in certain embodiments, the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 and the nucleic acid sequence encoding the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 and the nucleic acid sequence encoding the intracellular effector domain of CD3ζ. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0113] In certain embodiments, the nucleic acid molecules provided herein encode (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, (c) a transmembrane region of CD8α or a portion or variant thereof, and (d) a hinge region of CD8α or a portion or variant thereof. In certain ones of these embodiments, the nucleic acid molecule comprises, consists of, or consists essentially of: (a) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof; (b) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12 or a portion thereof, or to the nucleotide sequence shown in SEQ ID NO: 12 or a portion thereof; (c) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7 or a portion thereof, or to the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, and (d) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8 or a portion thereof, or to the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the effector domain of CD3ζ.In other aspects, the nucleic acid sequence encoding the effector domain of CD3ζ is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain. In certain aspects, the nucleic acid sequence encoding the transmembrane region of CD8α is directly adjacent to the nucleic acid sequence encoding the effector domain of CD3ζ or the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain, and in other aspects, there are one or more nucleotides separating the nucleic acid sequence encoding the transmembrane region of CD8α from the nucleic acid sequence encoding the effector domain of CD3ζ or the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain. Similarly, in certain aspects, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the effector domain of CD3ζ are directly adjacent to each other, and in other aspects, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain from the nucleic acid sequence encoding the intracellular effector domain of CD3ζ. In certain aspects, the nucleic acid sequence encoding the hinge region of CD8α is directly adjacent to the nucleic acid sequence encoding the transmembrane region of CD8α, and in other aspects, there are one or more nucleotides separating the nucleic acid sequence encoding the hinge region from the nucleic acid sequence encoding the transmembrane region. In certain aspects, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0114] In certain embodiments, the nucleic acid molecules provided in this specification encode (a) a TMIGD2 co-stimulatory domain, (b) a transmembrane region of CD8α or a portion or variant thereof, and (c) a hinge region of CD8α or a portion or variant thereof. In certain ones of these embodiments, the nucleic acid molecule contains, consists of, or consists essentially of: (a) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 - 837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 - 837 of SEQ ID NO: 10 or a portion thereof; (b) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7 or a portion thereof, or to the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof; and (c) a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8 or a portion thereof, or to the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the effector domain of CD3ζ. In other embodiments, the nucleic acid sequence encoding the effector domain of CD3ζ is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain.In certain embodiments, the nucleic acid sequence encoding the transmembrane region of CD8α is directly adjacent to the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the transmembrane region of CD8α from the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2. Similarly, in certain embodiments, the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 and the nucleic acid sequence encoding the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 from the nucleic acid sequence encoding the intracellular effector domain of CD3ζ. In certain embodiments, the nucleic acid sequence encoding the hinge region of CD8α is directly adjacent to the nucleic acid sequence encoding the transmembrane region of CD8α, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the hinge region from the nucleic acid sequence encoding the transmembrane region. In certain embodiments, the nucleic acid molecule further comprises, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0115] In certain embodiments, the nucleic acid molecules provided in this specification encode (a) a TMIGD2 co-stimulatory domain, (b) an effector domain of intracellular CD3ζ or a portion or variant thereof, (c) a transmembrane region of CD8α or a portion or variant thereof, and (d) an extracellular region containing an antigen-binding domain. In certain of these embodiments, the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to (a) nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 - 837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514 - 837 of SEQ ID NO: 10 or a portion thereof; (b) SEQ ID NO: 12 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 12 or a portion thereof; (c) SEQ ID NO: 7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, and comprises, consists of, or consists essentially of, and (d) the antigen-binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the effector domain of CD3ζ. In other embodiments, the nucleic acid sequence encoding the effector domain of CD3ζ is located between the nucleic acid sequence encoding the transmembrane region of CD8α and the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain.In certain embodiments, the nucleic acid sequence encoding the transmembrane region of CD8α is directly adjacent to the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the transmembrane region of CD8α from the nucleic acid sequence encoding the effector domain of CD3ζ or the co-stimulatory domain of TMIGD2. Similarly, in certain embodiments, the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 and the nucleic acid sequence encoding the effector domain of CD3ζ are directly adjacent to each other, and in other embodiments, there are one or more nucleotides separating the nucleic acid sequence encoding the co-stimulatory domain of TMIGD2 from the nucleic acid sequence encoding the intracellular effector domain of CD3ζ. Similarly, in certain embodiments, the antigen-binding domain is directly adjacent to the transmembrane region of CD8α, and in other embodiments, there are one or more nucleotides separating the antigen-binding domain from the transmembrane region of CD8α. In certain of these embodiments, the nucleotides separating the antigen-binding domain from the transmembrane region of CD8α constitute a hinge region, and in certain of these embodiments, the hinge region contains, consists of, or consists essentially of a nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof, which is the hinge region of CD8α. In certain embodiments, the nucleic acid molecule further contains, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0116] In certain embodiments, the nucleic acid molecules provided in this specification encode an extracellular region containing (a) a TMIGD2 co-stimulatory domain, (b) a transmembrane region of CD8α or a portion or variant thereof, and (c) an antigen-binding domain. In certain of these embodiments, the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to (a) nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof, or nucleotides 514-849 of SEQ ID NO: 9 or a portion thereof, nucleotides 154-489 of SEQ ID NO: 11 or a portion thereof, or nucleotides 514-837 of SEQ ID NO: 10 or a portion thereof; (b) SEQ ID NO: 7 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO: 7 or a portion thereof, and (c) the antigen-binding domain is a scFv that specifically binds to a tumor-associated antigen. In certain embodiments, the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the transmembrane region of CD8α are directly adjacent to each other, and in another embodiment, there are one or more nucleotides separating the nucleic acid sequence encoding the TMIGD2 co-stimulatory domain and the nucleic acid sequence encoding the transmembrane region of CD8α. In certain embodiments, the antigen-binding domain is directly adjacent to the transmembrane region of CD8α, and in another embodiment, there are one or more nucleotides separating the antigen-binding domain and the transmembrane region of CD8α.In certain of these embodiments, the nucleotides separating the antigen-binding domain from the transmembrane region of CD8α constitute the hinge region, and in certain of these embodiments, the hinge region contains, consists of, or consists essentially of the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 8 or a portion thereof. In certain embodiments, the nucleic acid molecule further contains, consists of, or consists essentially of SEQ ID NO: 16 or a portion thereof, SEQ ID NO: 17 or a portion thereof, SEQ ID NO: 18 or a portion thereof, both SEQ ID NO: 16 and SEQ ID NO: 18 or a portion thereof, or both SEQ ID NO: 17 and SEQ ID NO: 18 or a portion thereof.

[0117] In certain embodiments, the nucleic acid molecules provided herein contain the nucleotide sequence shown in nucleotides 514 - 849 of SEQ ID NO: 9 or a portion thereof, the nucleotide sequence shown in SEQ ID NO: 12, the nucleotide sequence shown in SEQ ID NO: 7, the nucleotide sequence shown in SEQ ID NO: 8, and a nucleic acid sequence encoding an antigen-binding domain. In certain embodiments, the nucleic acid molecules provided herein contain the nucleotide sequence shown in nucleotides 514 - 837 of SEQ ID NO: 10 or a portion thereof, the nucleotide sequence shown in SEQ ID NO: 12, the nucleotide sequence shown in SEQ ID NO: 7, the nucleotide sequence shown in SEQ ID NO: 8, and a nucleic acid sequence encoding an antigen-binding domain. In certain embodiments, the nucleic acid molecules provided herein contain the nucleotide sequence shown in nucleotides 154 - 489 of SEQ ID NO: 11 or a portion thereof, the nucleotide sequence shown in SEQ ID NO: 12, the nucleotide sequence shown in SEQ ID NO: 7, the nucleotide sequence shown in SEQ ID NO: 8, and a nucleic acid sequence encoding an antigen-binding domain.

[0118] [Table 2]

[0119]

Table 3

[0120] In any of the embodiments described in this specification, the nucleic acid molecule encoding the CAR may be codon-optimized for a particular cell using known techniques (Scholten et al., 2006). Codon optimization can be performed, for example, using the GenScript® OptimumGene™ tool. The codon-optimized sequences include sequences that are partially or fully codon-optimized.

[0121] The nucleic acid molecule encoding the CAR of this application can be inserted into an expression vector such as a viral vector for transduction into cells such as T cells. In some embodiments, the expression construct of this application encodes the CAR provided in this specification and, optionally, further encodes a self-cleaving peptide and / or an EGFRt marker operably linked to an expression control sequence such as a promoter, and contains a nucleic acid molecule.

[0122] In certain embodiments, the nucleic acid molecule of this application may be operably linked to a particular element of the vector. For example, a polynucleotide sequence required to affect the expression and processing of the ligated coding sequence may be operably linked. Expression control sequences may include appropriate transcription start, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and sequences that enhance protein secretion. Expression control sequences may be operably linked when adjacent to the gene of interest, and expression control sequences act in trans or at a distance to control the gene of interest.

[0123] In certain embodiments, the expression construct is included in a vector such as a viral vector, where the vector may be integrated into the genome of the cell or promote integration of the nucleic acid molecule insert when introduced into the cell, and thereby replicated with the genome of the cell. Viral vectors include retroviruses, adenoviruses, parvoviruses, coronaviruses, negative-strand RNA viruses, positive-strand RNA viruses, and double-stranded DNA viruses. (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0124] The genetic manipulation of the CAR of interest and the construction of the expression vector used for production can be achieved by using suitable molecular biology manipulation techniques known in the art. To obtain efficient transcription and translation, the polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence such as a promoter operably linked to a leader sequence and in particular a nucleotide sequence encoding an immunogen. The methods for making the CARs of this application are described, for example, in U.S. Patent No. 6,410,319; No. 7,446,191; U.S. Patent Application Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; International Publication No. 2014 / 031687; U.S. Patent No. 7,514,537; Brentjens et al., 2007 and Walseng et al., 2017 (these techniques are incorporated herein by reference).

[0125] In certain embodiments, the nucleic acid molecules of this application are used for transfection / transduction into cells such as T cells, NK cells, macrophages or other immune cells in adoptive cell therapy. Cells may be induced to incorporate the vector or other material using viral vectors, transformation by calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection or other methods. Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989). T cells and / or NK cells can be collected using known techniques, and various subsets or combinations thereof can be enriched or depleted by known techniques such as affinity binding to antibodies, flow cytometry or immunomagnetic selection. In certain embodiments, the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - double negative T cells, naive T cells, central memory T cells, effector memory T cells, stem cell memory T cells or combinations thereof. Methods for transfecting / transducing polynucleotides into T cells have been described previously (U.S. Patent Application Publication No. 2004 / 0087025), and adoptive transfer procedures using T cells with the desired target specificity have also been described (Schmitt et al. 2009; Dossett et al. 2009; Till et al. 2008; Wang et al. 2007; Kuball et al., 2007; Leen et al., 2007; U.S. Patent Application Publication No. 2011 / 0243972; No. 2011 / 0189141), and applying these techniques to the CARs disclosed in this application is within the scope of this application.

[0126] The functional characterization of the CARs described in this specification may be performed according to methods acceptable in the art for assaying the activity of T cells and / or NK cells, including determination of binding, activation or induction of T cells and / or NK cells, and also including determination of responses of antigen-specific T cells and / or NK cells. Examples include intracellular calcium, T cell proliferation, T cell and / or NK cell cytokine release, antigen-specific T cell and / or NK cell stimulation, MHC-restricted T cell and / or NK cell stimulation, cytotoxic activity, changes in T cell and / or NK cell phenotype marker expression, phosphorylation of specific T cell and / or NK cell proteins, and determination of other measures of T cell and / or NK cell function. Procedures for performing these assays and similar assays are described in this specification and / or, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998), and the references cited in these documents.

[0127] Kit In some embodiments, there is provided a kit comprising (a) a CAR vector disclosed herein, (b) a CAR nucleic acid molecule polynucleotide disclosed herein that may encode a marker peptide and / or a self-cleaving peptide, and / or (c) one or more reagents for transducing a cell with the vector or nucleic acid molecule. In certain embodiments, the kit further comprises instructions for use.

[0128] Method of use This disclosure provides a method of treating a disease or condition, the method comprising administering to a subject in need thereof an effective amount of a composition, cell, or unit dose of this application, wherein the disease or condition expresses an antigen to which the CAR provided in this specification specifically binds or is otherwise associated with said antigen. In certain embodiments, the disease or condition is a hyperproliferative or proliferative disease such as cancer, autoimmune disease, or infectious disease (e.g., virus, bacterium, fungus, or parasite).

[0129] In certain embodiments, the subject to be treated by the methods provided in this specification is human. In other embodiments, the subject is a non-human animal, e.g., in a veterinary or medical research setting. In embodiments where the subject is human, the subject may be male or female and may be of a suitable age, including pediatric, adolescent, young adult, adult, and geriatric subjects. The cells according to the present disclosure may be administered in a manner appropriate for the disease, condition, or disorder to be treated, as determined by one of ordinary skill in the medical arts.

[0130] The composition, cell, or unit dose of this application may be administered intravenously, intraperitoneally, intratumorally, into the bone marrow, lymph nodes, or cerebrospinal fluid so as to encounter the target antigen or cell. The appropriate dosage, preferred period, and frequency of administration of the composition are determined by factors such as the patient's symptoms; the degree, type, and severity of the disease, condition, or disorder; the unwanted type, level, or activity of the tagged cells; the specific form of the active ingredient; and the method of administration.

[0131] In some embodiments, the disease or condition is a malignant tumor. In some embodiments, the malignant tumor is cancer. Generally, cancers treatable by the methods and compositions of this application include carcinomas, sarcomas, gliomas, lymphomas, leukemias, myelomas, head and neck cancers, melanomas, pancreatic cancer, cholangiocarcinoma, hepatocellular carcinoma, breast cancer, gastric cancer, non-small cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small cell lung cancer, colorectal cancer, glioblastoma, Askin tumor, sarcoma botryoides, chondrosarcoma, Ewing sarcoma, PNET, malignant angioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, fibromatosis, round cell tumor, epitheliosarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), perivascular cell tumor, angiosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, linitis plastic, vip-producing tumor, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, epithelioma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, mixed glioma, Hodgkin lymphoma, B cell lymphoma, non-Hodgkin lymphoma (NHL), Burkitt lymphoma, small lymphocyte lymphoma (SLL), diffuse large B cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, Waldenstrom macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, adult T cell lymphoma, extranodal NK / T cell lymphoma, enteropathy-associated T cell lymphoma, nasal type, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, Sézary syndrome, angioimmunoblastic T cell lymphoma, undifferentiated large cell lymphoma, chondrosarcoma, fibrosarcoma (fibroblastic sarcoma), dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing sarcoma; gastrointestinal stromal tumor; leiomyosarcoma;Angiosarcoma (hemangiosarcoma); Kaposi's sarcoma; liposarcoma; pleomorphic sarcoma; synovial sarcoma; lung cancer (e.g., adenocarcinoma, squamous cell carcinoma (epidermoid carcinoma)); squamous cell carcinoma; adenocarcinoma; adenosquamous carcinoma; undifferentiated carcinoma; large cell carcinoma; small cell carcinoma; breast cancer (e.g., in situ ductal carcinoma (non-invasive), in situ lobular carcinoma (non-invasive), invasive ductal carcinoma, invasive lobular carcinoma, non-invasive carcinoma); liver cancer (e.g., hepatocellular carcinoma, cholangiocarcinoma or bile duct cancer); large cell undifferentiated carcinoma, bronchioloalveolar carcinoma); ovarian cancer (e.g., surface epithelial-stromal tumor (adenocarcinoma) or ovarian epithelial cancer (including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma), epidermoid (squamous cell carcinoma), fetal carcinoma and choriocarcinoma (germ cell tumor)); kidney cancer (e.g., renal adenocarcinoma, adrenal tumor, transitional cell carcinoma (renal pelvis), squamous cell carcinoma, Bellini duct carcinoma, clear cell adenocarcinoma, transitional cell carcinoma, carcinoid tumor of the renal pelvis); adrenal cancer (e.g., adrenal cortical carcinoma), testicular cancer (e.g., germ cell tumor (seminoma, choriocarcinoma, fetal carcinoma, teratocarcinoma), serous carcinoma); gastric cancer (e.g., adenocarcinoma); gastrointestinal cancer (e.g., duodenal adenocarcinoma); colorectal cancer; skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma); ovarian cancer, ovarian epithelial cancer, cervical adenocarcinoma, small cell carcinoma, pancreatic cancer, colorectal cancer (e.g., adenocarcinoma or squamous cell carcinoma), lung cancer, ductal carcinoma of the breast or prostatic adenocarcinoma is included.;

[0132] In some embodiments, the cancer is one or more of prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, gastric cancer, thyroid cancer, anal cancer, bone cancer, cervical cancer, endometrial cancer, esophageal cancer, eye tumor, gallbladder cancer, thymic cancer, sarcoma and osteosarcoma.

[0133] In some embodiments, the cancer includes hematological malignancies.

[0134] In some embodiments, the disease or condition is an "excessive proliferative disorder" and a "proliferative disorder". In some embodiments, the excessive proliferative disorder and the proliferative disorder are one or more of a tumor, cancer, neoplastic tissue, carcinoma, sarcoma, malignant cell, pre-malignant cell. In some embodiments, the cancer includes solid tumors.

[0135] In certain embodiments, the methods provided herein include administering a cell that expresses the CAR of this application, a composition containing the cell, or a unit dose thereof. The number of cells in the composition is at least one (e.g., one CAR-modified CD8 + T cell subset; one CAR-modified CD4 + T cell subset; one CAR-modified NK cell subset), or more generally more than 10 2 cells per square meter, e.g., up to 10 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells or 10 10 cells or more (e.g., about 10 11 cells). In certain embodiments, the cells are administered at about 10 5 to about 10 11 cells / m 2 , preferably about 10 5 or about 10 6 to about 10 9 or about 10 10 cells / m 2 . The number of cells depends on the intended final use of the composition and the type of cells contained therein. For example, cells modified to contain a CAR specific for one or more antigens contain a cell population that includes at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more such cells. For the uses provided herein, the cells are generally 1 L or less, 500 mL or less, 250 mL or less, or 100 mL or less. In multiple embodiments, the desired cell density typically exceeds 10 4 cells / mL, generally exceeds 10 7 cells / mL, and generally 10 8is above cells / mL. The cells may be administered as a single injection or as multiple injections over a period of time. A clinically relevant number of immune cells is, in total, 10 6 or more, 10 7 or more, 10 8 or more, 10 9 or more, 10 10 or more, or may be divided into multiple injections of 10 11 or more cells. In any of the aspects of this application, the cells are allogeneic cells, syngeneic cells or autologous cells.

[0136] This specification also contemplates a pharmaceutical composition containing cells expressing the CAR disclosed herein, and a pharmaceutically acceptable carrier, diluent and / or excipient. Preferred excipients include water, physiological saline, dextrose, glycerol, etc. and combinations thereof. In a plurality of aspects, the composition containing the cells disclosed herein further contains a preferred injection medium.

[0137] The pharmaceutical composition may be administered in a manner appropriate for the disease or condition to be treated (or prevented) as determined by one of ordinary skill in the medical arts. The appropriate dosage, preferred period, and frequency of administration of the composition are determined by factors such as the patient's health status, the patient's size (weight, mass, or body surface area), the type and severity of the patient's symptoms, the undesired type, level, or activity of the tagged cells, the specific form of the active ingredient, and the method of administration. Generally, the appropriate dosage and treatment regimen provide the composition in an amount sufficient to provide a therapeutic and / or prophylactic benefit (including an improvement in clinical prognosis such as an increase in the frequency of complete or partial remission, an extension of disease-free survival and / or overall survival, or a reduction in the severity of symptoms, as described in this specification). For prophylactic use, the dosage needs to be sufficient to prevent, delay the onset of, or reduce the severity of a disease associated with the disease or disorder. The prophylactic benefits of the immunogenic composition administered according to the methods described in this specification can be determined by conducting preclinical studies (including in vitro and in vivo animal experiments) and clinical studies and analyzing the data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by one of ordinary skill in the art.

[0138] The specific methods of treatment or prevention contemplated in the specification include administering cells (which may be autologous, allogeneic, or syngeneic) that contain the desired polynucleotide described in this specification that is stably integrated into the chromosomes of the cells. For example, such cell compositions may be generated ex vivo using autologous, allogeneic, or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, NK cells) for administration to a subject as an adoptive immunotherapy with the desired CAR-expressing T cell composition. In certain embodiments, the cells are hematopoietic progenitor cells or human immune cells. In certain embodiments, the immune system cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 -It is a double-negative T cell, NK cell, or a combination thereof. In certain embodiments, the immune system cell is a naive T cell, central memory T cell, stem cell memory T cell, effector memory T cell, NK cell, or a combination thereof. In certain embodiments, the cell is a CD4 + T cell. In certain embodiments, the cell is a CD8 + T cell. In certain embodiments, the cell is an NK cell.

[0139] As used herein, administration of a composition refers to delivering it to a subject, regardless of the route or mode of delivery. Administration may be performed parenterally, either continuously or intermittently. Administration may be for treating a subject that has already been confirmed to have a recognized symptom, disease, or disease state, or for treating a subject that is susceptible to or at risk of developing such a symptom, disease, or disease state. Co-administration with adjuvant therapy may include simultaneous and / or sequential delivery of multiple agents (e.g., CAR-expressing recombinant (modified) cells and one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, or combinations thereof) in any order and any dosing schedule.

[0140] In certain embodiments, multiple doses of the cells described herein are administered to a subject, and the interval between administrations may be about 2 to about 4 weeks.

[0141] In still further embodiments, the subject being treated is further receiving immunosuppressive therapy, such as a calcineurin inhibitor, corticosteroid, microtubule inhibitor, low-dose mycophenolic acid prodrug, or a combination thereof. In still further embodiments, the subject being treated has undergone non-myeloablative or myeloablative hematopoietic cell transplantation, and this treatment can be administered at least 2 months to at least 3 months after non-myeloablative hematopoietic cell transplantation.

[0142] An effective amount of a pharmaceutical composition (e.g., a cell, a CAR, a unit dose, or a composition) refers to an amount sufficient to achieve the desired clinical result or beneficial treatment described in this specification at the required dosage and for the required period. The effective amount may be delivered in one or more administrations.

[0143] The methods according to the present disclosure may further include administering one or more additional agents to treat a disease or disorder in a combination therapy. For example, in certain embodiments, the combination therapy includes administering a CAR (or a modified cell expressing it) together with (in parallel, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, the combination therapy includes administering the CAR (or a modified cell expressing it) of this application together with an agonist of a stimulatory immune checkpoint agent. In further embodiments, the combination therapy includes administering the CAR (or a modified cell expressing it) of this application together with a secondary therapy such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or a combination thereof.

[0144] Cytokines are used to manipulate the host immune response against anti-cancer activity (see, e.g., Floros & Tarhini, 2015). Cytokines useful for promoting an anti-cancer or anti-tumor immune response include, alone or in combination with the binding protein or cell expressing it of this application, for example, IFNα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-24, IFNγ, TNFα, and GM-CSF.

[0145] Various aspects of the technology have been described. It should be understood that the details shown above are provided to describe the aspects in a manner sufficient to enable one of ordinary skill in the relevant art to make and use the disclosed aspects. However, some of the details and advantages may not be essential to implementing some of the aspects. Additionally, some well-known structures or functions may not be shown in detail or described to avoid unnecessarily obscuring the relevant descriptions of the various aspects. Some aspects may be within the scope of the technology but may not be described in detail with respect to the drawings. Further, the characteristics, structures, or features of the various aspects may be combined in a suitable manner. Additionally, one of ordinary skill in the art will recognize that there are several other techniques that can be used to perform similar functions to those described above. The processes or blocks are presented in a given order, but alternative aspects may perform a routine having multiple steps in a different order or utilize a system having multiple blocks, and some processes or blocks may be absent, moved, added, further divided, combined, and / or modified. Each of these processes or blocks may be implemented in a variety of different ways. Also, the processes or blocks may be shown as being performed sequentially, but these processes or blocks may instead be performed in parallel or at different times. The headings used in the specification are for convenience only and do not interpret the scope or meaning of the technology described.

[0146] Various aspects of the present invention are set forth in the following paragraphs 0147 - 0199 herein:

[0147] A chimeric antigen receptor (CAR) comprising an extracellular region containing an antigen-binding domain, a transmembrane region, and an intracellular region containing an effector domain and a TMIGD2 co-stimulatory domain.

[0148] The CAR described in paragraph 0147, wherein the TMIGD2 co-stimulatory domain contains the intracellular region of TMIGD2.

[0149] The CAR described in paragraph 0148, wherein the TMIGD2 co-stimulatory domain contains a sequence selected from the group consisting of residues 172 to 282 of SEQ ID NO: 3, 172 to 278 of SEQ ID NO: 4, and residues 52 to 162 of SEQ ID NO: 5.

[0150] The CAR described in paragraph 0149, wherein the TMIGD2 co-stimulatory domain contains an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of residues 172 to 282 of SEQ ID NO: 3, 172 to 278 of SEQ ID NO: 4, and residues 52 to 162 of SEQ ID NO: 5.

[0151] The CAR described in any one of paragraphs 0147 to 0150, wherein the antigen-binding domain specifically binds to a tumor-associated antigen.

[0152] The tumor-associated antigen is selected from the group consisting of HHLA2, CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (including, for example, MAGE-A1, MAGE-A3, and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; a tumor- or pathogen-associated peptide bound to HLA; an hTERT peptide bound to HLA; a tyrosinase peptide bound to HLA; a WT-1 peptide bound to HLA; LTβR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME, and the SSX-2 antigen, the CAR described in paragraph 0151.

[0153] The antigen-binding domain contains an scFv, the CAR described in any one of paragraphs 0147 to 0152.

[0154] The antigen-binding domain contains a linker, the CAR described in any one of paragraphs 0147 to 0153.

[0155] The linker is a glycine-serine linker, the CAR described in paragraph 0154.

[0156] The glycine-serine linker is (Gly x Ser y )z containing, wherein x and y are each independently an integer from 0 to 10, except when both x and y are 0, and z is an integer from 1 to 10, the CAR described in paragraph 0155.

[0157] The extracellular region further contains an N-terminal leader sequence, the CAR described in any one of paragraphs 0147 to 0156.

[0158] The extracellular region further contains a hinge region, the CAR described in any one of paragraphs 0147 to 0157.

[0159] The hinge region contains the amino acid sequence shown in SEQ ID NO: 2, the CAR described in paragraph 0158.

[0160] The hinge region contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 2, the CAR described in paragraph 0159.

[0161] The transmembrane region contains the transmembrane region of CD8α, the CAR described in any one of paragraphs 0147 to 0160.

[0162] The transmembrane region contains the amino acid sequence shown in SEQ ID NO: 1, the CAR described in paragraph 0161.

[0163] The transmembrane region contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 1, the CAR described in paragraph 0161.

[0164] The effector domain is the effector domain of CD3ζ, the CAR described in any one of paragraphs 0147 to 0163.

[0165] The effector domain contains the amino acid sequence shown in SEQ ID NO: 6, the CAR described in paragraph 0164.

[0166] The CAR according to paragraph 0164, wherein the effector domain contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 5.

[0167] The CAR according to any one of paragraphs 0147 - 0166, containing: (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, residues 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 1.

[0168] The CAR according to any one of paragraphs 0147 - 0166, containing: (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, residues 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO: 6.

[0169] The CAR according to any one of paragraphs 0147 - 0166, containing: (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, residues 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5; (b) the sequence shown in SEQ ID NO: 1; and (c) the sequence shown in SEQ ID NO: 6.

[0170] The CAR according to paragraph 0168 or 0169, further containing the sequence shown in SEQ ID NO: 2.

[0171] An isolated nucleic acid molecule containing a nucleic acid sequence encoding the CAR according to any one of paragraphs 0147 - 0169.

[0172] A vector containing a nucleic acid sequence encoding the CAR according to any one of paragraphs 0147 - 0169.

[0173] The vector according to paragraph 0172, wherein the nucleic acid sequence encoding the CAR is operably linked to an expression control sequence.

[0174] The vector according to paragraph 0173, wherein the expression control sequence is a promoter.

[0175] The vector according to any one of paragraphs 0172 to 0174, further comprising a nucleic acid sequence encoding a self-cleaving peptide.

[0176] The vector according to paragraph 0175, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0177] The vector according to paragraph 0176, wherein the 2A self-cleaving peptide is a P2A peptide.

[0178] The vector according to any one of paragraphs 0172 to 0177, further comprising a nucleic acid sequence encoding a transduction marker polypeptide.

[0179] The vector according to paragraph 0178, wherein the transduction marker polypeptide is a cleaved form of epidermal growth factor receptor (EGFRt) or GFP, or a part or variant thereof.

[0180] The vector according to any one of paragraphs 0176 to 0179, wherein the nucleic acid sequence encoding the self-cleaving peptide is located 3' to the nucleic acid sequence encoding the CAR.

[0181] The vector according to paragraph 0179 or 0180, wherein the vector contains a nucleic acid sequence encoding a self-cleaving peptide, and the nucleic acid sequence encoding the self-cleaving peptide is located 5' to the nucleic acid sequence encoding the marker polypeptide.

[0182] The vector according to any one of paragraphs 0172 to 0181, wherein the vector is a viral vector.

[0183] An isolated cell expressing a CAR according to any one of paragraphs 0147 to 0171.

[0184] The cell according to paragraph 0183, wherein the cell contains the nucleic acid molecule according to paragraph 0164.

[0185] The cell is a cell as described in paragraph 0183 or 0184, containing the vector described in any one of paragraphs 0171 to 0180.

[0186] The cell is a cell as described in any one of paragraphs 0183 to 0185, which is a T cell, a natural killer (NK) cell, a macrophage or other immune cell.

[0187] The T cell is CD4 + T cell, CD8 + T cell, CD4 - CD8 - The cell is a cell as described in paragraph 0186, which is a double negative T cell, an NK cell, a macrophage, other immune cells or a combination thereof.

[0188] The T cell is a cell as described in paragraph 0186, which is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, an NK cell, a macrophage, other immune cells or a combination thereof.

[0189] The cell is a cell as described in any one of paragraphs 0183 to 0188, which further expresses a transduction marker on its surface.

[0190] The transduction marker is a cleaved form of epidermal growth factor receptor (EGFRt) or GFP, or a part or variant thereof, as described in paragraph 0189.

[0191] A method for treating a disease or condition in a subject in need of treatment, the method comprising administering to the subject an effective amount of the cell as described in any one of paragraphs 0183 to 0190.

[0192] The disease or condition is a malignant tumor, as described in paragraph 0191.

[0193] The malignant tumor is cancer, as described in paragraph 0192.

[0194] The cancer is the method according to paragraph 0193, selected from the group consisting of prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, stomach cancer, small intestine cancer, bone cancer, cervical cancer, endometrial cancer, eye tumor, gallbladder cancer, thyroid cancer, thymus cancer, sarcoma and osteosarcoma.

[0195] The cancer includes solid tumors, and is the method according to paragraph 0193 or 0194.

[0196] The cancer includes hematological malignancies, and is the method according to any one of paragraphs 0193 to 0195.

[0197] A method for inducing an immune response against a tumor-associated antigen that specifically binds to the CAR described in any one of paragraphs 0147 to 0171 in a subject, the method comprising administering to the subject an effective amount of the cells described in any one of paragraphs 0183 to 0190.

[0198] A composition comprising the CAR described in any one of paragraphs 0147 to 0171, and a pharmaceutically acceptable excipient, carrier or diluent.

[0199] A composition comprising the cells described in any one of paragraphs 0183 to 0190, and a pharmaceutically acceptable excipient, carrier or diluent.

[0200] The patents, applications and other references cited herein are incorporated herein by reference. Aspects of the described technology may be modified to provide further aspects, as necessary, using the various reference systems, functions and concepts described above.

[0201] These changes and other changes can be made based on the above detailed description. The above description details specific embodiments and describes the contemplated best mode, but various changes can be made even if it is detailed as much as possible. The implementation details may vary significantly while still being encompassed by the technology disclosed in this specification. As described above, the specific terms used when describing a particular characteristic or aspect of the present technology are not to be construed as being redefined so as to be limited to the particular feature, characteristic or aspect of the present technology to which the term is related in the specification.

[0202] The foregoing is only intended to illustrate various aspects of the present invention. The specific modifications discussed above are not to be regarded as limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present invention, and such equivalent embodiments are understood to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

[0203] The following examples are illustrative of some aspects of the present technology.

Example

[0204] Example 1 CAR-T cell therapy using CAR-T cells containing TMIGD2 as a co-stimulatory molecule The following example shows the generation of a new CAR-T vector containing the cytoplasmic tail of TMIGD2 (transmembrane and immunoglobulin domain containing 2), where the CAR-T vector can be transduced into normal T cells and can kill human tumor cells.

[0205] Generation of a CAR-T Vector with TMIGD2 CAR-T vectors using TMIGD2 as a costimulatory molecule are being developed (Figure 1). The new vector contains an antibody signal leader, VH, VL, the hinge and transmembrane regions of human CD8a, the intracellular tail of human TMIGD2, human CD3ζ, P2A, the human GMCSFR signal leader, and human EGFRt.

[0206] Killing of human tumor cells by TGMID2 CAR-T To prove the principle, the VH and VL of an mAb against human CD19 were cloned into the TMIGD2 vector shown in Figure 1, and experiments on the killing of human tumor cells by CAR-T were conducted. T cells derived from normal PBMCs were transfected with the TMIGD2 vector to generate CAR-T cells. Then, human Raji tumor cells expressing the CD19 antigen were incubated alone, together with non-transduced T cells, and together with T cells transduced with CAR-T. The results showed that CAR-T cells killed almost all of the Raji tumor cells, while non-transduced T cells did not (Figure 2), indicating that CAR-T cells containing the TMIGD2 costimulatory domain can efficiently kill tumor cells.

[0207] In vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells This example shows the in vivo therapeutic efficacy of CD19-TMIGD2 CAR-T cells in a Raji lymphoma model. On day 0, 0.25×10 6 individual Raji tumor cells were injected intravenously (I.V.) into NSG™ mice, and 10 7 individual non-transduced T cells or CD19-TMIGD2 CAR-T cells were injected intravenously on days 3 and 10, respectively. The survival of the mice was monitored. As shown in Figure 3, the survival of mice administered CD19-TMIGD2 CAR-T cells was significantly improved compared to non-transduced control mice.

[0208] Example 2 CAR-T cell therapy using anti-B7-H3 CAR-T cells containing TMIGD2 as a co-stimulatory molecule The following examples demonstrate the generation of anti-B7-H3 CAR-T cells incorporating a vector containing the cytoplasmic tail of TMIGD2 (transmembrane and immunoglobulin domain-containing 2), where the vector is transduced into normal T cells and kills human tumor cells expressing B7-H3 in vitro and in vivo.

[0209] Experimental methods Cell lines NIH 3T3 cells (mouse fibroblasts) were obtained from the American Type Culture Collection (ATCC); NSO cells (mouse multiple myeloma) were obtained from the Department of Cell Biology, Albert Einstein College of Medicine, HEK293T cells (human epithelial kidney) were obtained from the Department of Cell Biology, Albert Einstein College of Medicine, U118 cells (human glioblastoma) were obtained from ATCC, HCC827 cells (human lung adenocarcinoma) were obtained from ATCC, THP-1 cells (human acute monocytic leukemia) were obtained from ATCC, Raji cells (B-cell Burkitt lymphoma) were obtained from ATCC, Jurkat (NFAT) cells (T-cell leukemia) were obtained from BPS Bioscience, AsPC-1 cells (pancreatic adenocarcinoma) were obtained from ATCC, PANC-1 cells (pancreatic ductal adenocarcinoma) were obtained from ATCC, and Phoenix-AMPHO cells (human epithelial kidney) were obtained from ATCC. The cell lines were grown in DMEM or RPMI 1640 medium supplemented with 10% FBS, 1% penicillin, and 1% streptomycin and cultured at 37 °C and 5% CO2 in a humidified incubator.

[0210] Transfection of tumor cell lines Phoenix-AMPHO cells were co-transfected with an MSCV-YFP plasmid containing the protein of interest (mouse B7-H3, human B7-H3, cynomolgus monkey B7-H3, or luciferase-tdTomato (-Luc)) and a pCMV-VSV-G plasmid. Viral supernatants were harvested at 48 and 72 hours and used to transfect tumor cell lines using polybrene. Transfected cells were sorted at least twice using a BD FACS Aria II cell sorter to ensure population purity.

[0211] Generation of anti-B7-H3 monoclonal antibody C57BL / 6 mice were immunized with a recombinant protein consisting of the IgV domain of human B7-H3 fused to the human Fc fragment. After immunization, hybridomas were generated by fusing NSO myeloma cells and mouse spleen cells using standard methods. Hybridomas producing antibodies were screened by flow cytometry to ensure specific binding to mouse and human B7-H3. Subsequently, the hybridomas were subcloned three times by single cell dilution and grown in DMEM high glucose medium (supplemented with 10% ultra-low IgG FBS, 10% NCTC-109, 1% penicillin, 1% streptomycin, and 1% non-essential amino acids) in the cell compartment of a bioreactor flask. The medium compartment of the bioreactor flask contained DMEM high glucose medium supplemented with 1% penicillin and 1% streptomycin. Antibody supernatants were recovered from the cell compartment and stored at 4°C until purification by a column packed with protein G resin. The purified B7-H3 mAb was analyzed by SDS-PAGE and then subjected to affinity determination, isotype determination, and VH and VL sequencing.

[0212] Determination of B7-H3 mAb affinity The affinity of anti-B7-H3 mAbs for mouse and human B7-H3 was determined by biolayer interferometry. Recombinant mouse and human B7-H3-Fc proteins were loaded onto mouse or human capture biosensors, respectively. Subsequently, the biosensors loaded with the protein were placed in solutions containing serial dilutions of anti-B7-H3 mAbs. Kon, Koff, and KD were determined by analysis using a 1:1 binding model.

[0213] Generation of CAR constructs An anti-B7-H3 single-chain variable fragment (scFv) was generated by cloning the native mAb signal peptide sequence into the VH and VL regions of the anti-B7-H3 mAb connected by a G4S linker. The scFv was connected to the hinge domain and transmembrane domain of human CD8α, the intracellular regions of various co-stimulatory proteins (CD28, 4-1BB, TMIGD2, CD28-4-1BB, or TMIGD2-4-1BB), and the intracellular domain of human CD3ζ. Subsequently, a self-cleaving P2A peptide sequence was inserted, followed by the insertion of a signal peptide derived from the granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) α chain and the truncated human epidermal growth factor receptor (hEGFRt) protein for CAR detection. For the in vivo CAR-T cell persistence experiment, the hEGFRt protein was followed by a self-cleaving T2A peptide sequence and further followed by the firefly luciferase gene sequence. The entire CAR sequence was cloned into the pLVX-Zsgreen lentiviral expression plasmid under the control of the EF1α promoter. A commercially available CD19-CD28-4-1BB CAR was also modified to contain the P2A-EGFRt and T2A-luciferase sequences.

[0214] Production from CAR lentivirus and determination of virus titer HEK293T cells were co-transfected with the psPAX packaging plasmid, the pMD2.G envelope plasmid, and the CAR plasmid. Viral supernatants were harvested at 48 and 72 hours, concentrated 100-fold, and pooled to ensure equal titers. Viral titers were determined by transducing activated human T cells with the CAR lentivirus as described below. Viral titers were calculated based on EGFR+ T cells.

[0215] Isolation of Human T Cells Leukopak from healthy human donors was obtained, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep. T cells were purified by negative enrichment. T cells were either frozen in freezing medium and stored in liquid nitrogen or used immediately.

[0216] Generation of CAR-T Cells Fresh or frozen T cells were activated for 24 hours in CTS OpTmizer medium supplemented with OpTmizer T cell expansion supplement, 10% FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin, IL-7, and IL-15 in 24-well plates coated with OKT3 (1 μg / mL) and CD28 (1 μg / mL) antibodies. Activated T cells were transduced on non-tissue culture-treated plates coated with RetroNectin reagent (19 μg / mL) and the CAR lentivirus (approximate MOI of 10). CAR-T cells were grown for at least 7 days before use in experiments. Prior to all experiments, CAR-T cell transduction efficiency was normalized to 50% CAR + cells (for in vitro cytotoxicity screening assays) or to the least efficient donor by adding non-transduced T cells. If necessary, CAR-T cells were purified with anti-phycoerythrin (PE) microbeads after anti-EGFR-PE staining.

[0217] In Vitro Co-Culture Killing Assay 0.1×10 6Individual CAR-T cells and either HCC827-Luc (0.01×10 6 cells), U118-Luc (0.01×10 6 cells), or THP-1 (0.01×10 6 cells) were seeded in T cell medium without adding cytokines. Three to five days later, tumor cells were counted by flow cytometry. For the Incucyte time-lapse cytotoxicity assay, 5×10 3 individual HCC827-Luc or U118-Luc cells were seeded 1 day before imaging. Then, the plates coated with tumors were placed in the Incucyte instrument and imaged for 112 hours at 4-hour intervals at 4 positions per well. Sixteen hours after the first imaging, 0.1×10 6 individual CAR-T cells were gently added to the wells. Imaging data were analyzed using Incucyte software.

[0218] Flow cytometry Cells were stained using antibodies conjugated to the following fluorophores: fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), APC-Fire750, PE-cyanine (Cy)7, peridinin-chlorophyll-protein (PerCP)-Cy5.5, brilliant violet (BV)-421, BV-711, alexa fluor (AF)-532, AF-488, brilliant ultra violet (BUV)-496, BUV-395, BUV-496 and BUV-737. Expression of tdTomato and YFP was also used to distinguish cell populations. Antibody targets included CD45, CD3, CD4, CD8, CD45RA, CCR7, EGFR, G4S linker, PD-1, TIM-3, LAG-3, B7-H3, CD69 and CD33. Viability was determined using 7-aminoactinomycin D (7-AAD), zombie NIR, ghost violet 510 and 4',6-diamidino-2-phenylindole (DAPI). In some experiments, cells were fixed using 2% paraformaldehyde (PFA) solution prior to analysis. All samples were acquired using a BD LSRII or Cytek Aurora flow cytometer. Data analysis was performed in FlowJo (or SpectroFlo). T-distributed stochastic neighbor embedding (t-SNE) plots were generated in FlowJo using the t-SNE plugin.

[0219] Cytokine analysis by flow cytometry 0.4×10 6 CAR-T cells and 0.1×10 6Individual HCC827 tumor cells were co-cultured for 24 hours in 24-well plates. The supernatant was collected from the co-culture and stored at -80°C until analysis. The concentrations of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFNγ, and TNFα were determined for undiluted or 1:5 diluted supernatant samples using a flow cytometry-based LEGENDplex human Th cytokine panel kit. The data were analyzed using LEGENDplex online data analysis software.

[0220] RNA Isolation For in vitro RNA isolation, CAR-T cells were gently removed from the co-culture wells and seeded into new wells for 30 minutes to allow an excess of tumor cells to bind to the plate. The CAR-T cells were gently removed, and viable cells were concentrated by Lymphoprep density gradient centrifugation. T cells were further purified and isolated using CD3+ selection beads. RNA was extracted and stored at -80°C. RNA sequencing (40 million paired-end reads) after library preparation (using the NEBNext Ultra II kit) was performed at Admera Health. For in vivo RNA isolation, lung infiltrating T cells were isolated from the lungs of mice. Briefly, a single cell suspension was prepared from the lungs by enzymatic digestion (collagenase IV (200 IU / mL), dispase (0.5 IU / mL), and DNaseI (100 U / mL)) in RPMI 1640 medium. Next, the single cell suspension was overlaid on a discontinuous density gradient Percoll solution (40% and 80%), and immune cells at the 40% and 80% interface were collected. The population of viable cells was further concentrated by a second density gradient centrifugation using Lymphoprep. Mouse cells were then depleted twice. Subsequently, T cells from the same donor transduced with the same CAR were pooled, and RNA was isolated as described above. RNA sequencing (40 million paired-end reads) after library preparation (using SMARTseq V4 and NExtera XT kits) was performed at Admera Health.

[0221] B7-H3 CAR vs CD19 CAR RNA Sequencing Analysis The reads obtained from RNA sequencing were aligned to the reference human genome (hg38; downloaded from the UCSC genome browser in November 2019) using the STAR alignment software (version 2.6.1b). Gene-mapped fragments were counted using the HTseq software (version 0.6.1). Genes with an average expression count of 1 or more in any group were considered expressed. Differentially expressed genes (DEGs) were determined by an adjusted p-value of less than 0.05 and a log2 fold change of more than 0.5. Then, the DESeq2 software (version 3.10) was used to perform principal component analysis and differential expression analysis. Gene set enrichment analysis (GSEA) was performed using the Hallmark, Kegg, and Reactome databases with a ranked gene list determined by multiplying the sign of -log 10 (p-value) by the sign of log2 (fold change).

[0222] B7-H3 CAR in vitro RNA and in vivo RNA Sequencing Analysis The paired-end reads obtained from RNA sequencing were aligned to the human reference genome (hg38) using the STAR aligner (version 2.7.9a). Then, the alignment data was used to quantify the expression levels of individual genes (both coding and non-coding) in the GENCODE annotation (version 41) using the RSEM software (version 1.3.3), and estimated read counts and transcripts per million (TPM) were obtained. Genes with a TPM of less than 1 in all samples were excluded from further analysis. Differentially expressed genes (DEGs) were determined by an adjusted p-value of less than 0.05 and a log2 fold change of more than 0.5. Then, the DESeq2 software (version 1.38.3) was used to perform principal component analysis (PCA) and differential expression analysis. Gene set enrichment analysis (GSEA) was performed for each gene using -log 10Genes were ranked by multiplying the sign of the (p-value) and log2(fold change), and then enrichment analysis was performed using gene sets from Hallmark, KEGG, and Reactome. Gene sets with a false discovery rate (FDR) less than 0.05 were considered enriched. For DEGs or significantly differentially expressed genes (adjusted p-value < 0.05), overrepresentation analysis was also performed using the enrichGO function of clusterProfiler (version 4.6.2). Pathways with an adjusted p-value less than 0.05 were considered enriched with genes regulated either up or down. Gene sets indicating T cell dysfunction and metabolism were obtained from the literature, and the list of inhibitory proteins was curated manually.

[0223] Seahorse metabolic assay 0.1×10 6 Individual CAR-T cells were co-cultured alone or with 5×10 3 HCC827 tumor cells for 1 day or 7 days. The CAR-T cells were recovered from the wells, transferred to new wells for 30 minutes to allow tumor cell attachment, and then recovered again and analyzed using the Seahorse T cell metabolism profiling kit on plates coated with Seahorse PDL. After transferring to the plates coated with PDL, the CAR-T cells were rested for 5 minutes and then centrifuged to allow uniform cell distribution at the bottom of the wells. The Seahorse plates were measured using a Seahorse XF96 analyzer at baseline and after injection of oligomycin A, Bam15, and rotenone / antimycin A. The data were analyzed using Agilent Seahorse Analytics online software. The percentage of ATP from mitochondria was obtained by the following formula: 100% - percentage of ATP by glycolysis (%).

[0224] Jurkat (NFAT) T cell activation experiment Jurkat (NFAT) cells were transfected with the above-described CAR construct. OKT3 (1 μg / mL) or 0.2 × 10 5 either of 5 individual tumor cells (U118, HCC827, or AsPC-1) was coated overnight in three wells of a 96-well plate. 0.2 × 10 5 5 individual Raji tumor cells were added to another three wells. 0.1 × 10 6 either non-transfected or CAR-transfected Jurkat (NFAT) cells were added to each well and incubated for 6 hours. Jurkat (NFAT) luciferase was detected using the Bio-Glo luciferase assay system. The luciferase signal was acquired with a plate reader.

[0225] Chronic antigen exposure 0.2 × 10 6 6 individual CAR-T cells were seeded in three wells of a 96-well plate using cytokine-free T cell medium. 0.1 × 10 5 5 individual HCC827 tumor cells were added to each well. Every 3 - 4 days, the CAR-T cells were gently removed from the wells and transferred to new wells. Then, a small cell aliquot was analyzed by flow cytometry. The CAR-T cells not being analyzed were centrifuged, a portion of the supernatant was removed, and half of the original volume was left in the well. Then, 0.1 × 10 5 5 individual HCC827 parental cells in fresh T cell medium were added in an equal volume. This was repeated for 17 days.

[0226] Mouse In all in vivo experiments, female NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice, 8 - 12 weeks old, were used. NSG mice were purchased from the Jackson Laboratory and bred at the Albert Einstein College of Medicine. The mice were housed in a specific pathogen-free animal facility and allowed free access to food and water under a 12-hour light / dark cycle. All experimental procedures were approved by the Albert Einstein Institutional Animal Care and Use Committee.

[0227] Bioluminescence imaging Bioluminescence imaging was performed using an IVIS Spectrum in vivo imaging system and analyzed using Living Image software (version 3.0). Images were acquired 10 - 15 minutes after intraperitoneal (I.P.) injection of D-luciferin (150 μg / g mouse body weight).

[0228] In vivo lung cancer model 0.5×10 6 cells of HCC827-Luc in 100 μL of sterile PBS were injected into the tail vein of NSG mice. Three days later, tumor engraftment was confirmed by IVIS imaging, and the experimental groups were normalized to ensure equivalent baseline tumor burdens. Then, 10×10 6 cells of CAR-T were administered via the tail vein and reinjected after one week. Tumor burden was tracked by IVIS imaging for 100 days. Survival was also tracked during this period.

[0229] In vivo glioblastoma model U118-Luc tumor cells, 5×10 3Resuspended in sterile PBS at cells / μL. NSG mice anesthetized with continuous isoflurane (2%) were used to make a small hole freehand 1 mm anterior and 1 mm lateral to bregma of the skull using an 18-gauge needle. A flat-tip Hamilton syringe fitted with a sterile pipette tip pre-cut to expose 1 mm of the tip was used to inject 2 μL of tumor cells into the hole perpendicular to the bench top surface over 1 minute. Meloxicam (5 mg / kg) was administered 2 days before and after tumor injection. Seven days later, IVIS imaging was used to confirm tumor engraftment. Then, the baseline tumor volume was normalized and the mice were assigned to experimental groups. Thereafter, 1×10 6 CAR-T cells in 2 μL of PBS were injected intratumorally (I.T.) using the surgical supplies described above. Tumor volume and survival were tracked by IVIS imaging for 100 days.

[0230] in vivo pancreatic cancer model PANC-1-Luc tumor cells were resuspended in 50 μL of 1×10 4 cells / sterile PBS. The pancreas was exposed through a 1-cm incision in the skin and muscle of NSG mice. 50 μL of tumor cells were injected into the head of the pancreas using a 27-gauge needle. Meloxicam was administered 3 days before and after tumor injection. Seven days later, IVIS imaging was used to confirm tumor engraftment. Then, the baseline tumor volume was normalized and the mice were assigned to experimental groups. Then, 10×10 6 CAR-T cells were injected into the tail vein and reinjected 7 days later. Tumor volume and survival were tracked by IVIS imaging for 100 days.

[0231] in vivo CAR-T cell persistence model 0.5×10 6 parent HCC827 tumor cells were injected into the tail vein. Three days later, a single dose of 10×10 6 CAR-Luc T cells were injected into the tail vein. The CAR-luciferase T cell signal was tracked by IVIS imaging for 46 days.

[0232] Isolation of immune cells from mouse blood and organs To obtain immune cells from mouse blood, blood was collected from the tail vein into heparinized capillary tubes. Subsequently, red blood cells (RBCs) were lysed. The remaining cells were washed twice with PBS and subjected to flow cytometry analysis. The mice were euthanized, and the target organs were either directly excised (spleen) or excised after perfusion with PBS (lung). Subsequently, the spleen was transferred to a C tube and mechanically dissociated. The cell suspension was filtered through a 40 μM filter. To obtain immune cells from the lung, a cell suspension was prepared using enzymatic digestion, and immune cells were isolated using a discontinuous Percoll gradient. RBCs were removed with RBC lysis buffer.

[0233] Statistics Statistical analysis was performed using GraphPad prism software (version 9.4.1). As indicated in the legends of each figure, the tests between groups were performed using unpaired two-sided Student's t-test, one-way ANVOA, Kruskal–Wallis test, two-way ANOVA or Mantel–Cox log-rank test. The data are shown as individual values or as individual values and mean ± SEM. A P value of less than 0.05 was considered statistically significant.

[0234] Generation and validation of anti-B7-H3 monoclonal antibodies Using biolayer interferometry (BLI), six anti-B7-H3 mAb clones designated 1G5, 15F9, 23B2, 8B12, 12B4 and 24D12 were obtained that bound to both mouse and human B7-H3 with affinities ranging from 0.32 nM to 11.08 nM. All anti-B7-H3 mAbs that bind to mouse and human B7-H3 were verified by flow cytometry to stably express on 3T3 cells, respectively. In addition, each anti-B7-H3 mAb was tested and confirmed to be able to bind to cynomolgus monkey B7-H3, a non-human primate. All anti-B7-H3 mAbs were able to detect cynomolgus monkey B7-H3 stably expressed on 3T3 cells by flow cytometry. The anti-B7-H3 mAbs were able to recognize human, mouse and cynomolgus monkey B7-H3.

[0235] In vitro screening of anti-B7-H3 CAR-T cells Using a single-chain variable fragment (scFv) derived from the anti-B7-H3 mAb clone 8B12, the scFv was sequentially linked to the hinge domain and transmembrane (H / TM) domain of human CD8α, the intracellular domains of various costimulatory proteins, and the intracellular domain of CD3ζ to construct an anti-B7-H3 CAR. After the CD3ζ sequence, a self-cleaving P2A peptide and a truncated human epidermal growth factor receptor (hEGFRt) were linked (Figure 4A). The hEGFRt protein was modified to prevent antigen binding and signal transduction by removing two of the four extracellular domains and all of the intracellular domains. It still functioned as a marker of transduction efficiency because the anti-EGFR antibody could still recognize it. The following costimulatory domains were utilized in the CAR construct: CD28 (B7-H3.28.ζ CAR), 4-1BB (B7-H3.BB.ζ CAR), TMIGD2 (B7-H3.TMI.ζ CAR), CD28-4-1BB (B7-H3.28.BB.ζ CAR), or TMIGD2-4-1BB (B7-H3.TMI.BB.ζ CAR). Using an anti-CD19 scFv together with the CD28-4-1BB costimulatory domain, an anti-CD19 CAR (CD19.28.BB.ζ) that functions as an irrelevant target control was also generated in the same way (Figure 4A). In primary human T cells, all CAR constructs were efficiently expressed with a transduction efficiency (determined by the expression of the hEGFRt protein) always exceeding 85%, and there were no differences in memory phenotype or proliferation among the CAR-T cell constructs (Figure 4H-K). +

[0236] ​The in vitro antitumor responses of B7-H3 CAR-T cells were tested against multiple tumors that were B7-H3+. To more accurately reflect the transduction efficiency in other clinical trials, non-transduced T cells were used to reduce the transduction efficiency of CAR-T cells to 50%. To enable discrimination of tumor cells, the U118 glioblastoma (GBM) and HCC827 lung cancer cell lines were stably transfected with plasmids expressing luciferase and tdTomato (-Luc) protein. B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed tumor cell killing against U118-Luc cells (Figure 4B). B7-H3.28.ζ, B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed tumor cell killing against HCC827-Luc cells (Figure 4C). To confirm that the CAR of this application is also effective against non-solid tumors, it was also tested whether the CAR-T cells of this application could target the THP-1 acute monocytic leukemia (AML) cell line. B7-H3.TMI.ζ, B7-H3.28.BB.ζ and B7-H3.TMI.BB.ζ CAR-T cells showed significant tumor cell killing against THP-1 cells (Figure 4D). Although the variability between donors was high, it was consistently found that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed antitumor responses in all cell lines tested.

[0237] By repeating the imaging of U118-Luc or HCC827-Luc tumor cells cultured alone or co-cultured with B7-H3.TMI.ζ, B7-H3.28.BB.ζ or CD19.28.BB.ζ CAR-T cells, a time-lapse imaging cytotoxicity assay was performed to determine the difference in tumor cell killing kinetics between these two CARs. Based on the tdTomato signal and the morphological exclusion of dead cells, the proliferation of live tumor cells was tracked. In U118-Luc tumor cells, the control CD19.28.BB.ζ CAR-T cells showed tumor growth equivalent to that of tumor cells alone, while both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells eliminated the tumor cells (Figure 4E). This was also reproduced in the HCC827-Luc co-culture (Figure 4F).

[0238] Using a multiplex flow cytometry bead assay to measure the following cytokines: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFNγ and TNFα, the cytokine release profiles of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were evaluated. After co-culture with HCC827 tumor cells, both B7-H3.TMI.ζ CAR-T cells and B7-H3.28.BB.ζ CAR-T cells released significantly more cytokines than the control CD19.28.BB.ζ CAR-T cells in all cytokines tested (Figure 4G). Except for IL-6, the cytokines released by B7-H3.TMI.ζ CAR-T cells were significantly less than those released by B7-H3.28.BB.ζ CAR-T cells.

[0239] These results demonstrated that in vitro, the cytotoxic responses and killing kinetics of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were equivalent. The cytokine secretion profiles of these CAR-T cells were similar, but the amount of cytokines secreted by B7-H3.TMI.ζ CAR-T cells was less in most cases compared to B7-H3.28.BB.ζ CAR-T cells.

[0240] The B7-H3.TMI.ζ CAR-T cells exhibit antitumor responses in vivo The in vivo antitumor responses of the lead B7-H3 CARs, B7-H3.TMI.ζ and B7-H3.28.BB.ζ, were evaluated in three solid tumor models, together with the control CD19.28.BB.ζ. To enable in vivo tracking by bioluminescence imaging, all tumor cell lines (HCC827, U118, and PANC-1) were stably transfected with a plasmid containing luciferase and tdTomato (-Luc). The B7-H3.TMI.ζ and B7-H3.28.BB.ζ CARs were tested in a metastatic lung cancer model. HCC827-Luc tumor cells were intravenously (I.V.) injected into NSG mice, followed by intravenous injection of B7-H3.TMI.ζ, B7-H3.28.BB.ζ, or control CD19.28.BB.ζ CAR-T cells 3 days and 10 days later (Figure 5A). The B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells reduced the tumor burden and simultaneously increased the overall survival compared with CD19.28.BB.ζ CAR-T (Figures 5B-5D). There was no significant difference in tumor cell signal or survival between the two B7-H3 CAR-T cells (Figures 5B-5D).

[0241] CAR-T cell therapy was investigated in an orthotopic GBM model. Since the antitumor response of CAR-T cells injected intratumorally (I.T.) has been shown to be superior to that of CAR-T cells injected intravenously (I.V.) at equivalent low doses, U118-Luc cells were injected intracranially into the right cerebral hemisphere of NSG mice, and 7 days later, CAR-T cells were injected intratumorally (Figure 5E). Both B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells showed antitumor responses against U118-Luc cells compared to CD19.28.BB.ζ CAR-T cells (Figures 5F and 5G). There was no difference in tumor burden between B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells (Figures 5F and 5G). All B7-H3 CAR-T cells improved the overall survival compared to CD19 CAR-T cells, and the survival prognosis of B7-H3.TMI.ζ CAR-T cells was superior to that of B7-H3.28.BB.ζ CAR-T cells (Figure 5H). In mice treated with B7-H3.28.BB.ζ CAR-T cells, no significant tumor burden was observed before death, suggesting that the cause of death in these mice was not due to the tumor.

[0242] CAR-T cell therapy was investigated in an orthotopic model of pancreatic cancer. NSG mice were orthotopically injected with PANC-1-Luc tumor cells into the pancreas, and CAR-T cells were injected I.V. 7 days and 14 days later (Figure 5I). All B7-H3 CAR-T cells reduced the tumor burden and improved the overall survival compared to the control CD19.28.BB.ζ CAR-T cells (Figures 5J - 5L). The tumor burden of B7-H3.TMI.ζ CAR-T cells was less than that of B7-H3.28.BB.ζ CAR-T cells, but it reached statistical significance only on day 21 (Figure 5K). Similarly, B7-H3.TMI.ζ CAR-T cells showed an improvement in survival compared to B7-H3.28.BB.ζ CAR-T cells, with 4 out of 7 mice surviving for 100 days after tumor cell injection compared to 1 out of 7 mice treated with B7-H3.28.BB.ζ CAR-T cells.

[0243] In summary, these experiments demonstrate that B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells exhibit antitumor responses against multiple tumor models in vivo. Furthermore, B7-H3.TMI.ζ CAR-T cells show comparable or superior results depending on the tumor.

[0244] B7-H3.TMI.ζ CAR-T cells persist in vivo Since B7-H3 CAR-T cells were determined to be cytolytic in multiple in vivo tumor models, the proliferation and persistence of B7-H3 CAR-T cells in vivo were tested. To enable in vivo tracking of CAR-T cells by bioluminescence imaging of previous CAR constructs, they were modified to include a self-cleaving T2A peptide followed by luciferase (CAR-Luc) (Figure 6A). NSG mice were injected with parental HCC827 cells that do not express luciferase, followed by a single subtherapeutic I.V. injection of B7-H3.TMI.ζ-Luc, B7-H3.28.BB.ζ-Luc, or CD19.28.BB.ζ-Luc CAR-T cells (Figure 6B). The CAR-Luc signal was detectable in all constructs on day 7, and there was no difference between any of the groups; after day 21, the two B7-H3 CAR-Luc T cells showed a significantly higher signal than the CD19 CAR-Luc T cells; however, by day 46, the B7-H3.TMI.ζ-Luc CAR-T cells showed a significantly higher CAR-Luc signal than the CD19.28.BB.ζ-Luc CAR-T cells, while the B7-H3.28.BB.ζ-Luc CAR-T cells did not, suggesting that B7-H3.TMI.ζ-Luc CAR-T cells can persist longer in vivo than B7-H3.28.BB.ζ-Luc CAR-T cells, although the latter approached statistical significance (Figures 6C-6E).

[0245] By testing the peak CAR-Luc signals of each mouse throughout the experiment, it was found that B7-H3.TMI.ζ-Luc and B7-H3.28.BBζ-Luc CAR-T cells showed comparable proliferation superior to that of CD19.28.BBζ-Luc CAR-T cells (Figure 6F). At the end of the experiment, by testing the number of T cells in the lungs, spleens, and blood of mice treated with CAR-Luc, it was found that B7-H3-CAR-Luc T cells were present in numbers superior to those of CD19.28.BBζ-Luc CAR-T cells and were present in comparable numbers among the B7-H3 CAR-Luc T cell constructs (Figures 6G-6I). Collectively, these data indicate that any B7-H3 CAR Luc-T cells can proliferate and persist in vivo in an antigen-dependent manner. Furthermore, B7-H3.TMI.ζ-Luc CAR-T cells showed moderately improved persistence compared to B7-H3.28.BBζ-Luc CAR-T at a later time, which is likely due to the persistence of the CAR at sites other than the lungs, spleens, and blood.

[0246] B7-H3.TMI.ζ and B7-H3.28.BBζ CAR-T cells show differences in transcriptome in vitro After 24-hour co-culture with HCC827 tumor cells in vitro, RNA sequencing was performed to compare B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells with CD19.28.BB.ζ CAR-T cells. Extensive differences were shown in the transcriptome between B7-H3.TMI.ζ CAR-T cells and B7-H3.28.BB.ζ CAR-T cells and CD19.28.BB.ζ CAR-T cells (Figure 7A). Among B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells, 307 differentially expressed genes (DEGs) were common, and the unique DEGs were 198 and 153, respectively (Figure 7B). Gene set enrichment analysis (GSEA) revealed that for both B7-H3 CAR-T cells, compared with CD19 CAR-T cells, a number of enriched pathways were identified, and the expressed pathways were highly similar, but the degree of enrichment was different (Figure 7C). Interestingly, among the top enriched pathways, the "oxidative phosphorylation" pathway in B7-H3.TMI.ζ CAR-T cells was found to be equivalent to the "glycolysis" pathway in B7-H3.28.BB.ζ CAR-T cells (Figure 7C), suggesting a difference in metabolism between these two CAR constructs.

[0247] Using the Seahorse T cell metabolic profiling assay, the changes in the rates of glycolysis and mitochondrial ATP production were measured at baseline (cultivation in the absence of tumor cells) and after co-culture to verify the mechanistic findings. In this assay, the sum of the rates of ATP production by glycolysis and mitochondrial ATP production is 100%, and thus these values are dependent on each other. During 24-hour acute stimulation, B7-H3.TMI.ζ CAR-T cells had a decreased rate of ATP production by glycolysis (Figure 7D, left) and simultaneously an increased rate of mitochondrial ATP production (Figure 7D, right) compared to B7-H3.28.BB.ζ CAR-T cells at baseline and after co-culture. After chronic stimulation by repeated addition of tumor cells, B7-H3.TMI.ζ CAR-T cells showed no difference in the rate of ATP production by glycolysis or mitochondria at baseline (Figure 7E, left), but after co-culture, the rate of ATP production by glycolysis decreased and simultaneously the rate of mitochondrial ATP production increased (Figure 7E, right). It was observed that B7-H3.TMI.ζ CAR-T cells maintained their metabolic signature, while B7-H3.28.BB.ζ CAR-T cells moderately increased their glycolytic energy consumption and decreased their mitochondrial energy consumption (Figure 7D and Figure 7E). These functional metabolic assays reproduced the findings of the RNA sequencing experiments.

[0248] To directly compare B7-H3.TMI.ζ CAR-T cells and B7-H3.28.BB.ζ CAR-T cells, RNA sequencing was performed on RNA isolated after 72 hours of co-culture with HCC827 tumor cells. There were extensive transcriptome differences between these B7-H3 CARs, and a total of 1328 DEGs were identified (Figure 7F). By GSEA analysis, it was found that in B7-H3-TMIGD2 CAR-T cells, four metabolisms were negatively enriched, including "oxidative phosphorylation", "fatty acid metabolism" and "lipogenesis", and "glycolysis" was approaching significance (FDR = 0.05) (Figure 7G). These results suggest that after 72 hours of co-culture, B7-H3.28.BB.ζ CAR-T cells were overall more metabolically active than B7-H3.TMI.ζ CAR-T cells. To determine the most significantly contributing pathways, overrepresentation analysis (ORA) was performed by testing for overrepresented pathways among the most highly expressed DEGs (adjusted p < 0.05, fold change > 1.5). By ORA analysis, the "glycolytic process" was the only overrepresented metabolic pathway in B7-H3.28.BB.ζ CAR-T cells, and other pathways were found to broadly explain hypoxia or nucleotide processes. (Figure 7H). B7-H3.TMI.ζ CAR-T cells broadly showed changes in pathways related to transcription, mitosis and ubiquitination (Figure 7H).

[0249] Using publicly available gene sets that describe important enzymes, regulatory proteins, accessory proteins and other related genes in glycolysis and oxidative phosphorylation, in the comparison of B7-H3.TMI.ζ CAR-T cells and B7-H3.28.BB.ζ CAR-T cells, eight upregulated DEGs (HK1, PGAM1, TPI1, ALDOC, ALDOA, PFKFB3 and PFKFB4) related to classical glycolytic enzymes or regulation of glycolysis were found, while there were two DEGs (NFDFB1 and AK2) related to subunits of oxidative phosphorylation or other functions (Figure 7I). Overall, these data suggest that B7-H3.28.BB.ζ CAR-T cells utilize the glycolytic pathway more than other pathways for metabolism.

[0250] B7-H3.TMI.ζ CAR-T cells exhibit different transcriptional programs in vivo The tumor microenvironment was examined to determine the impact of transcriptional programs in B7-H3 CAR-T cells. RNA sequencing of lung infiltrating T cells collected from the lungs of lung tumor xenograft mice 7 days after B7-H3 CAR-T cell injection was performed. Extensive differences in the transcriptome were found among the B7-H3 CAR-T cells of this application, with 945 DEGs present (Figure 8A). By testing the top enriched pathways by GSEA analysis, it was found that the B7-H3.TMI.ζ CAR-T cells had pathways related to RNA and DNA related processes widely enriched in the positive direction (Figure 8B). In the ORA analysis, the B7-H3.28.BB.ζ CAR-T cells overrepresented pathways that widely included pathways such as cytokine and chemokine pathways, while the B7-H3.TMI.ζ CAR-T cells had few genes meeting the log2 fold change cutoff and no statistically significant overrepresented pathways (adjusted p-value < 0.05), but it was found that the lysosome and vacuole pathways were overrepresented pathways approaching significance (p < 0.1) (Figure 8C). Using the same metabolic gene set as before, the metabolic-related gene signature was analyzed in this dataset. Six DEGs were found to be associated with glycolytic enzymes and regulatory proteins upregulated in B7-H3.28.BB.ζ CAR-T cells (PFKM, GAPDH, PFKFB3, PFKFB2, HIF1A, and PFKFB4), but there were no genes associated with oxidative phosphorylation (Figure 8D).

[0251] Using the recently shown T cell dysfunction gene signature, 12 DEGs were found to be upregulated in B7-H3.28.BB.ζ CAR-T cells (IL2RA, PLS3, DUSP4, GZMB, PHLDA1, CSF1, TNFRSF18, NDFIP2, AHI1, CDK6, LAYN, and HAVCR2), while only one was found in B7-H3.TMI.ζ CAR-T cells (KLRC1) (Figure 8E). By testing a manually curated gene list of inhibitory proteins, six downregulated DEGs (BTLA, HAVCR2, PDCD1, CTLA4, PDCDLG2, and CD274) were found in B7-H3.TMI.ζ CAR-T cells compared to B7-H3.28.BB.ζ CAR-T cells (Figure 8F). Collectively, these data suggest that B7-H3.TMI.ζ and B7-H3.28.BB.ζ function differently within the in vivo tumor microenvironment, with the former having lower glycolytic, dysfunctional, and inhibitory phenotypes compared to the latter.

[0252] Common significant genes (adjusted p-value < 0.05) were analyzed between in vivo RNA and in vitro RNA sequencing experiments that directly compared the two B7-H3 CAR-T cells. 67 common genes were found in the B7-H3.28.BB.ζ CAR, and 85 genes were found in the B7-H3.TMI.ζ CAR (Figure 8G). ORA analysis of these common genes revealed numerous overrepresented pathways in both CAR constructs (Figure 8H). In the B7-H3.28.BB.ζ CAR, pathways widely related to hypoxia and nucleotide metabolism were shown, among others; notably, the "glycolytic process" was also found in this list. In the B7-H3.TMI.ζ CAR, pathways widely related to endosomes, lysosomes, autophagy, and others were present. Collectively, these results indicate that B7-H3.TMI.ζ CAR-T cells and B7-H3.28.BB.ζ CAR-T cells have different pathway signatures when testing common DEGs between experiments.

[0253] B7-H3.TMI.ζ CAR-T cells exhibit different phenotypic changes after chronic antigen exposure Sustained exposure to antigens can lead to a dysfunctional phenotype and a suboptimal effector response in CAR-T cells. In this context, different CAR constructs may have a significant impact on the expression of various cell surface markers. An in vitro model of chronic antigen exposure (CAE) was adopted, in which CAR T cells were continuously cultured with sufficient HCC827 tumor cells such that tumor cells were always present in the co-culture, to test whether the TMIGD2 and CD28-4-1BB co-stimulatory domains differentially alter the expression of cell surface proteins. CD3 + , CD4 + and CD8 + CAR + The phenotypes of B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were compared at each analysis time point during a 17-day co-culture period with HCC827 tumor cells. First, to account for the results of the entire CD3 + CAR + population, the overall changes between the two B7-H3 CAR constructs were observed by t-distributed stochastic neighbor embedding (t-SNE) analysis of all time points of CAR + cells (Figure 9A). CAR expression decreased in both B7-H3 CAR-T cell types, and the CAR + cells on day 17 in B7-H3.TMI.ζ CAR-T cells were fewer (Figure 9B). This finding indicates downregulation of CAR after tumor encounter. PD-1 + TIM-3 + LAG-3 +Focusing on exhausted CAR-T cells, it was found that the exhausted cells acquired by B7-H3.TMI.ζ CAR-T cells by the 13th and 17th days were fewer than those of B7-H3.28.BB.ζ CAR-T cells (Figure 9C). Since differences in the expression of PD-1, TIM-3, LAG-3, or combinations thereof may underlie this finding, each protein was subsequently tested individually. No difference in expression was detected for PD-1 or TIM-3 (Figure 9C). In contrast, LAG-3 expression in B7-H3.TMI.ζ CAR T cells was found to be significantly lower at the 13th and 17th days compared to B7-H3.28.BB.ζ CAR T cells (Figure 9C).

[0254] In the test of memory phenotype, significant enrichment of the central memory cell (CD45RA - CCR7 + ) population was found after the 10th day, but the opposite was true on the 6th day (Figure 9C). A high proportion of naive T cells (CD45R + CCR7 + ) in B7-H3.28.BB.ζ was observed on the 17th day, but there was no difference between groups for effector memory (CD45RA - CCR7 - ) or finally differentiated EMRA cells (CD45RA + CCR7 - ) (Figure 9C). The CD69+ expression of B7-H3.TMI.ζ CAR-T cells compared to B7-H3.28.BB.ζ CAR-T cells was also found to be high on the 3rd and 6th days, equivalent on the 10th day, and high on the 13th and 17th days (Figure 9C).

[0255] Both B7-H3 CAR-T cells showed a tendency of a decrease in CD4 + T cells and an increase in CD8 + T cells. However, after the 13th day, compared to B7-H3.28.BB.ζ CAR-T cells, B7-H3.TMI.ζ CAR-T cells had significantly lower CD4 + T cells and higher CD8 +T cells were found to be significantly higher (Figure 9C). Since the design of this experiment is more similar to CAE than other experiments, the DEGs of in vivo RNA sequencing data (Figure 8) were reexamined to verify this finding. The CD4 gene was found to be a significantly downregulated DEG (adjusted p-value < 0.05; log2 fold change > ±0.5). In B7-H3.TMI.ζ CAR T cells, the CD8A gene was significantly upregulated (adjusted p-value < 0.05; log2 fold change = 0.4206); in B7-H3.TMI.ζ CAR-T cells, the CD8B gene also showed an upregulation, although not significantly (adjusted p-value = 0.161; log2 fold change = 0.31). To confirm this finding at the protein level, lung infiltrating and splenic T cells obtained from lung tumor xenograft mice 46 days after tumor injection were analyzed. One donor was the same as in Figure 6, while the other was a different donor. Compared with B7-H3.28.BB.ζ CAR-T cells, B7-H3.TMI.ζ CAR-T cells had a higher proportion of CD8 + T cells and a lower proportion of CD4 + T cells in lung infiltrating T cells, while there was no difference in splenic T cells between either population.

[0256] This trend was almost reproduced with only slight changes over time in the CD4 + CAR + and CD8 + CAR + populations, except for the notable exceptions described below (Figures 9E - 9F). Compared with B7-H3.28.BB.ζCD4 + CAR + CAR-T cells, B7-H3.TMI.ζCD4 + CAR + T cells had more TIM-3 expression on day 10 and lower expression on days 13 and 17 (Figure 9E). They also had an equivalent proportion of CD69 + cells on day 17 due to a decrease in CD69 expression. Compared with B7-H3.28.BB.ζ CAR-T cells, B7-H3.TMI.ζCD8 + CAR +The proportion of TEMRA cells on days 10 and 13 was low in T cells (Figure 9F).

[0257] It has been reported that antigen affinity and T cell receptor (TCR) signaling intensity can alter T cell memory formation in CD8 + T cells, and that TCR signaling intensity can affect the expression of PD-1 and LAG-3. Since the B7-H3 CAR uses the same scFv and thus has the same antigen affinity, the TMIGD2 and CD28-4-1BB costimulatory domains were tested to determine whether they change the intensity of T cell activation as a mechanism underlying this phenotypic difference. Jurkat (NFAT) cells, a T cell activation reporter cell line that expresses firefly luciferase via the NFAT response element, were transduced with B7-H3.TMI.ζ, B7-H3.28.BB.ζ, and CD19.28.BB.ζ CARs. Non-transduced Jurkat (NFAT) cells or Jurkat (NFAT) cells transduced with CARs were cultured alone, or together with CD19+B7-H3- cells (Raji), or with CD19 - B7-H3 +Cultured with cell lines (HCC827, AsPC-1, and U118) (Figure 9D). All Jurkat (NFAT) cells signaled in response to OKT3 stimulation but did not signal when cultured alone. Jurkat (NFAT) cells transduced with B7-H3.TMI.ζ and B7-H3.28.BB.ζ signaled broadly equivalently in response to HCC827, AsPC-1, and U118 tumor cell lines but did not signal in response to Raji cells. Notably, the levels were nearly identical in the HCC827 cell line used for CAE stimulation and the HCC827 cell line used for all RNA sequencing experiments. Jurkat (NFAT) cells transduced with CD19.28.BB.ζ signaled in response to Raji cells but did not signal in response to HCC827, AsPC-1, and U118 tumor cell lines. Collectively, these results indicate that B7-H3.TMI.ζ CAR-T cells acquire differences in memory, exhaustion, activation, and CD4 / CD8 phenotype by CAE and that this effect is not due to differences in signal intensity.

[0258] Discussion B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were superior performing CARs in in vitro killing assays. B7-H3.TMI.ζ CAR-T cells were also effective in multiple solid tumor models and showed equivalent or better results than B7-H3.28.BB.ζ CAR-T cells. Additionally, B7-H3.TMI.ζ CAR-T cells showed a unique transcriptome, metabolomics, and phenotypic profile, indicating that costimulation by TMIGD2 has distinct advantages over CD28-4-1BB.

[0259] An in vitro killing assay was performed with low transduction efficiency, and it was found that only B7-H3.TMI.ζ and B7-H3.28.BB.ζ CAR-T cells were able to kill three different tumor cell lines. Co-stimulation by TMIGD2 may be superior to the currently FDA-approved CD28 and 4-1BB co-stimulation domains. When comparing the two lead constructs, the concentration of cytokines released was low in B7-H3.TMI.ζ CAR-T cells. Considering that cytokine release syndrome (CRS) may be partially mediated by cytokines released from CAR-T cells or cells activated by CAR-T cells (e.g., macrophages and monocytes), the CAR with TMIGD2 may also function as a safe co-stimulation domain.

[0260] Unexpectedly, even when combining the signaling of TMIGD2 and 4-1BB with a third-generation CAR, no cytotoxicity was shown in two solid tumor cell lines (HCC827 and U118). Jurkat (NFAT) cells transfected with the B7-H3.TMI.BB.ζ CAR of this application showed reduced activation compared to cells transfected with the B7-H3.28.BB.ζ CAR.

[0261] Although not observed in other tumor models, in the orthotopic GBM model, unexpected toxicity was observed in mice treated with B7-H3.28.BB.ζ CAR-T cells, while not in those treated with B7-H3.TMI.ζ CAR-T cells, suggesting that the latter is safer in this situation. Since these mice died without a significant increase in tumor mass, it is highly likely that the CAR-T cells rather than the tumor cells are the underlying cause. B7-H3.28.BB.ζ CAR-T cells release a significantly higher level of cytokines than B7-H3.TMI.ζ CAR-T cells, so local cytokine release syndrome may be a factor. Similar effects have been reported clinically and are described as "localized" or "compartmentalized" cytokine release syndrome. This toxicity may also be enhanced by IT injection of CAR-T cells in this model compared to IV administration.

[0262] The CAR co-stimulatory domain shows a significant impact on CAR-T cell metabolism. CAR-T cells with the CD28 co-stimulatory domain utilize glycolytic metabolism, while the 4-1BB co-stimulatory domain utilizes oxidative metabolism. Furthermore, analysis of CD19 CAR-T cell products from a certain clinical trial showed enrichment of the glycolytic gene signature in non-responders and partial responders. Based on RNA sequencing pathway analysis, overexpression analysis, acute and chronic stimulation Seahorse metabolic assays, and analysis of common DEGs among RNA sequencing experiments, B7-H3.28.BB.ζ CAR-T cells showed a metabolic profile more dependent on glycolysis than B7-H3.TMI.ζ CAR-T cells.

[0263] Compared with B7-H3.28.BB.ζ CAR-T cells, B7-H3.TMI.ζ CAR-T cells have reduced phenotypes related to dysfunction and exhaustion involved in the anti-tumor response of CAR-T cells, which can be modified by the selection of the co-stimulatory domain. Therefore, TMIGD2 co-stimulation is a new way to prevent T cell dysfunction and exhaustion.

[0264] B7-H3.TMI.ζ CAR-T cells were found to show a time-dependent enrichment of central memory cells not during primary generation but at CAE. A high proportion of this population is associated with better outcomes in CAR-T cell therapy. In addition, CAR-T cells generated from bulk CD8 + T cells have an increased risk of CRS compared to the central memory-enriched population. The TMIGD2 co-stimulatory domain can increase central memory cells and may be beneficial for improving treatment efficacy and safety.

[0265] In B7-H3.TMI.ζ CAR-T cells compared with B7-H3.28.BB.ζ CAR-T cells, at CAE, CD8 +It was found that T cells were unexpectedly enriched. This finding was consistent with the phenotypic analysis of in vivo lung-infiltrating T cells and the reanalysis of in vivo RNA sequencing gene expression data. CD8+ CAR-T cells form a larger population than CD4+ CAR-T cells after injection of the bulk CAR-T cell product and after injection of the CAR-T cell product using CD8:CD4 T cells in a 1:1 ratio. Compared with CD28-4-1BB co-stimulation, TMIGD2 co-stimulation improves the proliferation of CD8+ CAR-T cells and / or TMIGD2 co-stimulation improves the survival of CD8+ CAR-T cells.

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Claims

1. A chimeric antigen receptor (CAR) comprising: (a) an extracellular region containing an antigen-binding domain; (b) a transmembrane region; and (c) an intracellular region containing an effector domain and a TMIGD2 co-stimulatory domain. The CAR containing the above.

2. The CAR according to claim 1, wherein the TMIGD2 co-stimulatory domain contains the intracellular region of the TMIGD2.

3. The CAR according to claim 2, wherein the TMIGD2 co-stimulatory domain contains a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO:

5.

4. The CAR according to claim 3, wherein the TMIGD2 co-stimulatory domain contains an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of residues 172-282 of SEQ ID NO: 3, 172-278 of SEQ ID NO: 4, and residues 52-162 of SEQ ID NO:

5.

5. The CAR according to any one of claims 1 to 4, wherein the antigen-binding domain specifically binds to a tumor-associated antigen.

6. The tumor-associated antigen is selected from the group consisting of HHLA2, CD19; CD20; BCMA; CD22; CD3; CEACAM6; c-Met; EGFR; EGFRvIII; ErbB2; ErbB3; ErbB4; EphA2; IGF1R; GD2; O-acetyl GD2; O-acetyl GD3; GHRHR; GHR; FLT1; KDR; FLT4; CD44v6; CD151; CA125; CEA; CTLA-4; GITR; BTLA; TGFBR2; TGFBR1; IL6R; gp130; Lewis A; Lewis Y; TNFR1; TNFR2; PD1; PD-L1; PD-L2; HVEM; MAGE-A (e.g., including MAGE-A1, MAGE-A3 and MAGE-A4); mesothelin; NY-ESO-1; PSMA; RANK; ROR1; TNFRSF4; CD40; CD137; TWEAK-R; HLA; a tumor or pathogen-related peptide bound to HLA; hTERT bound to HLA; a tyrosinase peptide bound to HLA; a WT-1 peptide bound to HLA; LTβR; LIFRβ; LRP5; MUC1; OSMRβ; TCRα; TCRβ; CD25; CD28; CD30; CD33; CD52; CD56; CD79a; CD79b; CD80; CD81; CD86; CD123; CD171; CD276; B7-H3; B7H4; TLR7; TLR9; PTCH1; WT-1; HA1-H; Robo1; alpha-fetoprotein (AFP); Frizzled; OX40; PRAME and SSX-2 antigen, the CAR according to claim 5.

7. The CAR according to any one of claims 1 to 6, wherein the antigen-binding domain contains an scFv.

8. The CAR according to any one of claims 1 to 7, wherein the antigen-binding domain contains a linker.

9. The CAR according to claim 8, wherein the linker is a glycine-serine linker.

10. The glycine-serine linker is (Gly ｘ Ser ｙ ), ｚ wherein x and y are each independently an integer from 0 to 10, except when both x and y are 0, and z is an integer from 1 to 10, the CAR according to claim 9.

11. The CAR according to any one of claims 1 to 10, wherein the extracellular region further contains an N-terminal leader sequence.

12. The CAR according to any one of claims 1 to 11, wherein the extracellular region further contains a hinge region.

13. The CAR according to claim 12, wherein the hinge region contains the amino acid sequence shown in SEQ ID NO:

2.

14. The CAR according to claim 13, wherein the hinge region contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO:

2. **Claim 15** The CAR according to any one of claims 1 to 14, wherein the transmembrane region contains the transmembrane region of CD8α. **Claim 16** The CAR according to claim 15, wherein the transmembrane region contains the amino acid sequence shown in SEQ ID NO:

1. **Claim 17** The CAR according to claim 15, wherein the transmembrane region contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO:

1. **Claim 18** The CAR according to any one of claims 1 to 17, wherein the effector domain is the effector domain of CD3ζ. **Claim 19** The CAR according to claim 18, wherein the effector domain contains the amino acid sequence shown in SEQ ID NO:

6. **Claim 20** The CAR according to claim 18, wherein the effector domain contains an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO:

5. **Claim 21** The CAR according to any one of claims 1 to 20, which contains (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO:

1. **Claim 22** The CAR according to any one of claims 1 to 20, which contains (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5; and (b) the sequence shown in SEQ ID NO:

6. **Claim 23** The CAR according to any one of claims 1 to 20, which contains (a) a sequence selected from the group consisting of residues 172 - 282 of SEQ ID NO: 3, 172 - 278 of SEQ ID NO: 4, and residues 52 - 162 of SEQ ID NO: 5, (b) the sequence shown in SEQ ID NO: 1; and (c) the sequence shown in SEQ ID NO:

6. **Claim 24** The CAR according to claim 22 or 23, which further contains the sequence shown in SEQ ID NO:

2. **Claim 25** An isolated nucleic acid molecule containing a nucleic acid sequence encoding the CAR according to any one of claims 1 to 24. **Claim 26** A vector containing a nucleic acid sequence encoding the CAR according to any one of claims 1 to 24. **Claim 27** The vector according to claim 26, wherein the nucleic acid sequence encoding the CAR is operably linked to an expression control sequence.

28. The vector according to claim 27, wherein the expression control sequence is a promoter.

29. The vector according to any one of claims 26 to 28, further comprising a nucleic acid sequence encoding a self-cleaving peptide.

30. The vector according to claim 29, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

31. The vector according to claim 30, wherein the 2A self-cleaving peptide is a P2A peptide.

32. The vector according to any one of claims 26 to 31, further comprising a nucleic acid sequence encoding a transduction marker polypeptide.

33. The vector according to claim 32, wherein the transduction marker polypeptide is a cleaved form of epidermal growth factor receptor (EGFRt) or GFP, or a part or variant thereof.

34. The vector according to any one of claims 29 to 31, wherein the nucleic acid sequence encoding the self-cleaving peptide is 3' of the nucleic acid sequence encoding the CAR.

35. The vector according to claim 32 or 33, wherein the vector contains a nucleic acid sequence encoding a self-cleaving peptide, and the nucleic acid sequence encoding the self-cleaving peptide is 5' of the nucleic acid sequence encoding the marker polypeptide.

36. The vector according to any one of claims 26 to 35, wherein the vector is a viral vector.

37. An isolated cell expressing the CAR according to any one of claims 1 to 24.

38. The cell according to claim 37, wherein the cell contains the nucleic acid molecule according to claim 25.

39. The cell according to claim 37 or 38, wherein the cell contains the vector according to any one of claims 26 to 36.

40. The cell according to any one of claims 37 to 39, wherein the cell is a T cell, a natural killer (NK) cell, a macrophage or other immune cell.

41. The T cells are CD4 ＋ T cells, CD8 ＋ T cells, CD4 － CD8 － The cell according to claim 40, which is CD8 double-negative T cells, NK cells, macrophages, other immune cells, or a combination thereof.

42. The cell according to claim 40, wherein the T cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, an NK cell, a macrophage, other immune cells or a combination thereof.

43. The cell according to any one of claims 37 to 42, wherein the cell further expresses a transduction marker on its surface.

44. The cell according to claim 43, wherein the transduction marker is a cleaved form of epidermal growth factor receptor (EGFRt) or GFP, or a part or variant thereof.

45. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the cell according to any one of claims 37 to 44.

46. The method according to claim 45, wherein the disease or condition is a malignant tumor.

47. The method according to claim 46, wherein the malignant tumor is cancer.

48. The method according to claim 47, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer, melanoma, leukemia, lymphoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor, gastric cancer, small intestine cancer, bone cancer, cervical cancer, endometrial cancer, eye tumor, gallbladder cancer, thyroid cancer, thymus cancer, sarcoma, and osteosarcoma.

49. The method according to claim 47 or 48, wherein the cancer includes solid tumors.

50. The method according to any one of claims 47 to 49, wherein the cancer includes hematological malignancies.

51. A method for inducing an immune response against a tumor-associated antigen that specifically binds to the CAR according to any one of claims 1 to 24 in a subject, the method comprising administering to the subject an effective amount of the cell according to any one of claims 37 to 44.

52. A composition comprising the CAR according to any one of claims 1 to 24, and a pharmaceutically acceptable excipient, carrier or diluent.

53. A composition comprising the cell according to any one of claims 37 to 44, and a pharmaceutically acceptable excipient, carrier or diluent.

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