Compositions and methods for improved immunotherapies

Abstract

The present application provides recombinant fusion proteins, engineered chimeric antigen receptor fusions, cells engineered to express one or more recombinant fusion protein / chimeric antigen receptors, B2M fusion proteins, and uses thereof for the treatment of diseases, conditions and / or disorders, and uses thereof for manufacturing cell populations.

Description

WSGR Docket No.61078-716.601 COMPOSITIONS AND METHODS FOR IMPROVED IMMUNOTHERAPIES CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 419,884, filed on October 27, 2022, the contents of which are incorporated by reference herein in their entirety. BACKGROUND OF THE DISCLOSURE

[0002] Immunotherapeutic cell compositions may be generated for the treatment of diseases and / or conditions, for example, certain cancers and autoimmune conditions. Many existing immunotherapies comprise isolation of a subjects own T-cells, engineering and expansion of these cells ex vivo, and subsequently administering the engineered cells to the original subject. While these therapies have proven effective in the treatment of certain diseases, challenges of using the subject’s own cells for immunotherapies include uncontrollable interpatient variability, long wait times for subjects while cells are expanded and processed ex vivo, and high costs. Thus, the development of off-the-shelf allogeneic cells for these immunotherapies is an attractive alternative approach, however, major challenges still remain for these allogeneic immunotherapies, including graft rejection and conditions such as graft vs. host disease (GVHD). Host rejection of allogeneic cells occurs when host cells recognize the allogenic cells and direct an immune response against the cells, while GVHD occurs when a grafted cell recognizes the host as foreign and directs an immune response against the host. Recombinant fusion proteins may be expressed in grafted cells to improve the efficacy of grafted cell implantation, while decreasing the activation of host immune responses and / or the activation of graft cell immune responses. Alternative approaches to decrease these immune responses may include disruption of graft cell genes that encode for T-cell receptor and / or major histocompatibility complex proteins. Therefore, the promise of off-the-shelf or universal allogeneic cell immunotherapy products in effectively treating diseases such as cancer demonstrates an urgent medical need exists for strategies to engineer cells that do not activate the host / graft immune response. SUMMARY OF THE DISCLOSURE

[0003] Disclosed herein includes a composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that binds to a second extracellular domain of the MHC that is different than the first extracellular domain, wherein the first and second domains are operatively linked by a linker domain.WSGR Docket No.61078-716.601

[0004] Further disclosed herein includes a composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell.

[0005] Further disclosed herein includes a composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that binds to a second extracellular domain of the MHC that is different than the first extracellular domain, wherein the first and second domains are operatively linked by a linker domain.

[0006] Further disclosed herein includes a composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell . In some embodiments, the recombinant fusion protein further comprises a third domain that binds to an extracellular receptor on a second cell; wherein the first, second and third domains are operatively linked. In some embodiments, the first extracellular domain of the MHC is a domain that binds to B2M. In some embodiments, the first domain is B2M or an MHC-binding fragment or variant thereof. In some embodiments, the second domain is CD8 or an MHC-binding fragment or variant thereof, or LILRB1 variant thereof. In some embodiments, the second or third domain is CD160 or an MHC-binding fragment or variant thereof, wherein the CD160 or MHC-binding fragment or variant thereof is an and LCDR3 of QSVTNN (SEQ ID NO: 293), FAS (SEQ ID NO: 294) and HQDYSSPLT (SEQ ID NO: 295), respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GYTFTSNW (SEQ ID NO: 290), IAPGSGNT (SEQ ID NO: 291) scFv comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, orWSGR Docket No.61078-716.601 comprises an amino acid sequence set forth in SEQ ID NO. 252 or 253. In some embodiments, the second domain or third domain binds to a second extracellular domain of the MHC that is different than the first extracellular domain. In some embodiments, the second extracellular domain of the MHC embodiments, the MHC is an MHC class I. In some embodiments, the MHC is encoded by an HLA- A02:01 gene. In some embodiments, the second domain inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell. In some embodiments, the cell-surface receptor is CD8, or wherein the cell-surface receptor is not TCRalpha / beta or TCR delta / gamma. In some embodiments, the first cell is an allogeneic cell. In some embodiments, the first cell is a grafted cell. In some embodiments, the second cell is a T cell, an NK cell, an NKT cell, a monocyte, a myeloid cell, a macrophage, a dendritic cell, a hematopoietic stem cell or an iPSC In some embodiments, the second cell is a host cell. In some embodiments, the second cell is a T-cell. In some embodiments, the T cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the T cell is a chimeric antigen receptor T (CAR-T) cell. In some embodiments, the second cell is a CD8+ T-cell. In some embodiments, the second cell is a NK cell. In some embodiments, the second cell is a gamma delta T cell. In some embodiments, the second cell is an induced pluripotent stem cell (iPSC). In some embodiments, the second cell is an embryonic or adult hematopoietic stem cell (HSC). In some embodiments, the first domain is linked to the second domain. In some embodiments, the first domain is linked to the second domain by a first linker sequence. In some embodiments, the first domain is linked to the third domain. In some embodiments, the first domain is linked to the third domain by a second linker sequence. In some embodiments, the second domain is linked to the third domain. In some embodiments, the second domain is linked to the third domain by a third linker sequence. In some embodiments, the first domain is linked to the second domain and the first domain is linked to the third domain. In some embodiments, the second domain is linked to the first domain and the second domain is linked to the third domain. In some embodiments, the first, second, and or third linker sequence is at least 5 amino acids in length. In some embodiments, the first, second, and or third linker sequence is at most 30 amino acids in length. In some embodiments, the linker sequence is from 5 to 30 amino acids in length. In some embodiments, the third domain is a domain that binds to an immune checkpoint protein. In some embodiments, the third domain is a PDL1 or PDL2 domain. In some embodiments, the third domain is a domain that binds to PD1 or PD2. In some embodiments, the third domain is a domain that binds to CTLA-4, LAG-3, TIM-3, TIGIT OR VISTA. In some embodiments, the third domain isWSGR Docket No.61078-716.601 a domain that binds to a T cell receptor. In some embodiments, the third domain is an intracellular embodiments, the first or second domain is crosslinked to the MHC. In some embodiments, the crosslinking prevents MHC clustering.

[0007] Further disclosed herein includes a composition comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding the recombinant fusion protein of the composition described herein. In some embodiments, the recombinant fusion protein comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence any one of SEQ ID NOs. 42-56, 62-111, and 240-258. In some embodiments, the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain is an anti-CD22 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-WSGR Docket No.61078-716.601 4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a- fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY- CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2. In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. In some embodiments, the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.

[0008] Further disclosed herein includes a composition comprising a cell, wherein the cell comprises the composition of any of the pharmaceutical compositions disclosed herein. any one of claims 1-52. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell comprises a disruption of NLRC5, RFX5, RFXANK, RFXAP, a viral immunoevasin, ICAM1, CD80, CD58, OX40L, and any combination thereof. In some embodiments, the cell expresses a dominant negative protein, wherein the dominant negative (DN) protein is selected from the group consisting of DN-TCRalpha, DN- RFXANK, DN-RFXAP, a DN-viral immunoevasin, ICAM1, DN-CD80, DN-CD58, DN-CD2, DN- OX40L, and any combination thereof. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells comprises at least 1x10^5 cells.

[0009] Further disclosed herein includes a pharmaceutical composition comprising any of the compositions disclosed herein, and a pharmaceutically acceptable excipient or carrier.

[0010] Further disclosed herein includes a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of CAR-T cells. In some embodiments, the disease or condition is cancer, and or an auto- immune disease. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung cancer, liver cancer,WSGR Docket No.61078-716.601 pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and / or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and / or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0013] FIG. 1 coreceptor by a host T cell, thereby reducing TCR-pMHC avidity and recruitment of Lck to host TCR complex.

[0014] FIG.2A inhibit binding of CD8 coreceptor by a host T cell and tethered PD-L1 can engage with PD-1 on host T cell.WSGR Docket No.61078-716.601

[0015] FIG. 2B 1 on host T cell.

[0016] FIG.3 shows the % graft T cell count relative to the number of host cells after expression of the indicated B2M fusion proteins in graft T cells with or without RFX5 KO or RFXANK KO.

[0017] FIG.4 shows the % graft T cell count relative to the number of host cells after expression of the indicated B2M fusion proteins in graft T cells with or without RFX5 KO or RFXANK KO.

[0018] FIG. 5 shows the % TRAC KO edited graft T cell count relative to the host T cells after incubating the graft T cells or the graft T cells with B2M KO, RFX5 KO, or RFX5 / CD58 double KO with the anti-MHCI (clone TP25.99) antibody. The % graft T cell survival was measured via flow cytometry after the graft cells were mixed with primed allogeneic CD8+ T cells for 48 hours.

[0019] FIG.6A depicts an exemplary diagram of anti-MHCI (TP25.99) scFv as an alloreactive CD8+ T cell inhibitor incorporated into cells as a fusion to B2M, a fusion to a transmembrane domain or secreted as a soluble scFv.

[0020] FIG.6B shows the flow cytometry results of B2M expression of the various constructs of anti- MHCI-B2M fusions in B2M KO T cells.

[0021] FIGS.7A-7C show the % survival of the TRAC KO and RFX5 KO graft T cells. The % graft T cell survival was measured via flow cytometry after the graft cells were mixed with primed allogeneic CD8+ T cells for 48 hours. FIG. 7A shows the % survival of graft T cells expressing various constructs of the anti-MHCI scFv (clone TP25.99) – B2M fusions. FIG. 7B shows the % survival of graft T cells expressing various constructs of the membrane tethered anti-MHCI scFv (clone TP25.99). FIG.7C shows the % survival of graft T cells expressing various constructs of the soluble anti-MHCI scFv (clone TP25.99).

[0022] FIGS.8A-8C T cells. Host cells from 6 different donors were tested. FIG. 8A shows the % of graft survival on different days after the graft cells were mixed with allogeneic PBMCs. FIG.8B demonstrates the fold fusion relative to control. Allogeneic PBMCs were labeled with Cell Trace Far Red (CTFR), and fold change of the CD56+ NK cells was calculated based on the CTFR and CTFRlow events counted by flow cytometry. FIG. 8C demonstrates the CD8+ T cell proliferation elicited by graft T cells Far Red (CTFR), and fold change of the CD3+ host T cells was calculated based on the CTFR and CTFRlowevents counted by flow cytometry.WSGR Docket No.61078-716.601 DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] Disclosed herein include compositions and methods comprising recombinant polypeptides and / or recombinant nucleic acids encoding the recombinant polypeptides, comprising recombinant Beta-2 microglobulin (B2M) fusion proteins. Specifically, the present disclosure describes a recombinant B2M fusion protein comprising a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and a second domain that (i) binds to a second extracellular domain of the MHC that is different than the first extracellular domain or (ii) binds to an immune checkpoint protein, or (iii) inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain or (iv) activates a cell-surface receptor that is an immune checkpoint protein, wherein the first and second domains are operatively linked by a linker domain. The recombinant B2M fusion protein may further comprise a third domain that binds to an extracellular receptor on a second cell. In some aspects, the recombinant B2M fusion protein may be expressed in a cell. In some aspects, the cell may further comprise a chimeric antigen receptor (CAR). In some embodiments, the B2M fusion protein and / or CAR may comprise recombinant polypeptides and / or recombinant nucleic acids encoding the recombinant polypeptides. In some embodiments, the B2M fusion protein and / or CAR may be expressed in a cell. In some aspects, the compositions and methods disclosed herein my further comprise the disruption of a gene in the recombinant cells described herein. In some embodiments, the gene may be a gene that encodes a T- cell receptor. In some embodiments, the gene may be a gene that encodes an MHC. In some embodiments, the gene may be a gene that encodes an extracellular domain of a T-cell receptor or MHC. In some embodiments, the gene may be a gene that encodes an intracellular receptor of a T-cell receptor or MHC. In some embodiments, the disruption of a gene may comprise administering a gene editing molecule to a recombinant cell of the disclosure. Definitions

[0024] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0025] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included inWSGR Docket No.61078-716.601 the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0026] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0027] “Percent (%) sequence identity” or “homology” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0028] “Percent (%) identity” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0029] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong. Although any recombinant polypeptides, methods and other aspects similar or equivalent to those described herein can also be used in the practice or testing of the recombinant polypeptides, methods and other aspects, representative illustrative recombinant polypeptides, methods and other aspects are now described.WSGR Docket No.61078-716.601

[0031] As used herein, the terms “polynucleotides,” “nucleic acids,” and “oligonucleotide,” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides, or ribonucleotides, or analogs thereof, either in single-, double-, or multi- stranded form. Polynucleotides may have any three-dimensional structure and may perform any known function or unknown function. Various non-limiting examples of polynucleotides includes: non- coding or coding regions of a gene fragment, or a gene, locus (loci) that is defined from linkage analysis, exons or introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA or RNA of any sequences, nucleic acid probes or primers. There are many forms and descriptions denoting a polypeptide as disclosed herein. Therefore, a polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure, and may perform any function, known or unknown. A polynucleotide may comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.WSGR Docket No.61078-716.601

[0032] As used herein, the term “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Also “polypeptides” may denote an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. Variants of the amino acid sequences described herein may be included in various embodiments. The term "variant" refers to a protein or polypeptide in which one or more amino acid substitutions, deletions, and / or insertions are present as compared to the amino acid sequence of a protein or polypeptide, and the term includes naturally occurring allelic variants and alternative splice variants of a protein or polypeptide. The term "variant" includes the replacement of one or more amino acids in an amino acid sequence with a similar or homologous amino acid(s) or a dissimilar amino acid(s). Some variants include alanine substitutions at one or more amino acid positions in an amino acid sequence. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein.

[0033] A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.

[0034] A “fragment” is a truncated form of a native biologically active protein that, in some instances, retains at least a portion of the therapeutic and / or biological activity. A “variant” is a protein with sequence homology to the native biologically active protein that, in some instances, retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.

[0035] As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids. As used herein, the term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q),WSGR Docket No.61078-716.601 asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T), wherein the redundancy for the genetic code is included in its entirety for all amino acid codons.

[0036] As used herein, the term “non-naturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.

[0037] As used herein, the terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.

[0038] “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[0039] “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity to those sequences.

[0040] The term “binding domain”, as used herein, refers to a molecule, such as a protein, or polypeptide sequence, which specifically binds to a target.

[0041] As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.WSGR Docket No.61078-716.601

[0042] As used herein, the term “operably connected” or “operably linked” refers to positioning of components such that they function in their intended manner. For example, the components can be operably connected by a fusion, a linker, and / or a spacer.

[0043] As used herein, “specifically binds” means that the binding domain preferentially binds the corresponding target over other targets. In some embodiments, “specifically binds” means that the binding domains have a higher affinity for the target than for other targets.

[0044] As used herein, a “therapeutically effective amount” or “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of an agent means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

[0045] As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0046] It is appreciated that certain features of the recombinant polypeptides, fusion proteins, and / or recombinant nucleic acids encoding the recombinant polypeptides / fusion proteins, methods and other aspects, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the recombinant polypeptides, and / or recombinant nucleic acids encoding the recombinant polypeptides, methods and other aspects, which are, for brevity, described in the context of a single embodiment, may also be providedWSGR Docket No.61078-716.601 separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present recombinant polypeptides, methods and other aspects and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. B2M Fusion Proteins

[0047] In some aspects, disclosed herein are recombinant polypeptides and / or recombinant polynucleic acids encoding the recombinant polypeptides, comprising fusion proteins comprising a first domain, and a second domain, or comprising a first domain, a second domain, and / or a third domain. In some embodiments, the fusion proteins may be B2M fusion proteins. Some of the exemplary B2M fusion proteins are depicted in FIG.1, FIG.2A, and FIG.2B. First Domain

[0048] In some aspects, the first domain of the B2M fusion protein may be a domain that binds a first extracellular domain of an MHC of a first cell. In some embodiments, the first domain may be B2M or an MHC-binding fragment or variant thereof. In some embodiments, the first extracellular domain of the MHC is a domain that binds to B2M. The first domain can be any molecule that binds to the alpha chain of an MHC class 1 molecule.

[0049] In some embodiments, the first domain comprises an amino acid sequence MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1). In some embodiments, the first domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 1. In some embodiments, the first domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the first domain is a B2M domain. In some embodiments, the first domain is an anti-MHC binding domain.

[0050] In some embodiments, the first domain may comprise a portion of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids to 10 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 to 20 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 to 30 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 40 of SEQ ID NO: 1. In some embodiments, the first domain may compriseWSGR Docket No.61078-716.601 amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 50 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 60 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 70 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 80 of SEQ ID NO:. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 90 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 100 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 or 5 or 10 or 15 or 20 or 25 or 30 to 110 of SEQ ID NO: 1. In some embodiments, the first domain may comprise less than 119 amino acids of SEQ ID NO: 1.

[0051] In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 or 50 or 60 or 70 to 119 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 or 50 or 60 or 70 to 90 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 or 50 or 60 or 70 to 80 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 or 50 or 60 to 70 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 or 50 to 60 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 or 40 to 50 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 or 20 or 25 or 30 to 40 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 1 to 30 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 or 10 or 15 to 20 of SEQ ID NO: 1. In some embodiments, the first domain may comprise amino acids 5 to 10 of SEQ ID NO: 1.

[0052] In some embodiments, the first domain of the B2M fusion protein may be a binding domain 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. a KA of less than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of the B2MWSGR Docket No.61078-716.601 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the first domain of the B2M fusion protein may to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M- 1.

[0053] KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For KA of greater than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the first domain of the B2M fusion protein equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0054] conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0055] In some embodiments, the first domain of the B2M fusion protein may be a binding domain 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. a KA of less than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion proteinWSGR Docket No.61078-716.601 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of the B2M nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the first domain of the B2M fusion protein may to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M- 1.

[0056] KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For KA of greater than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the first domain of the B2M fusion protein equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0057] conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0058] In some embodiments, the first domain of the B2M fusion protein may be a domain that binds a KA of less than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. ForWSGR Docket No.61078-716.601 KA of less than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of the B2M nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the first domain of the B2M fusion protein may to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M- 1.

[0059] KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the first domain of the B2M fusion protein may bind to or associate 1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein may bind 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0060] conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0061] In some embodiments, the first domain of the B2M fusion protein may be a domain that binds to HLA-A. In some embodiments, the first domain of the B2M fusion protein binds to MHCI HLA-A with low affinity. For example, the first domain of the B2M fusion protein can bind to MHCI HLA-A with a KA of less than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM,WSGR Docket No.61078-716.601 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the first domain of the B2M fusion protein may bind to or associate with MHCI HLA-A with a KA of less than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein may bind to MHCI HLA-A with a KA of less than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the first domain of the B2M fusion protein can bind to MHCI HLA-A with a KD of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the first domain of the B2M fusion protein may bind to or associate with MHCI HLA-A with a KD of greater than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein may bind to MHCI HLA-A with a KD of greater than or equal to about 10^6 M- 1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0062] In some embodiments, the first domain of the B2M fusion protein binds to MHCI HLA-A with a high affinity. For example, the first domain of the B2M fusion protein can bind to MHCI HLA-A with a KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the first domain of the B2M fusion protein may bind to or associate with MHCI HLA-A with a KA of greater than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein may bind to MHCI HLA-A with a KA of greater than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M- 1. For example, the first domain of the B2M fusion protein can bind to MHCI HLA-A with a KD of less than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the first domain of the B2M fusion protein may bind to or associate with MHCI HLA-A with a KD of less than or equal to about 10^5 M-1. For example, the first domain of the B2M fusion protein may bind to MHCI HLA-A with a KD of less than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0063] The binding affinity of the first domain to MHCI HLA-A can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0064] In some aspects, the first domain may be expressed in an allogeneic and / or grafted T-cell. In some aspects, the first domain may inhibit killing of the grafted cells in which the first domain isWSGR Docket No.61078-716.601 expressed when bound to the MHC of the grafted cell via the first domain, and wherein the first domain is operatively linked to a second and / or third domain. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 5%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 10%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 15%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 20%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 30%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 40%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 50%, compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 60% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 70% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 80% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 90% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 95% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by at least about 99% compared to grafted cells in which the first domain is not expressed. In some embodiments, the first domain may inhibit killing of the grafted cells in which it is expressed by less than about 50% compared to a grafted cell in which the first domain is not expressed.

[0065] In some aspects, the first domain may be modified N-terminally or C-terminally. In some embodiments, the first domain may be operatively linked to a second domain by a linker. In some embodiments, the first domain may be operatively linked to a second domain by a first linker, and operatively linked to a third domain by a second linker. In some embodiments, the first domain mayWSGR Docket No.61078-716.601 be linked to a second domain, and / or a third domain, and / or a fourth domain by a third linker. In some embodiments, the first domain may be linked to three or more additional domains by additional linkers.

[0066] In some embodiments, the linker may include one or more intervening amino acid residues that are positioned between the first domain and second domain, and / or are positioned between the first domain and the third domain and / or are positioned between the first, second, or third domains and any additional domains. In principle, there are no particular limitations to the length and / or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues.

[0067] In some aspects, the first domain may anchor the recombinant B2M fusion protein to an MHC of a first cell. In some embodiments, the MHC may be a class 1 MHC molecule. In some embodiments, the class 1 MHC may be encoded by an HLA-A*02:01 gene. In some embodiments, the first cell may be an allogeneic cell. In some embodiments, the first cell may be an allogeneic and / or grafted cell. In some embodiments, the first cell may be a T-cell. Second Domain

[0068] In some aspects, the second domain of the recombinant B2M fusion protein may be a domain that binds to a second extracellular domain of an MHC that is different than a first extracellular domain. the second domain may be CD8 or an MHC-binding fragment or variant thereof, or LILRB1 or an thereof. In some embodiments, the second domain may inhibit binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell. In some embodiments, the first and second domains are operatively linked by a linker domain.

[0069] In some aspects, the second domain of the recombinant B2M fusion protein may be a domain that is expressed in a first cell and binds to an extracellular receptor expressed on a second cell. In some embodiments, the second domain may be a domain that bind to an immune checkpoint protein. In some embodiments, the second domain may be a PDL1 or PDL2 domain. In some embodiments, the second domain may be a domain that binds to PD1 or PD2.WSGR Docket No.61078-716.601

[0070] In some embodiments, the second domain comprises CD8 or an MHC binding fragment or variant thereof. For example, the second domain can comprise CD8alpha or an MHC binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPR GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 2). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the second domain is a CD8alpha domain or an MHC-binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2 and contains one or more mutations that increase the affinity to an alpha3 domain of an MHCI heavy chain. In some embodiments, the second domain comprises two or more CD8alpha domains or an MHC-binding fragments or variants thereof. In some embodiments, the second domain comprises two or more CD8alpha domains or an MHC-binding fragments or variants thereof that are linked together. In some embodiments, the second domain is linked to the first domain via a linker. In some embodiments, the second domain comprises two or more CD8alpha domains or an MHC-binding fragments or variants thereof and the second domain is linked to the first domain via a linker.

[0071] For example, the second domain can comprise CD8beta or an MHC binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQA PSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPEL TFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSL GVAIHLCCRRRRARLRFMKQFYK (SEQ ID NO: 2B). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2B. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 2B. In some embodiments, the second domain is a CD8beta domain or an MHC-binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2B and contains one or more mutations that increase the affinity to an alpha3 domain of an MHCI heavy chain. In some embodiments, the second domain comprises two or more CD8beta domains orWSGR Docket No.61078-716.601 an MHC-binding fragments or variants thereof. In some embodiments, the second domain comprises two or more CD8beta domains or an MHC-binding fragments or variants thereof that are linked together. In some embodiments, the second domain is linked to the first domain via a linker. In some embodiments, the second domain comprises two or more CD8beta domains or an MHC-binding fragments or variants thereof and the second domain is linked to the first domain via a linker.

[0072] In some embodiments, the second domain comprises a CD8beta domain or an MHC-binding fragment or variant thereof and a CD8alpha domain or an MHC-binding fragment or variant thereof. In some embodiments, the second domain comprises a CD8beta domain or an MHC-binding fragment or variant thereof and a CD8alpha domain or an MHC-binding fragment or variant thereof that are linked together. In some embodiments, the second domain comprises a CD8beta domain or an MHC- binding fragment or variant thereof and a CD8alpha domain or an MHC-binding fragment or variant thereof and the second domain is linked to the first domain via a linker.

[0073] In some embodiments the second domain is linked to the C terminus of the first domain. In some embodiments the second domain is linked to the N terminus of the first domain. In some embodiments the second domain is linked to a linker at the C terminus of the first domain. In some embodiments the second domain is linked to a linker at N terminus of the first domain.

[0074] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC-binding fragment or variant thereof, (ii) a linker, and (iii) CD8alpha or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHC binding fragment or variant thereof, (ii) a linker, and (iii) B2M or an MHC-binding fragment or variant thereof.

[0075] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC-binding fragment or variant thereof, (ii) a linker, and (iii) CD8beta or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8beta or an MHC binding fragment or variant thereof, (ii) a linker, and (iii) B2M or an MHC-binding fragment or variant thereof.

[0076] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHC binding fragment or variant thereof, (ii) a linker, (iii) B2M or an MHC-binding fragment or variant thereof, (iv) a linker, and (v) CD8alpha or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC- binding fragment or variant thereof, (ii) a linker, (iii) CD8alpha or an MHC binding fragment or variant thereof, (iv) a linker, and (v) CD8alpha or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHCWSGR Docket No.61078-716.601 binding fragment or variant thereof , (ii) a linker, (iii) CD8alpha or an MHC binding fragment or variant thereof, (iv) a linker, and (v) B2M or an MHC-binding fragment or variant thereof.

[0077] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8beta or an MHC binding fragment or variant thereof, (ii) a linker, (iii) B2M or an MHC-binding fragment or variant thereof, (iv) a linker, and (v) CD8beta or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC- binding fragment or variant thereof, (ii) a linker, (iii) CD8beta or an MHC binding fragment or variant thereof, (iv) a linker, and (v) CD8beta or an MHC binding fragment or variant thereof. In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8beta or an MHC binding fragment or variant thereof , (ii) a linker, (iii) CD8beta or an MHC binding fragment or variant thereof, (iv) a linker, and (v) B2M or an MHC-binding fragment or variant thereof.

[0078] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHC binding fragment or variant thereof, (ii) a linker, (iii) B2M or an MHC-binding fragment or variant thereof, (iv) a linker, and (v) CD8beta or an MHC binding fragment or variant thereof.

[0079] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8beta or an MHC binding fragment or variant thereof, (ii) a linker, (iii) B2M or an MHC-binding fragment or variant thereof, (iv) a linker, and (v) CD8alpha or an MHC binding fragment or variant thereof.

[0080] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC-binding fragment or variant thereof, (ii) a linker, (iii) CD8alpha or an MHC binding fragment or variant thereof, (iv) a linker, and (v) CD8beta or an MHC binding fragment or variant thereof.

[0081] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) B2M or an MHC-binding fragment or variant thereof, (ii) a linker, (iii) CD8beta or an MHC binding fragment or variant thereof, (iv) a linker, and (v) CD8alpha or an MHC binding fragment or variant thereof.

[0082] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHC binding fragment or variant thereof , (ii) a linker, (iii) CD8beta or an MHC binding fragment or variant thereof, (iv) a linker, and (v) B2M or an MHC-binding fragment or variant thereof.

[0083] In some embodiments, the recombinant protein comprises, from N to C terminus: (i) CD8beta or an MHC binding fragment or variant thereof , (ii) a linker, (iii) CD8alpha or an MHC binding fragment or variant thereof, (iv) a linker, and (v) B2M or an MHC-binding fragment or variant thereof.

[0084] In some embodiments, the second domain comprises LILRB1 or MHC binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYR EKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPWSGR Docket No.61078-716.601 TLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVS PSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDA GYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAP SDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRS TYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGP TSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQR KADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDP QAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQL HSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 3). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 3. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the second domain is a LILRB1 domain or an MHC-binding fragment or variant thereof.

[0085] an antibody (e.g. W6 / 32) that can bind broadly to the alpha3 domains across MHCI alleles. For example, the second domain can comprise an scFv having a VH and a VL domain linked by a linker, such as a linker with a sequence of GSTSGSGKPGSGEGSTKG or GGGGSGGGGSGGGGS. For example, the second domain can comprise an scFv with a VH domain having an amino acid sequence with at least about 80% sequence identity to QVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWSGGSTDYNA AFISRLSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFTTSTSAWFA. For example, the second domain can comprise an scFv with a VL domain having an amino acid sequence with at least about 80% sequence identity to IVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRF TGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPW. For example, the second domain can comprise a domain that binds to a sequence with at least 80% sequence identity to DPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQK WAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPT (SEQ ID NO: 4). In someWSGR Docket No.61078-716.601 embodiments, the second domain comprises a domain that binds to a sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 4.

[0086] SIVMTQTPKFLLVSAGDRVTITCKASQSVTNNVAWYQQKPGQSPKLLIYFASNRYTGVPDRF TGSGYGTDFTFTISTVQAEDLAVYFCHQDYSSPLTFGGGTKLELKGSTSGSGKPGSGEGSTK GQVQLQQSGDDLVKPGASVKLSCKASGYTFTSNWINWIKQRPGQGLEWIGRIAPGSGNTYY NEIFKAKATLTVDTSSSTVYIQLSSLSSEDSAVYFCARLLRGALDYWGPGTSVTVSS (SEQ ID comprises an amino acid sequence with at least about 80, 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 252.

[0087] QVQLQQSGDDLVKPGASVKLSCKASGYTFTSNWINWIKQRPGQGLEWIGRIAPGSGNTYYN EIFKAKATLTVDTSSSTVYIQLSSLSSEDSAVYFCARLLRGALDYWGPGTSVTVSSGSTSGSG KPGSGEGSTKGSIVMTQTPKFLLVSAGDRVTITCKASQSVTNNVAWYQQKPGQSPKLLIYFA SNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCHQDYSSPLTFGGGTKLELK (SEQ ID scFv comprises an amino acid sequence with at least about 80, 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 253.

[0088] In some embodiments, the second domain comprises CD160 or an MHC binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEEAEGFVVFLCKD RSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGTYQCCARSQKSGIRLQGH FFSILFTETGNYTVTGLKQRQHLEFSHNEGTLSSGFLQEKVWVMLVTSLVALQAL (SEQ ID NO: 5). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 5. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID fragment or variant thereof.

[0089] In some embodiments, the second domain comprises E3-19K or an MHC binding fragment thereof. In some embodiments, the second domain comprises an amino acid sequence MRYMILGLLALAAVCSAAKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKWSGR Docket No.61078-716.601 YAVYAIWQPGDTNDYNVTVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLEN TGTFCSTALLITALALVCTLLYLKYKSRRSFIDEKKMP (SEQ ID NO: 6). In some embodiments, the second domain comprises a portion of the amino acid sequence according to SEQ ID NO: 6. For example, the second domain can comprise an amino acid sequence AKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKYAVYAIWQPGDTNDYNV TVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLEN, or a sequence with at least about 80, 85, 90, 95, 97, 98, or 99% sequence identity to AKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGKYAVYAIWQPGDTNDYNV TVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLEN. In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 6. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the second domain is a E3-19K or an MHC-binding fragment or thereof.

[0090] In some aspects, the second domain of the recombinant B2M fusion protein may be a domain that binds to an immune checkpoint protein. In some aspects, the second domain of the recombinant B2M fusion protein may be a domain that inhibits an immune checkpoint protein.

[0091] In some aspects, the recombinant B2M fusion protein may comprise a third domain that binds to an immune checkpoint protein. some aspects, the recombinant B2M fusion protein may comprise a third domain that inhibits an immune checkpoint protein.

[0092] In some embodiments, the second domain comprises PDL1 or a PD1 binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMED KNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGAD YKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNS KREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILL CLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 7). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 7. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the second domain is a domain that binds to PD1.

[0093] In some embodiments, the second domain comprises PDL2 or a PD1 binding fragment or variant thereof. In some embodiments, the second domain comprises an amino acid sequence MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVEWSGR Docket No.61078-716.601 NDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRK INTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGR NFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYS SKDTTKRPVTTTKREVNSAI (SEQ ID NO: 8). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 8. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the second domain is a domain that binds to PD1.

[0094] In some embodiments, the second domain may be a domain that binds to CTLA-4. In some embodiments, the second domain comprises CD80 or a CTLA-4 binding fragment or variant thereof. In some embodiments, the domain that binds to CTLA-4 comprises an amino acid sequence MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINT TVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWA ITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV(SEQ ID NO: 9). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 9. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 9.

[0095] In some embodiments, the second domain may be a domain that binds to CTLA-4. In some embodiments, the second domain comprises CD86 or a CTLA-4 binding fragment or variant thereof. In some embodiments, the domain that binds to CTLA-4 comprises an amino acid sequence MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQE NLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRI HQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQ KSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVL PTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFK SSKTSSCDKSDTCF (SEQ ID NO: 10). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 10. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 10.

[0096] In some embodiments, the second domain may be a domain that binds to LAG-3. In someWSGR Docket No.61078-716.601 protein 1 domain or a LAG-3 binding fragment or variant thereof. In some embodiments, the domain

[0097] or variant thereof. In some embodiments, the second domain that binds to LAG3 comprises an amino acid sequence MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEK TKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPV DPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 11). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 11. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 11.

[0098] In some embodiments, the second domain comprises Galectin-3 or a LAG-3 binding fragment or variant thereof. In some embodiments, the second domain that binds to LAG3 comprises an amino acid sequence MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAP PGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLP LPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWG REERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSA SYTMI (SEQ ID NO: 12). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 12. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 12.

[0099] In some embodiments, the second domain comprises L-SECtin or a LAG-3 binding fragment or variant thereof. In some embodiments, the second domain that binds to LAG3 comprises an amino acid sequence MDTTRYSKWGGSSEEVPGGPWGRWVHWSRRPLFLALAVLVTTVLWAVILSILLSKASTER AALLDGHDLLRTNASKQTAALGALKEEVGDCHSCCSGTQAQLQTTRAELGEAQAKLMEQE SALRELRERVTQGLAEAGRGREDVRTELFRALEAVRLQNNSCEPCPTSWLSFEGSCYFFSVP KTTWAAAQDHCADASAHLVIVGGLDEQGFLTRNTRGRGYWLGLRAVRHLGKVQGYQWV DGVSLSFSHWNQGEPNDAWGRENCVMMLHTGLWNDAPCDSEKDGWICEKRHNC (SEQ ID NO: 13). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 13. In some embodiments, the second domainWSGR Docket No.61078-716.601 comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 13.

[0100] In some embodiments, the second domain comprises fibrinogen-like protein 1 domain or a LAG-3 binding fragment or variant thereof. In some embodiments, the second domain that binds to LAG3 comprises an amino acid sequence MAKVFSFILVTTALTMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQ FLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVI QRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNS RYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHD NYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVV MKIRPNDFIPNVI (SEQ ID NO: 14). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 14. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 14.

[0101] In some embodiments, the second domain may be a domain that binds to TIM3. In some embodiments, the second domain comprises galectin 9, phosphatidylserine (PtdSer), CEACAM1 or high mobility group protein B1 (HMGB1) domain or a TIM3 binding fragment or variant thereof. In some embodiments, the domain that binds to TIM3 may be a galectin 9, phosphatidylserine (PtdSer), CEACAM1 or high mobility group protein B1 (HMGB1) domain.

[0102] In some embodiments, the second domain comprises galectin 9 or a TIM3 binding fragment or variant thereof. In some embodiments, the second domain that binds to TIM3 comprises an amino acid sequence MAFSGCQAPYLSPAVPFSGTIQGGLQDGFQITVNGAVLSCSGTRFAVDFQTGFSGNDIAFHF NPRFEDGGYVVCNTRQKGTWGPEERKMHMPFQKGMPFDLCFLVQSSDFKVMVNGSLFVQ YFHRVPFHRVDTISVNGSVQLSYISFQNPRAVPVQPAFSTVPFSQPVCFPPRPRGRRQKPPSV RPANPAPITQTVIHTVQSASGQMFSQTPAIPPMMYPHPAYPMPFITTIPGGLYPSKSIILSGTVL PSAQRFHINLCSGSHIAFHMNPRFDENAVVRNTQINNSWGSEERSLPRKMPFVRGQSFSVWI LCEAHCLKVAVDGQHVFEYYHRLRNLPTINKLEVGGDIQLTHVQT (SEQ ID NO: 15). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 15. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 15.

[0103] In some embodiments, the second domain comprises phosphatidylserine (PtdSer) or a TIM3 binding fragment or variant thereof. In some embodiments, the second domain that binds to TIM3WSGR Docket No.61078-716.601 comprises an amino acid sequence MASCVGSRTLSKDDVNYKMHFRMINEQQVEDITIDFFYRPHTITLLSFTIVSLMYFAFTRDDS VPEDNIWRGILSVIFFFLIISVLAFPNGPFTRPHPALWRMVFGLSVLYFLFLVFLLFLNFEQVKS LMYWLDPNLRYATREADVMEYAVNCHVITWERIISHFDIFAFGHFWGWAMKALLIRSYGL CWTISITWELTELFFMHLLPNFAECWWDQVILDILLCNGGGIWLGMVVCRFLEMRTYHWAS FKDIHTTTGKIKRAVLQFTPASWTYVRWFDPKSSFQRVAGVYLFMIIWQLTELNTFFLKHIF VFQASHPLSWGRILFIGGITAPTVRQYYAYLTDTQCKRVGTQCWVFGVIGFLEAIVCIKFGQ DLFSKTQILYVVLWLLCVAFTTFLCLYGMIWYAEHYGHREKTYSECEDGTYSPEISWHHRK GTKGSEDSPPKHAGNNESHSSRRRNRHSKSKVTNGVGKK (SEQ ID NO: 16). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 16. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 16.

[0104] In some embodiments, the second domain comprises CEACAM1 or a TIM3 binding fragment or variant thereof. In some embodiments, the second domain that binds to TIM3 comprises an amino acid sequence MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQ LFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTL QVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQS LPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDT YYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCN RTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERM KLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENGLSPGAIAGIVI GVVALVALIAVALACFLHFGKTGRASDQRDLTEHKPSVSNHTQDHSNDPPNKMNEVTYSTL NFEAQQPTQPTSASPSLTATEIIYSEVKKQ (SEQ ID NO: 17). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 17. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 17.

[0105] In some embodiments, the second domain comprises high mobility group protein B1 (HMGB1) domain or a TIM3 binding fragment or variant thereof. In some embodiments, the second domain that binds to TIM3 comprises an amino acid sequence MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSI GDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKWSGR Docket No.61078-716.601 SKKKKEEEEDEEDEEDEEEEEDEEDEDEEEDDDDE (SEQ ID NO: 18). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 18. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 18.

[0106] In some embodiments, the second domain may be a domain that binds to TIGIT. In some embodiments, the second domain comprises CD155, CD112, or CD113 or a TIGIT binding fragment or variant thereof. In some embodiments, the domain that binds to TIGIT may be a CD155, CD112, or CD113 domain.

[0107] In some embodiments, the second domain comprises CD155 or a TIGIT binding fragment or variant thereof. In some embodiments, the second domain that binds to TIGIT comprises an amino acid sequence MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVT HVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEG NYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWH SDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVY YPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPV DKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRNAIIFLVLGILVFLILLGIGIYFYW SKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR (SEQ ID NO: 19). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 19. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 19.

[0108] In some embodiments, the second domain comprises CD112 or a TIGIT binding fragment or variant thereof. In some embodiments, the second domain that binds to TIGIT comprises an amino acid sequence MARAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPPVPG LYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDA TLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCI SKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFE EPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAV AQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGGIIGGIIAAIIA TAVAATGILICRQQRKEQTLQGAEEDEDLEGPPSYKPPTPKAKLEAQEMPSQLFTLGASEHS PLKTPYFDAGASCTEQEMPRYHELPTLEERSGPLHPGATSLGSPIPVPPGPPAVEDVSLDLED EEGEEEEEYLDKINPIYDALSYSSPSDSYQGKGFVMSRAMYV (SEQ ID NO: 20). In someWSGR Docket No.61078-716.601 embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 20. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 20.

[0109] In some embodiments, the second domain comprises CD113 or a TIGIT binding fragment or variant thereof. In some embodiments, the second domain that binds to TIGIT comprises an amino acid sequence MARTLRPSPLCPGGGKAQLSSASLLGAGLLLQPPTPPPLLLLLFPLLLFSRLCGALAGPIIVEP HVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKN YSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETV AAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIISQYKLFPTRFARGRRITCVVKHPA LEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPD GLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADI EDLATEPKKLPFPLSTLATIKDDTIATIIASVVGGALFIVLVSVLAGIFCYRRRRTFRGDYFAK NYIPPSDMQKESQIDVLQQDELDSYPDSVKKENKNPVNNLIRKDYLEEPEKTQWNNVENLN RFERPMDYYEDLKMGMKFVSDEHYDENEDDLVSHVDGSVISRREWYV (SEQ ID NO: 21). In some embodiments, the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 21. In some embodiments, the second domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 21.

[0110] In some embodiments, the second domain may be a domain that binds to a T-cell receptor.

[0111] In some embodiments, the second domain of the B2M fusion protein may be a domain that 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For example, the second domain of the B2M fusion protein may bind to or associate with M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, greater than or equal to about 10^5 M-1. For example, the second domain of the B2M fusion proteinWSGR Docket No.61078-716.601 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0112] KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM. For a KA of greater than or equal to about 10^5 M-1. For example, the second domain of the B2M fusion M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the second domain nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, 75µM, 100µM, 125µM, 140µM or 150µM or greater than 150µM. For example, the second domain with a KD of less than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0113] conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0114] In some aspects, the second domain may inhibit binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell. In some embodiments, the second domain may inhibit binding of the cell- surface receptor of the second cell by at least about 80% compared to a cell that does not express the second domain. In some embodiments, the second domain may inhibit binding of the cell-surface receptor of the second cell by at least about 90% compared to a cell that does not express the second domain. In some embodiments, the second domain may inhibit binding of the cell-surface receptor of the second cell by at least about 95% compared to a cell that does not express the second domain. In some embodiments, the second domain may inhibit binding of the cell-surface receptor of the second cell by at least about 97% compared to a cell that does not express the second domain. In some embodiments, the second domain may inhibit binding of the cell-surface receptor of the second cell by at least about 98% compared to a cell that does not express the second domain. In some embodiments,WSGR Docket No.61078-716.601 the second domain may inhibit binding of the cell-surface receptor of the second cell by at least about 99% compared to a cell that does not express the second domain. In some embodiments, the second domain may inhibit binding of the cell-surface receptor of the second cell by less than 80% compared to a cell that does not express the second domain. In some embodiments, the second domain that may inhibit binding of the cell-surface receptor may be a CD8 domain or an MHC-binding fragment or MHC-binding fragment or variant thereof. In some embodiments, the second cell may be a host cell. In some embodiments the first cell may be an allogeneic and / or grafted cell.

[0115] In some embodiments, the cell surface receptor of the second cell may be a CD8 receptor. In some embodiments, the cell-surface receptor is not TCRalpha / beta or TCR delta / gamma. In some embodiments the cell-surface receptor of the second cell is a cell-surface receptor that binds to an MHC class 1 molecule on a first cell.

[0116] In some aspects, the second domain may be expressed in an allogeneic and / or grafted T-cell. In some aspects, the second domain may inhibit killing of the grafted cells in which the second domain is expressed when bound to the MHC of the grafted cells via the first domain. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 5% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 10% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 15% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 20% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 30% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 40% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 50%, compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the grafted cells in which it is expressed by at least about 60% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the cell in which it is expressed by at least about 70% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domainWSGR Docket No.61078-716.601 may inhibit killing of the cell in which it is expressed by at least about 80% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the cell in which it is expressed by at least about 90% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the cell in which it is expressed by at least about 95% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the cell in which it is expressed by at least about 99% compared to grafted cells in which the second domain is not expressed. In some embodiments, the second domain may inhibit killing of the cell in which it is expressed by less than about 50% compared to grafted cells in which the second domain is not expressed.

[0117] In some aspects, the second domain may induce minimal host NK cell proliferation. In some embodiments, the graft cells expressing the second domain may elicit less than 0.1 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 0.2 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 0.5 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 0.8 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 1 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 1.2 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 1.3 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 1.5 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 1.8 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 2 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain may elicit less than 3 fold change in NK proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domainWSGR Docket No.61078-716.601 may elicit less than 4 fold change in NK proliferation compared to the graft cells not expressing the second domain.

[0118] In some aspects, the graft cells expressing the second domain may elicit reduced level of CD8+ T cell proliferation than the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.1 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.2 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.3 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.4 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.5 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.6 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.7 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.8 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 0.9 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain. In some embodiments, the graft cells expressing the second domain elicit less than 1 fold change in host CD8+ T cell proliferation compared to the graft cells not expressing the second domain.

[0119] In some embodiments, the second domain may be operatively linked to a first domain by a linker. In some embodiments, the second domain may be operatively linked to the first domain by a first linker, and operatively linked to a third domain by a second linker. In some embodiments, the second domain may be linked to a first domain, and / or a third domain, and / or a fourth domain by a third linker. In some embodiments, the second domain may be linked to three or more additional domains by additional linkers.

[0120] In some embodiments, the linker may include one or more intervening amino acid residues that are positioned between the second domain and first domain, and / or are positioned between the second domain and the third domain and / or are positioned between the first, second, or third domains and anyWSGR Docket No.61078-716.601 additional domains. In principle, there are no particular limitations to the length and / or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. Third Domain

[0121] In some aspects, the recombinant B2M fusion protein may comprise a third domain that binds to an immune checkpoint protein. In some aspects, the recombinant B2M fusion protein may comprise a third domain that inhibits an immune checkpoint protein. Immune checkpoint proteins are a family of inhibitory immunoreceptors that function to regulate the activation of immune cells, for example, T-cells. Activation of immune checkpoint receptors such as PD-1, CTLA-4, LAG3, TIM3, TIGIT, VISTA, and / or BTLA delivers inhibitory signals to the T-cell receptor. These inhibitory signals may result in reduced immune responses directed against, for example, allogeneic / grafted cells expressing ligands for immune checkpoint proteins.

[0122] In some aspects, the third domain of the recombinant B2M fusion protein may be a domain that is expressed in a first cell and binds to an extracellular receptor expressed on a second cell. In some embodiments, the third domain may be a domain that bind to an immune checkpoint protein. In some embodiments, the third domain may be a PDL1 or PDL2 domain. In some embodiments, the third domain may be a domain that binds to PD1 or PD2.

[0123] In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 7. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the third domain is a domain that binds to PDL1.

[0124] In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 8. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the third domain is a domain that binds to PDL2.

[0125] In some embodiments, the third domain may be a domain that binds to CTLA-4. In some embodiments, the domain that binds to CTLA-4 comprises an amino acid sequence of SEQ ID NO: 9.WSGR Docket No.61078-716.601 In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 9. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 9.

[0126] In some embodiments, the third domain may be a domain that binds to CTLA-4. In some embodiments, the domain that binds to CTLA-4 comprises an amino acid sequence to SEQ ID NO: 10. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 10. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 10.

[0127] In some embodiments, the third domain may be a domain that binds to LAG-3. In some fibrinogen-like protein 1 domain. In some embodiments, the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 11. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 11. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 11.

[0128] In some embodiments, the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 12. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 12. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 12.

[0129] In some embodiments, the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 13. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 13. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 13.

[0130] In some embodiments, the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 14. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 14. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 14.

[0131] In some embodiments, the third domain may be a domain that binds to TIM3. In some embodiments, the domain that binds to TIM3 may be a galectin 9, phosphatidylserine (PtdSer), CEACAM1 or high mobility group protein B1 (HMGB1) domain. In some embodiments, the thirdWSGR Docket No.61078-716.601 domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 15. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 15. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 15.

[0132] In some embodiments, the third domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 16. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 16. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 16.

[0133] In some embodiments, the third domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 17. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 17. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 17.

[0134] In some embodiments, the third domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 18. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 18. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 18.

[0135] In some embodiments, the third domain may be a domain that binds to TIGIT. In some embodiments, the domain that binds to TIGIT may be a CD155, CD112, or CD113 domain. In some embodiments, the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 19. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 19. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 19.

[0136] In some embodiments, the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 20. In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 20. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 20.

[0137] In some embodiments, the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 21. In some embodiments, the third domain comprises an amino acid sequence with atWSGR Docket No.61078-716.601 least about 80% sequence identity to SEQ ID NO: 21. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 21.

[0138] In some embodiments, the third domain may be a domain that binds to a T-cell receptor.

[0139] In some aspects, the third domain may be an intracellular signaling domain. In some domain may comprise a stalk domain. In some embodiments, the stalk domain comprises an amino acid sequence MAAPAVSGLSRQVRCFSTSVVRPFAKLVRPPVQVYGIEGRYATALYSAASKQNKLEQVEKE LLRVAQILKEPKVAASVLNPYVKRSIKVKSLNDITAKERFSPLTTNLINLLAENGRLSNTQGV VSAFSTMMSVHRGEVPCTVTSASPLEEATLSELKTVLKSFLSQGQVLKLEAKTDPSILGGMI VRIGEKYVDMSVKTKIQKLGRAMREIV(SEQ ID NO: 22). In some embodiments, the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 22. In some embodiments, the third domain comprises an amino acid sequence with at least about 85, 90, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 22.

[0140] In some embodiments, the third domain of the B2M fusion protein may be a domain that binds to an immune checkpoint protein. In some embodiments, the third domain of the B2M fusion protein binds to an immune checkpoint protein with low affinity. For example, the third domain of the B2M fusion protein can bind to an immune checkpoint protein with a KA of less than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, and / or 50µM. For example, the third domain of the B2M fusion protein may bind to or associate with an immune checkpoint protein with a KA of less than or equal to about 10^5 M-1. For example, the third domain of the B2M fusion protein may bind to an immune checkpoint protein with a KA of less than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the third domain of the B2M fusion protein can bind to an immune checkpoint protein with a KD of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, or greater than 50µM. For example, the third domain of the B2M fusion protein may bind to or associate with an immune checkpoint protein with a KD of greater than or equal to about 10^5 M-1. For example, the third domain of the B2M fusion protein may bind to an immune checkpoint protein with a KD of greater than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M- 1, or 10^13 M-1.WSGR Docket No.61078-716.601

[0141] In some embodiments, the third domain of the B2M fusion protein binds to an immune checkpoint protein with a high affinity. For example, the third domain of the B2M fusion protein can bind to an immune checkpoint protein with a KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, and / or 50µM. For example, the third domain of the B2M fusion protein may bind to or associate with an immune checkpoint protein with a KA of greater than or equal to about 10^5 M-1. For example, the third domain of the B2M fusion protein may bind to an immune checkpoint protein with a KA of greater than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1. For example, the third domain of the B2M fusion protein can bind to an immune checkpoint protein with a KD of less than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1µM, 5 µM, 10 µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM, 50µM, or greater than 50µM. For example, the third domain of the B2M fusion protein may bind to or associate with an immune checkpoint protein with a KD of less than or equal to about 10^5 M-1. For example, the third domain of the B2M fusion protein may bind to an immune checkpoint protein with a KD of less than or equal to about 10^6 M-1, 10^7 M-1, 10^8 M-1, 10^9 M-1, 10^10 M-1, 10^11 M-1, 10^12 M-1, or 10^13 M-1.

[0142] The binding affinity of the third domain to an immune checkpoint protein can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0143] In some aspects, the third domain may be expressed in an allogeneic and / or grafted T-cell. In some aspects, the third domain may inhibit killing of the grafted cells in which the third domain is expressed when bound to the MHC of the grafted cells via the first domain. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 5% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 10% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 15% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 20% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 30% compared to grafted cells inWSGR Docket No.61078-716.601 which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 40% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 50%, compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 60% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by at least about 70% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by at least about 80% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by at least about 90% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by at least about 95% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by at least about 99% compared to grafted cells in which the third domain is not expressed. In some embodiments, the third domain may inhibit killing of the cell in which it is expressed by less than about 50% compared to grafted cells in which the third domain is not expressed.

[0144] In some embodiments, the third domain may be operatively linked to a first domain by a linker. In some embodiments, the third domain may be operatively linked to the first domain by a first linker, and operatively linked to a second domain by a second linker. In some embodiments, the third domain may be linked to a first domain, and / or a second domain, and / or a fourth domain by a third linker. In some embodiments, the third domain may be linked to three or more additional domains by additional linkers.

[0145] In some embodiments, the linker may include one or more intervening amino acid residues that are positioned between the second domain and first domain, and / or are positioned between the second domain and the third domain, and / or are positioned between the first, second, or third domains and any additional domains. In principle, there are no particular limitations to the length and / or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments,WSGR Docket No.61078-716.601 the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. Additional Domain

[0146] In some aspects the recombinant B2M fusion protein may comprise the first domain of the disclosure, the second domain of the disclosure, the third domain of the disclosure, and an additional domain. In some embodiments, the additional domain comprises a domain that binds to a T-cell receptor. In some embodiments, the additional domain is an intracellular signaling domain. In some Cross-Linking B2M

[0147] In some aspects, a recombinant polypeptide or recombinant nucleic acid encoding the recombinant polypeptide may further comprise a first domain of a first recombinant fusion protein cross-linked to a first domain of a second recombinant fusion protein. In some embodiments, the first domain of a first recombinant fusion protein is crosslinked to a first domain of a second recombinant fusion protein via a leucine zipper. In some embodiments, the cross-linking ties MHC / B2M complexes together irrespective to the peptide presented by the complex. In some embodiments, the cross-linking inhibits or prevents cognate TCRs from clustering MHCI / B2M complexes presenting a same peptide into a functional cell-to-cell synapse. In some embodiments, the first domain may be B2M or an MHC- binding fragment or variant thereof. In some embodiments, the first domain of a first recombinant fusion protein comprises B2M or an MHC-binding fragment or variant thereof and is cross-linked to a second recombinant fusion protein that comprises B2M or an MHC-binding fragment or variant thereof. In some embodiments, the first domain of a plurality of recombinant fusion proteins may be cross-linked. In some embodiments, the recombinant fusion protein may be a recombinant fusion protein of the disclosure described herein, for example, comprising a first domain, a second domain, and / or a third domain. In some embodiments, the first domain may be cross-linked to a cell membrane. In some embodiments, the cell membrane may be the cell membrane of the cell in which the first domain is expressed. In some embodiments, the second domain may be cross-linked to a cell membrane. In some embodiments, the cell membrane may be the cell membrane of the cell in which the first domain is expressed. In some embodiments, the third domain may be cross-linked to a cell membrane. In some embodiments, the cell membrane may be the cell membrane of the cell in which the first domain is expressed. In some embodiments, the additional domain may be cross-linked to a cell membrane. In some embodiments, the cell membrane may be the cell membrane of the cell in which the first domain is expressed.WSGR Docket No.61078-716.601

[0148] In some aspects, cross-linking the first domain may comprise cytoskeletal anchoring modifications. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain. In some embodiments, the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.

[0149] In some aspects, cross-linking the second domain may comprise cytoskeletal anchoring modifications. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain. In some embodiments, the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.

[0150] In some aspects, cross-linking the third domain may comprise cytoskeletal anchoring modifications. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain. In some embodiments, the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.

[0151] In some aspects, cross-linking the additional domain may comprise cytoskeletal anchoring modifications. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin. In some embodiments, the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain. In some embodiments, the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.

[0152] In some aspects, a cross-linker may include any type of reagent, molecule, or process that can induce a strong chemical bond between two members that are to be bound. For example, a cross linker can create / form a strong chemical bond (for example, a covalent bond) between biopolymer chains, such as nucleic acids and proteins.

[0153] In some aspects, a cross-linker may include reactive groups to specific protein functional groups, such as, for example, primary amines, sulfhydryls, carboxyl, carbonyl, photoreactive groups, chemoselective groups, arginine specific, and / or bioorthagonal groups.

[0154] In some aspects, cross-linking molecules may include, p-Azidobenzoyl hydrazide, 3-([2-WSGR Docket No.61078-716.601 nitrobenzyloxy-succinimide, N-(4-[p-Azidosalicylamido]butyl)-3'-(2'-pyridyldithio) propionamide, p- azidosalicylamido]ethyl) disulfide, 1,4-Bis-Maleimidobutane, 1,4-Bis-Maleimidyl-2,3- Maleimidotriethylene-glycol, 1,11-Bis-Maleimidotetraethyleneglycol, Bis(2- [succinimidoxycarbonyloxy]ethyl)sulfone, Bis(sulfosuccinimidyl)glutarate-d0, Bis(sulfosuccinimidyl)2,2,4,4-glutarate-d4, Bis(sulfosuccinimidyl)suberate, Bis(sulfosuccinimidyl)suberate-d0, Bis(sulfosuccinimidyl)2,2,7,7-suberate-d4, C6-Succinimidyl 4- hydrazinonicotinate acetone hydrazone, C6-Succinimidyl 4-formylbenzoate, N,N- Dicyclohexylcarbodiimide, 1-5-Difluoro-2,4-dinitrobenzene, Dimethyl adipimidate•2HCI, Dimethyl pimelimidate•2HCI, Dimethyl suberimidate•2HCl, 1,4-Di-(3'-[2'pyridyldithio]propionamido) butane, Disuccinimidyl glutarate, Dithiobis(succimidylpropionate) (Lomant’s Reagent), Disuccinimidyl suberate, Disuccinimidyl tartarate, Dimethyl 3,3'-dithiobispropionimidate•2HC, Dithiobis- maleimidoethane, 3,3'-Dithiobis(sulfosuccinimidylpropionate), 1-Ethyl-3-(3-dimethylaminopropyl) 1-carboxy-(6-amidocaproate), Succinimidyl 6-(3'-[2-pyridyl-dithio]propionamido) hexanoate, m- Maleimidobenzoyl-N-hydroxysuccinimide ester, 4-(4-N-Maleimidophenyl)- butyric acid hydrazide•HCI, Methyl N-succinimidyl adipate, 2-[N 2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N 6-(6- biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate, 2-{N 2-[N6-(4-Azido-2,3,5,6- tetrafluorobenzoyl)-N 6-(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate, N- Hydroxysuccinimidyl-4-azidosalicylic acid, 3-(2-Pyridyldithio)propionylhydrazide, N-(p- Maleimidophenyl)isocyanate, N-Succinimidyl (4'-azidophenyl)1, 3'-dithiopropionate, Sulfosuccimidyl 2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-1,3'-dithiopropionate, Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido) ethyl 1,3'-dithiopropionate, Succinimidyl 4- hydrazinonicotinate acetone hydrazone, N-Succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, Sulfosuccinimidyl 2-(p-azido- salicylamido)ethyl 1,3'-dithiopropionate, N-Succinimidyl S- acetylthioacetate, N-Succinimidyl S-acetylthiopropionate, Succinimdyl 3- (bromoacetamido)propionate, Sulfosuccinimidyl(perfluoroazidobenzamido) ethyl 1,3'- dithiopropionate, Succinimidyl 4-formylbenzoate, Succinimidyl 4-hydrazidoterephthalateWSGR Docket No.61078-716.601 hydrochloride, N-succinimidyl iodoacetate, N-Succinimidyl(4-iodoacetyl)aminobenzoate, Succinimidyl 4-(N-maleimido- methyl)cyclohexane-1-carboxylate, Succinimidyl 4-(p-maleimido- phenyl)butyrate, Succinimidyloxycarbonyl- yloxy)butyrate, N-Succinimidyl 3-(2-pyridyldithio)propionate, Sulfodisuccinimidyl tartarate, [2-pyridyldithio]-toluamido)hexanoate, Sulfosuccinimidyl 6-(3'-[2-pyridyl- dithio]propionamido)hexanoate, m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Sulfosuccinimidyl(4-azido-salicylamido)hexanoate, Sulfosuccinimidyl(4-azido- phenyldithio)propionate, Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate, Sulfosuccinimidyl 4-(N-maleimido- methyl)cyclohexane-1-carboxylate, Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, Sulfo-NHS- (2-6-[Biotinamido]-2-(p-azidobezamido) hexanoamido)ethyl-1,3'-dithiopropionate (Trifunctional), (betaine), Tris-(2-Maleimidoethyl)amine (Trifunctional), or Tris-(succimimidyl aminotricetate) (Trifunctional).

[0155] In some aspects, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 10% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 20% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 30% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 40% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 50% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 60% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 70% compared to aWSGR Docket No.61078-716.601 first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 80% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 90% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 95% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 99% compared to a first domain that is not cross-linked. In some embodiments, the cross-linking of the first domain of the recombinant B2M fusion protein may prevent pMHC clustering by at least about 100% compared to a first domain that is not cross-linked.

[0156] In some aspects, the cross-linked first domain may be expressed in an allogeneic and / or grafted T-cell. In some aspects, the cross-linked first domain may inhibit killing of the grafted cells in which the cross-linked first domain is expressed when bound to the MHC of the grafted cells via the cross- linked first domain. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 5%, compared to grafted cells in which the cross- linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 10%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 15%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 20%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 30%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 40%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 50%, compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 60% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross- linked first domain may inhibit killing of the grafted cells in which it is expressed by at least aboutWSGR Docket No.61078-716.601 70% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 80% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 90% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 95% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by at least about 99% compared to grafted cells in which the cross-linked first domain is not expressed. In some embodiments, the cross-linked first domain may inhibit killing of the grafted cells in which it is expressed by less than about 50% compared to grafted cells in which the cross-linked first domain is not expressed. Linker Domain

[0157] In some aspects, a recombinant B2M fusion protein may comprise a linker domain. In some embodiments, a recombinant B2M fusion protein may comprise a plurality of linker domains.

[0158] In some aspects, the first domain may be modified N-terminally or C-terminally. In some embodiments, the first domain may be operatively linked to a second domain by a linker. In some embodiments, the first domain may be operatively linked to a second domain by a first linker, and operatively linked to a third domain by a second linker. In some embodiments, the first domain may be linked to a second domain, and / or a third domain, and / or a fourth domain by a third linker. In some embodiments, the first domain may be linked to three or more additional domains by additional linkers.

[0159] In some embodiments, the second domain may be operatively linked to a first domain by a linker. In some embodiments, the second domain may be operatively linked to the first domain by a first linker, and operatively linked to a third domain by a second linker. In some embodiments, the second domain may be linked to a first domain, and / or a third domain, and / or a fourth domain by a third linker. In some embodiments, the second domain may be linked to three or more additional domains by additional linkers.

[0160] In some embodiments, the third domain may be operatively linked to a first domain by a linker. In some embodiments, the third domain may be operatively linked to the first domain by a first linker, and operatively linked to a second domain by a second linker. In some embodiments, the third domain may be linked to a first domain, and / or a second domain, and / or a fourth domain by a third linker. In some embodiments, the third domain may be linked to three or more additional domains by additional linkers.WSGR Docket No.61078-716.601

[0161] In some embodiments, the additional domain may be operatively linked to a first domain by a linker. In some embodiments, the additional domain may be operatively linked to the first domain by a first linker, and operatively linked to a second domain by a second linker. In some embodiments, the additional domain may be linked to a first domain, and / or a second domain, and / or a third domain by a third linker. In some embodiments, the additional domain may be linked to three or more additional domains by additional linkers.

[0162] In some embodiments, the linker may include one or more intervening amino acid residues that are positioned between the second domain and first domain, and / or are positioned between the second domain and the third domain, and / or are positioned between the first, second, or third domains and any additional domains. In principle, there are no particular limitations to the length and / or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues.

[0163] Recombinant Fusion Protein Constructs

[0164] In some aspects, a recombinant B2m fusion construct comprises the amino acid or nucleic acid sequence of any one of the constructs, proteins, and / or nucleic acids disclosed in any one of Tables 3A, 3B, 4, 5, 8, and any combination thereof. In some embodiments, a recombinant B2m fusion construct comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid or nucleic acid sequence of any one of the constructs, proteins, and / or nucleic acids disclosed in any one of Tables 3A, 3B, 4, 5, 8, and any combination thereof. Chimeric Antigen Receptors (CARs)

[0165] In some aspects, a recombinant polypeptide or recombinant nucleic acid encoding the recombinant polypeptide further comprises a sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain. In some aspects, a cell expressing a B2M fusion protein further comprises the CAR. In some aspects, a recombinant nucleic acid encoding a recombinant polypeptide comprising a B2M fusion protein, and a recombinant nucleic acid encoding a recombinant polypeptide encoding a CAR may both be delivered to a cell. In some aspects, a recombinant nucleic acid encoding a recombinant polypeptide comprising a B2MWSGR Docket No.61078-716.601 fusion protein, and a recombinant nucleic acid encoding a recombinant polypeptide encoding a CAR may both be delivered to a cell and expressed by the cell. CAR Extracellular Domain

[0166] The extracellular domain of the CAR comprises an antigen binding domain. The antigen binding domain can be any domain that specifically binds to an antigen. In some embodiments, the antigen is an antigen expressed by a tumor cell. In some embodiments, the antigen binding domain comprises a scFv, a nanobody, a ligand, or a receptor.

[0167] The antigen binding domain can be any molecule that binds to the selected antigen with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. Alternatively, the antigen binding domain can be a receptor or a receptor fragment that binds specifically to the target antigen. The antigen binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners). Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See for example, D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L. Smith et al, Mol Ther (2018)26(6): 1447-56 (scFv); S.R. Banihashemi et al., Iran J Basic Med Sci (2018) 21(5):455-64 (CD19 nanobody); F. Rahbarizadeh et al Adv Drug Deliv Rev (2019) 141:41-46 (sdAb);S.M. Kipriyanov et al., Int J Cancer (1998) 77(5):763-72 (diabody); F. Le Gall et al., FEBS Lett (1999) 453(1-2): 164-68 (triabody); M.A. Ghetie et al., Blood (1994) 83(5): 1329-36 (F(ab')2); and M.A. Ghetie et al., Clin Cancer Res (1999) 5(12):3920-27 (F(ab')2 and Fab'). Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody. Antibody derivatives can also be designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0168] In some embodiments, the antigen binding domain specifically binds to CD19. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity toWSGR Docket No.61078-716.601 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0169] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0170] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.

[0171] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9%WSGR Docket No.61078-716.601 identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 32. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 32.

[0172] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an aminoWSGR Docket No.61078-716.601 acid sequence with at least 96% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 33. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 33.

[0173] In some embodiments, the antigen binding domain specifically binds to CD22. In some embodiments, the antigen binding domain is an anti-CD22 binding domain.

[0174] In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFS GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEI.

[0175] In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SS.

[0176] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK.

[0177] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSWSGR Docket No.61078-716.601 GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKGGGGSQVQLQQSGPGLVK PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINP DTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS. In some embodiments, the antigen binding domain comprises an scFv with at least about 85, 90, 95, 97, 98, or 99% sequence identity to QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK. In some embodiments, the antigen binding domain comprises an scFv with 100% sequence identity QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK.

[0178] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an aminoWSGR Docket No.61078-716.601 acid sequence with at least 96% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 34. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 34.

[0179] In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN- CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7- H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY- CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2, or a mixture thereof.

[0180] In some embodiments, the antigen binding domain of a CAR provided herein is operatively linked to a transmembrane domain by a hinge domain. In some embodiments, the antigen binding domain of a CAR provided herein is directly linked to a transmembrane domain by a hinge domain. In some embodiments, the hinge domain of a CAR provided herein is from CD28. In some embodiments, the hinge domain of a CAR provided herein has the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In some embodiments, the hinge domain of a CAR or provided herein is from CD8. In some embodiments, the hinge domain of a CAR provided herein has the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY.WSGR Docket No.61078-716.601

[0181] In certain embodiments, a hinge or spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and / or CH3 of IgG 1, lgG4, or IgD. Illustrative spacer domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD28, which may be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain includes a CD8a or CD28 hinge region. In some embodiments, the hinge is a PD-1 hinge or CD152 hinge.

[0182] In some embodiments, the CAR further includes an extracellular spacer domain, which may include a hinge domain. The hinge domain is generally a flexible polypeptide connector region disposed between the targeting moiety and the transmembrane domain. Exemplary hinge domain sequences include those from IgG subclasses (such as IgGl and IgG4), IgD, CD28, and CD8 domains. In some embodiments, the hinge domain provides structural flexibility to flanking polypeptide regions. The hinge domain may consist of natural or synthetic polypeptides. It will be appreciated by those skilled in the art that hinge domains may improve the function of the CAR by promoting optimal positioning of the antigen binding domain in relationship to the portion of the antigen recognized by it. In some embodiments, a hinge domain may not be required for optimal CAR activity. In some embodiments, a hinge domain comprising a short sequence of amino acids promotes CAR activity by facilitating antigen-binding by, for example, relieving steric constraints that could otherwise alter antibody binding kinetics. In some embodiments, the hinge domain is linked downstream of the antigen-binding domain of a CAR and upstream of the transmembrane domain of a CAR.

[0183] Non-limiting examples of suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4. In some embodiments, the hinge domain can include regions derived from a human CD8a (also known as CD8a) molecule, a CD28 molecule, and any other receptors that provide a similar function in providing flexibility to flanking regions. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain. In some embodiments, the hinge domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4 hinge domain.

[0184] In some embodiments, the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and theWSGR Docket No.61078-716.601 extracellular hinge domain. In some embodiments, the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain. In principle, there are no particular limitations to the length and / or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and / or proximity of the antigen binding domain and the extracellular hinge domain to one another to achieve a desired activity of the CAR. In some embodiments, the orientation and / or proximity of the antigen binding domain and the extracellular hinge domain to one another can be varied and / or optimized as a “tuning” tool or effect to enhance or reduce the efficacy of the CAR. In some embodiments, the orientation and / or proximity of the antigen binding domain and the hinge domain to one another can be varied and / or optimized to create a partially functional version of the CAR. In some embodiments, the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.

[0185] Alternatively, the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGSn”) sequence, or a variation thereof such as (SGG)n, (GGGS)n, (SGGG)n, (GSGGG)n, and the like, where n can range from about 1 to about 15. The synthetic polypeptide spacer domain can also include a naturally occurring sequence, such as a hinge domain derived from CD8a, IgG, and the like. CAR Transmembrane Domain

[0186] The extracellular domain of the CAR is operably connected to the transmembrane domain. In some embodiments, the extracellular domain is connected to the transmembrane domain by a spacer. The transmembrane domain of the CAR serves to transduce the external signal received by the extracellular domain to the intracellular domain. The transmembrane domain can be any proper CD28 transmembrane domain, CD8 transmembrane domain, CD8H transmembrane domain, and transmembrane and immunoglobulin domain containing 2 protein (CD28H). The transmembrane domain can be selected from a transmembrane region of a transmembrane protein such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof.WSGR Docket No.61078-716.601 Examples of the transmembrane domain include the transmembrane regions of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0187] In some embodiments, the CAR comprises a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8a, CD28, CD154, and PD-1; and one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: 4-1BB, CD28, CD134, and CD137; and an intracellular signaling domain from a polypeptide CD79a, CD79, and CD665. Such a CAR may further include a spacer domain between the antigen- binding portion and the transmembrane domain, e.g., a CD8a hinge. In some embodiments, the CAR comprises a transmembrane domain from CD28. In some embodiments, the CAR comprises a transmembrane domain with the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV. In some embodiments, the CAR comprises a transmembrane domain from CD8. In some embodiments, the CAR comprises a transmembrane domain with the sequence IWAPLAGTCGVLLLSLVITLYC.

[0188] The transmembrane domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the TM domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and / or PD-1.

[0189] The transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3zeta chain), CD28, CD2, CD4, OX40, 4-1BB (CD137), ICOS (CD278), ILRB (CD122), IL-2RG (CD132), CTLA-4, PD-1, or CD40, or a sequence derived from such a transmembrane domain. The cytoplasmic signaling domain in general comprises a domain that transduces the event of ligand binding into an intracellular signal that activates the T cell. The CD3z intracellular domain / activating domain is frequently used, although others such as MyD88 can be used. In an embodiment, the transmembrane domain is the transmembrane domain from CD3eta, CD2, CD8, or CD28. In an embodiment, the transmembrane domain is derived from the transmembrane domain from CD2 or CD28. In some embodiments, the transmembrane domain has about 70, 75, 80, 85, 90,WSGR Docket No.61078-716.601 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD3zeta, CD28, CD2, CD4, OX40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.

[0190] According to some embodiments, a CAR includes a transmembrane domain derived from CD8a or CD28 and a short polypeptide linker, e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the transmembrane domain and the intracellular signaling domain of the CAR or anti-CD2 fusion protein. A glycine-serine linker may be employed as such a linker, for example. CAR Intracellular Domain

[0191] The transmembrane domain of the CAR is operably connected to the intracellular domain. The intracellular domain serves to transduce the received external signal to kick-start the downstream signaling cascade. The intracellular domain comprises an intracellular signaling domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from CD2. In some embodiments, the intracellular domain comprises a truncated CD2 intracellular domain. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRP PPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN. In some embodiments, the intracellular signaling domain consists of an amino acid sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRP PPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN. In some TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and / or ZAP70.

[0192] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB (CD137). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0193] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. In someWSGR Docket No.61078-716.601 embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0194] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).

[0195] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPAT. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH.

[0196] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI. In some embodiments, the CAR comprises an intracellular domain comprising a truncated CD3epsilon intracellular domain.

[0197] Signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of the T cell and a secondary or costimulatory signal may also be required. Thus, T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR / CD3 complex) and costimulatory signaling domains that act in an antigen- independent manner to provide a secondary or costimulatory signal. As such, the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.

[0198] Primary signaling domains can regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable CD22, CD79a, CD79b, and CD665. In certain embodiments, a CAR includes a CD3zeta primary signaling domain and one or more costimulatory signaling domains. In certain embodiments, a CAR includes a 4-1BB costimulatory signaling domain. The intracellular primary signaling andWSGR Docket No.61078-716.601 costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain. In certain embodiments, a CAR lacks a CD2 intracellular signaling domain.

[0199] In some embodiments, the CAR includes one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR. Exemplary costimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and / or ZAP70. In some embodiments, the costimulatory signaling domain has at least about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a costimulatory signaling domain from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and / or ZAP70 domain. In some embodiments, a CAR includes one or more costimulatory signaling domains selected from the group consisting of CD2, 4-1BB, CD28, CD137, and CD134, and a CD3zeta primary signaling domain. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7- H3, and a ligand that specifically binds with CD83, and the like. In certain embodiments, the CAR comprises two or more intracellular signaling domains. For example, the CAR may comprise a first signaling domain and a second signaling domain or fragments thereof independently selected from a CD3zeta intracellular signaling domain, a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX-40 intracellular signaling domain, an inducible co-stimulator (ICOS) intracellular signaling domain, a CD27 intracellular signaling domain, and a MyD88 / CD40 intracellular signaling domain. By way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a CD28 intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a 4-1BB intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain, a second intracellular signaling domain or fragment thereof that is a 4-1BB intracellularWSGR Docket No.61078-716.601 signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.

[0200] TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and / or ZAP70 cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain has CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and / or ZAP70 cytoplasmic signaling domain. CARs of the disclosure may comprise a CD2 co-stimulatory domain, and one or more additional co-stimulatory domains to increase cytokine production or sensitivity, reduce or prevent anergy, and / or to increase proliferation and cytotoxic activity. These additional co-stimulatory domains can be derived from co- stimulatory proteins such as B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, OX40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4b1, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and / or HLA- DR. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, OX40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB- A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4b1, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and / or HLA-DR domains.

[0201] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and anWSGR Docket No.61078-716.601 intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0202] In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 35. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 36. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 37. In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 35. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 36. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 37. CAR Localization Tag

[0203] In some embodiments, the CAR further comprises a protein localization tag. The protein localization tag can be operably linked to the intracellular domain of the CAR. The protein localization tag can be operably linked to the extracellular domain of the CAR. The protein localization tag can be an ER localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasmaWSGR Docket No.61078-716.601 membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, or a nuclear localization tag. In some embodiments, the protein localization tag is an ER localization tag. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 23. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 24. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 25. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 26. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 27. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 28. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 29. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 30. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 23. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 23. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 24. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 24. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 25. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 25. In some embodiments, the ER localization tagWSGR Docket No.61078-716.601 comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 25. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 25. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 26. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 26. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 27. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 27. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 28. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 28. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 29. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 29. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 30. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 30.

[0204] In some embodiments, the ER localization tag comprises an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 40). In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 40. In someWSGR Docket No.61078-716.601 embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 40. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 40. In some embodiments, the ER localization tag comprises the amino acid sequence KKMP (SEQ ID NO: 41). In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 41.

[0205] In some embodiments, the protein localization tag is a Golgi localization tag. In some embodiments, the Golgi localization tag comprises the amino acid sequence YQRL (SEQ ID NO: 38). In some embodiments, the Golgi localization tag consists of the amino acid sequence YQRL (SEQ ID NO: 38). In some embodiments, the protein localization tag is a lysosome localization tag. In some embodiments, the lysosome localization tag comprises the amino acid sequence KFERQ (SEQ ID NO: 39). In some embodiments, the lysosome localization tag consists of the amino acid sequence KFERQ (SEQ ID NO: 39).

[0206] In some embodiments, a protease cleavage site is disposed between the protein localization tag and the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the intracellular domain of the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the extracellular domain of the CAR. Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and / or amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional amino acids can be present at the N-terminus and / or C-terminus of the cleavage site. A protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized / cleaved by the protease.

[0207] Various protease cleavage sites include, but are not limited to protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and / or the aspartic acid protease family, and / or the glutamic acid protease family. In some embodiments, serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and / or subtilisin-like proteases, and / or alpha / beta hydrolases, and / or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some embodiments, the protease cleavage site is TEV protease cleavage site. Disruption of GenesWSGR Docket No.61078-716.601

[0208] In some aspects, the recombinant cells of the disclosure comprising recombinant polypeptides and / or recombinant nucleic acids encoding the recombinant polypeptides may further comprise a disruption of a gene. In some embodiments, the gene may be a gene that encodes a T-cell receptor. In some embodiments, the gene may be a gene that encodes an MHC. In some embodiments, the gene may be a gene that encodes an extracellular domain of a T-cell receptor or MHC. In some embodiments, the gene may be a gene that encodes an intracellular receptor of a T-cell receptor or MHC. In some embodiments, the gene may be selected from the group consisting of TCRalpha, immunoevasin, ICAM1, CD80, CD58, OX40L, SUGT1, TAP1, TAP2, TAPBP, HLA-A, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD74, US11, K3, ICP47, and any combination thereof. In some embodiments, the disruption of the gene may include administering a gene editing platform to a recombinant cell of the disclosure. In some embodiments, the disruption of the gene may include knock-down (KD), knock-out (KO), and / or knock-in and or overexpression of the gene. In some embodiments, disruption of the gene may comprise a modification of the gene, such as, for example, insertion, deletion, substitution, inversion, duplication, translocation, and / or frameshift or the like.

[0209] In some aspects, the gene editing technology may include, for example, CRISPR / Cas9 systems, TALENS, and / or Zinc finger Nucleases (ZFNs), meganucleases, type IIS restriction endonucleases (Fokl and Fokl fusions) and the like. In some embodiments, gene editing technology may include RNA interference (RNAi), including small-interfering RNAs (siRNAs). In some embodiments, gene editing technologies may include microRNAs (miRNAs). Gene editing enables the possibility of permanently modifying a genomic sequence of interest by enabling targeted disruption, insertion, excision, and correction in both ex vivo and in vivo settings. Exemplary gene editing systems and methods for modulating gene expression are described in detailed herein. CRISPR / cas Gene Editing Systems

[0210] “CRISPR” or “CRISPR / Cas” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas”, as used herein, refers to a CRISPR-associated protein. A “CRISPR / Cas system” refers to a system derived from CRISPR and Cas which can be used to silence or modify a target gene.

[0211] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.WSGR Docket No.61078-716.601

[0212] The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in gene editing applications in eukaryotic cells, the spacers are derived from the eukaryotic target gene sequence.

[0213] RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341: 833-836.

[0214] As these naturally occur in many different types of bacteria, the exact arrangements of the CRISPR and structure, function and number of Cas genes and their product differ somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol.1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J. Mol. Evol.60: 174-182; Bolotin et al. (2005) Microbiol.151: 2551-2561; Pourcel et al. (2005) Microbiol.151: 653-663; and Stern et al. (2010) Trends. Genet.28: 335-340. For example, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. Brouns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR- based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341: 833-836. In some aspects, the Cas9 is derived from a S. pyogenes Cas9.

[0215] The CRISPR / Cas systems can thus be used to edit a target gene (adding, replacing or deleting one or more base pairs), or introducing a premature stop which thus decreases expression of a target gene. The CRISPR / Cas system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a target promoter, sterically blocking RNA polymerases. TALEN Gene Editing System

[0216] “TALEN” refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene.WSGR Docket No.61078-716.601

[0217] TALENs are produced artificially by fusing a TAL effector (“TALE”) DNA binding domain, e.g., one or more TALEs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TALEs to a DNA-modifying domain, e.g., a FokI nuclease domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence. Zhang (2011), Nature Biotech. 29: 149-153. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech.29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501. TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence. Zhang (2011), Nature Biotech.29: 149-153.

[0218] To produce a TALEN, a TALE protein is fused to a nuclease (N), e.g., a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech.29: 143-8; Hockemeyer et al. (2011) Nature Biotech.29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech.25: 786-793; and Guo et al. (2010) J. Mol. Biol.200: 96.

[0219] The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech.29: 143-8.

[0220] TALEN can be used inside a cell to produce a double-stranded break (DSB) in a target nucleic acid, e.g., a site within a gene. A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16. For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene. Miller, J. C., (2011) Nat. Biotechnol.29, 143-148 and Hockemeyer, D. (2011) Nat. Biotechnol.29, 731-734. Zinc Finger Nuclease Gene Editing SystemWSGR Docket No.61078-716.601

[0221] “ZFN” or “Zinc Finger Nuclease” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene.

[0222] Like a TALEN, a ZFN comprises a DNA-modifying domain, e.g., a nuclease domain, e.g., a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0223] A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. Sera (2002), Biochemistry, 41:7074-7081; Liu (2008) Bioinformatics, 24:1850-1857.

[0224] A ZFN using a FokI nuclease domain or other dimeric nuclease domain functions as a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5.

[0225] Also like a TALEN, a ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the ZFN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene, e.g., as described in WO2013 / 169802. Meganuclease Gene Editing System

[0226] “Meganuclease” refers to a meganuclease, an artificial nuclease which can be used to edit a target gene.

[0227] Meganucleases are derived from a group of nucleases which recognize 15-40 base-pair cleavage sites. Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and / or DNA recognition.WSGR Docket No.61078-716.601

[0228] Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art. E.g., Chevalier et al. (2002), Mol. Cell., 10:895-905; Epinat et al. (2003) Nucleic Acids Res 31: 2952-62; Silva et al. (2006) J Mol Biol 361: 744-54; Seligman et al. (2002) Nucleic Acids Res 30: 3870-9; Sussman et al. (2004) J Mol Biol 342: 31-41; Rosen et al. (2006) Nucleic Acids Res; Doyon et al. (2006) J Am Chem Soc 128: 2477-84; Chen et al. (2009) Protein Eng Des Sel 22: 249-56; Arnould S (2006) J Mol Biol.355: 443-58; Smith (2006) Nucleic Acids Res.363(2): 283-94.

[0229] A meganuclease can create a double-stranded break in the DNA, which can create a frame- shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the Meganuclease; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene, e.g., as described in Silva et al. (2011) Current Gene Therapy 11:11-27. Targeting RNA

[0230] In some aspects, the gene editing systems of the disclosure may include a targeting RNA. The targeting RNA is any ribonucleotide sequence having sufficient complementarity with a target polynucleotide sequence, e.g., a target DNA sequence, to hybridize with the target sequence. In some aspects, the targeting RNA is capable of directing sequence-specific cleavage of DNA at or adjacent to the target DNA sequence by a polypeptide comprising a cleavage domain. The targeting RNA is typically about 20 nucleotides. In some embodiments, a targeting RNA is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a targeting RNA is fewer than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. In some embodiments, the targeting RNA comprises a sequence of 10 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 11 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 12 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 13 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 14 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 15 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 16 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 17 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 18 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 19WSGR Docket No.61078-716.601 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 20 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 21 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 22 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 23 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 24 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 25 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 26 nucleic acids.

[0231] In some embodiments, the degree of complementarity between a targeting RNA and its corresponding target DNA sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non- limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a targeting RNA to direct by the polypeptide comprising a cleavage domain at or adjacent to the target sequence may be assessed by any suitable assay. For example, the components of an gene editing system as described herein, including the targeting RNA to be tested, may be provided to a host cell having the complimentary target DNA sequence, such as by transfection with vectors encoding the components of gene editing system, followed by an assessment of preferential cleavage within the target DNA sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target DNA sequence may be evaluated in a test tube by providing the target DNA sequence, components of a gene editing system, including the targeting RNA to be tested, and a control targeting RNA different from the test targeting RNA, and comparing binding or rate of cleavage at the target DNA sequence between the test and control targeting RNA reactions. Other assays are possible, and will occur to those skilled in the art.

[0232] A targeting RNA may be selected to target any target DNA sequence. In some embodiments, the target DNA sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, a unique target sequence in a genome may include a sequence NNNNNNNNNNNNNNN, where N is A, G, T, or C, and has a single occurrence in the genome.

[0233] In some embodiments, a targeting RNA is selected to reduce the degree of secondary structure within the targeting RNA. Secondary structure may be determined by any suitable polynucleotideWSGR Docket No.61078-716.601 folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0234] In embodiments, the targeting RNA hybridizes to a continuous stretch of nucleic acids within the target DNA. In other embodiments the targeting RNA hybridizes to a discontinuous stretch of nucleic acids within the target DNA. In embodiments, the targeting RNA may hybridize to a single- stranded target DNA sequence, for example, though base pairing. In embodiments, the targeting RNA may hybridize to a double-stranded target DNA sequence, for example by hybridizing to the major- or minor-groove edges of the base pairs of the target DNA sequence.

[0235] In embodiments, the targeting RNA hybridizes to a target DNA sequence that is actively transcribed, e.g., actively transcribed in the cell type being studied. In embodiments, the targeting RNA hybridizes to a target DNA sequence that does not comprise condensed chromatin, e.g., does not comprise condensed chromatin in the cell type being studied.

[0236] Non-limiting examples of targeting RNAs and / or target DNA sequences are described in, for example: Wang T, et al., Science (2013), vol.343, pp.80-84 and WO2015 / 048577, which are hereby incorporated by reference in their entirety. It will be understood by one of ordinary skill that the targeting RNAs of the genome editing systems of the present invention are not limited to those disclosed, for example, in Wang, et al. It will be appreciated that unlike other known gene editing systems, targeting RNAs to virtually any sequence of target DNA can be designed. Guide RNA

[0237] In some aspects, the gene editing systems of the disclosure may comprise a guide RNA.

[0238] The Guide RNA refers to ribonucleic acid sequence that is capable of binding to a guide RNA- binding domain, e.g., a guide RNA-binding domain as described herein. For example, the Guide RNA refers to an RNA aptamer. Such aptamers, and their corresponding polypeptide-based guide RNA- binding domains are known in the literature, any of which are suitable for use in the present invention. Non-limiting examples of guide RNA / guide RNA-binding domain pairs are described in detail herein.

[0239] In some embodiments, the guide RNA is between 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1- 30, 1-20 or 1-10 nucleotides. In some embodiments, the guide RNA is between about 20 and about 100 nucleotides, e.g., between about 30 and about 90, e.g., between about 40 and about 80, e.g., between about 50 and about 70 nucleotides. In some embodiments, the guide RNA is 1, 2, 3, 4, 5, 6,WSGR Docket No.61078-716.601 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or more nucleotides.

[0240] Additional guide RNA molecules may be discovered using RNA-based combinatorial libraries, screened against any guide RNA-binding molecule of interest. Those of ordinary skill will appreciate how to design and identify guide RNAs to any guide RNA-binding domain of interest. By way of example, libraries, e.g., phage display libraries, of RNA aptamers may be designed according to known methods, and those libraries may be screened for specific binding to a useful guide RNA-binding domains, also according to known methods. For example, RNA molecules that bind to specific targets of interest, e.g., guide RNA-binding domains, may be identified using SELEX (E.g., Fitzwater et al., Methods Enzymol., vol.267, pp.275-301 (1996), which is incorporated herein in its entirety).

[0241] Guide RNAs capable of specifically binding to guide RNA-binding domains may also be generated by rational design based on computer modeling and or structural biology. RNA Interference

[0242]

[0162] In some aspects, the gene editing systems of the disclosure may include a system for RNA interference. The phrase "RNA interference" and the term "RNAi" are synonymous and refer to the process by which a polynucleotide or siRNA comprising at least one ribonucleotide unit exerts an effect on a biological process. The process includes, but is not limited to, gene silencing by degrading mRNA, attenuating translation, interactions with tRNA, rRNA, hnRNA, cDNA and genomic DNA, as well as methylation of DNA with ancillary proteins.

[0243] In some embodiments, RNAi may comprise siRNA. The term "siRNA" and the phrase "short interfering RNA" refer to unimolecular nucleic acids and to nucleic acids comprised of two separate strands that are capable of performing RNAi and that have a duplex region that is between 18 and 30 base pairs in length. Additionally, the term siRNA and the phrase "short interfering RNA" include nucleic acids that also contain moieties other than ribonucleotide moieties, including, but not limited to, modified nucleotides, modified internucleotide linkages, non-nucleotides, deoxynucleotides and analogs of the aforementioned nucleotides.

[0244] siRNAs can be duplexes, and can also comprise short hairpin RNAs, RNAs with loops as long as, for example, 4 to 23 or more nucleotides, RNAs with stem loop bulges, micro-RNAs, and short temporal RNAs. RNAs having loops or hairpin loops can include structures where the loops are connected to the stem by linkers such as flexible linkers. Flexible linkers can be comprised of a wide variety of chemical structures, as long as they are of sufficient length and materials to enable effectiveWSGR Docket No.61078-716.601 intramolecular hybridization of the stem elements. Typically, the length to be spanned is at least about 10-24 atoms. When the siRNAs are hairpins, the sense strand and antisense strand are part of one longer molecule. In some embodiments, RNAi may include shRNA.

[0245] In some embodiments, RNAi may include MicroRNAs. MicroRNAs (miRNAs) belong to a class of small non-coding RNAs. miRNAs regulate gene expression by interacting with mRNAs via base-pairing and reducing the production of proteins from these mRNAs by affecting their translation. A miRNA may bind to and regulate the translation of up to several hundred target messenger RNAs, which enables the coordinated expression of multiple related genes.

[0246] A microRNA in accordance with the present disclosure can have a conventional naturally occurring sequence, a chemically modified version or sequence, or homologue thereof. In some embodiments, microRNAs presented herein are 7-130 nucleotides long, double stranded RNA molecules, either having two separate strands or a hairpin structure. A microRNA can be 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 7-30, 7-25, 15-30, 15-25, 17- 30, or 17-25 nucleotides long. In some embodiments, one of the two strands, which is referred to as the “guide strand” or “active strand”, contains a sequence which is identical or substantially identical to the seed sequence (e.g. nucleotide positions 2-9) of the parent microRNA sequence. One strand of a miRNA can be identical or substantially identical across the entire length of the guide strand of a naturally occurring miRNA. The second of the two strands, which is referred to as a “passenger strand”, contains a sequence that is complementary or substantially complementary to the seed sequence of the corresponding given microRNA. In some embodiments the double stranded RNA comprises a sequence that is at least 50-60%, 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95% or more identical to the sequence of a microRNA (miRNA) active strand or a complement thereof. A double stranded RNA may comprise a sequence, or comprise a strand consisting of a sequence, that is at least 90% identical to the sequence of a micro-RNA (miRNA) active strand or a complement thereof. A passenger strand will typically be at least partially complementary (e.g. at least about 50%, 75%, 80%, 85%, 90%, 95%, or 100% complementary) to its cognate guide strand. In some embodiments, the oligonucleotide is a mimic of a naturally occurring miRNA, or an analog or homolog thereof. In general, a “miRNA mimic” is a double-stranded RNA molecule that retains at least a portion of the biological activity of the miRNA it is said to mimic, e.g. at least 25%, 50%, 75%, 85%, 95%, or more of the biological activity. In some cases, a miRNA mimic has an enhanced biological activity as compared to the reference miRNA and as measured by a suitable assay, such as activity that is at least 10%, 25%, 50%, 75%, 90%, 100%, 200%, 300%, or more increased.WSGR Docket No.61078-716.601

[0247] In some aspects, a gene editing system, and / or gene expression modulator of the present disclosure may be provided to a cell. In some embodiments, the gene editing system, and / or gene expression modulator may be provided to a recombinant cell of the disclosure. In some embodiments, the recombinant cell of the disclosure may comprise a B2M fusion protein, a CAR, and / or a gene editing system, and / or gene expression modulator.

[0248] In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of a gene that encodes a T-cell receptor (TCR). In some embodiments, the gene may be a gene that encodes an MHC. In some embodiments, the gene may be a gene that encodes an extracellular domain of a T-cell receptor or MHC. In some embodiments, the gene may be a gene that encodes an intracellular receptor of a T-cell receptor or MHC. In some embodiments, the gene may be selected from the group consisting of TCRalpha, TCRbeta, CD80, CD58, OX40L, and any combination thereof. shRNA

[0249] In some aspects, T-cell receptor expression may be decreased using small-hairpin RNAs chains (e.g., CD3 zeta) T cells. In some non-limiting embodiments, targeting shRNAs have been designed for key components of the TCR complex. For example, an shRNA targeting CD3 epsilon can comprise the sequence ctggaggcttgctgaaggctgtatgctgAACGCCAACTGATAAGAGGCAgttttggccactgactgacTGCCTCTTC AGTTGGCGTTcaggacacaaggcctgttactagcactcacatggaacaaatggccca.

[0250] In some aspects, MHC expression may be decreased using small-hairpin RNAs (shRNAs) that target nucleic acids encoding MHCI and / or MHCII proteins in T cells (e.g., B2M, CIITA, NLRC5, RFX5, RFXANK, RFXAP, viral immunoevasins and the like). In some non-limiting embodiments targeting shRNAs have been designed for key components of the MHC complex For example, an shRNA targeting B2M can comprise the sequence: ctggaggcttgctgaaggctgtatgctgAATCTTTGGAGTACGCTGGATgttttggccactgactgacATCCAGCGCT CCAAAGATTcaggacacaaggcctgttactagcactcacatggaacaaatggccca. Dominant Negative Inhibition

[0251] In some aspects, over-expression of a dominant-negative inhibitor protein may be capable of interrupting T cell receptor expression and / or function. In some embodiments, a minigene that incorporates part, or all, of a polynucleotide encoding for one of the TCR components (e.g., TCR- alpha, TCR-beta, CD3-gamma, CD3-delta, CD3-epsilon, or CD3-zeta) is prepared, but is modi-fied soWSGR Docket No.61078-716.601 that: (1) it lacks key signaling motifs (e.g., an ITAM) required for protein function; (2) is modified so it does not associate properly with its other natural TCR components; or (3) can as-sociate properly but does not bind ligands (e.g., a truncated TCR beta minigene). In addition, the minigene may be altered to include an inhibitory signal motif, e.g., a cytoplasmic domain from a KIR protein, which alters cell signaling and promotes inhibitory signals through the recruitment of phosphatases, e.g., SHP1 and SHP2.

[0252] These minigenes may also encode a portion of a protein that serves as a means to identify the over-expressed minigene. For example, polynucleotides encoding a truncated CD19 protein, which contains the binding site for anti-CD19 mAbs, can be operably linked to the minigene so that the resulting cell that expresses the minigene will express the encoded protein and can be identified with anti-CD19 mAbs. This identification enables one to determine the extent of minigene expression and isolate cells expressing this protein (and thus lack a functional TCR).

[0253] In some aspects, over-expression of a dominant-negative inhibitor protein may be capable of interrupting MHC expression and / or function. In some embodiments, a minigene that incorporates be used.

[0254] In some aspects, a recombinant cell of the disclosure may comprise expression of a dominant negative protein, wherein the dominant negative protein (DN) may be DN-TCRalpha, DN-TCRbeta, RFXAP, a DN-viral immunoevasin, ICAM1, DN-CD80, DN-CD58, DN-CD2, DN-OX40L, DN- SUGT1, DN-TAP1, DN-TAP2, DN-TAPBP, DN-HLA-A, DN-HLA-C, DN-HLA-DR, DN-HLA-DP, DN-HLA-DQ, DN-CD74, a DN-immunoevasin, DN-US11, DN-K3, or DN-ICP47, or any combination thereof.

[0255] In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 5%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 10%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 15%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 20%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 25%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 30%. In some embodiments, theWSGR Docket No.61078-716.601 gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 35%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 40%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 45%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 50%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 55%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 60%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 65%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 70%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 75%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 80%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 85%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 90%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 95%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the gene by at least about 100%.

[0256] In some aspects, the administration of the gene editing system, and / or gene expression modulator to a first cell may inhibit expression of a T-cell receptor on the cell. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 5%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 10%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 15%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 20%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least aboutWSGR Docket No.61078-716.601 25%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 30%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 35%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 40%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 45%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 50%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 55%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 60%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 65%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 70%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 75%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 80%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 85%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 90%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 95%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the T-cell receptor by at least about 100%.

[0257] In some aspects, the administration of the gene editing system, and / or gene expression modulator to a first cell may inhibit expression of an MHC on the surface of the cell. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 5%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 10%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 15%. In some embodiments, theWSGR Docket No.61078-716.601 gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 20%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 25%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 30%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 35%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 40%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 45%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 50%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 55%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 60%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 65%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 70%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 75%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 80%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 85%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 90%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 95%. In some embodiments, the gene editing system, and / or gene expression modulator may decrease and / or modulate expression of the MHC by at least about 100%. Compositions

[0258] In some aspects, the present disclosure describes compositions comprising any of the recombinant fusion proteins and / or chimeric antigen receptors disclosed herein. In another aspect, the present disclosure provides compositions comprising any of the recombinant polynucleic acids disclosed herein. In some embodiments, the composition may comprise any combination of the recombinant polypeptides and recombinant polynucleic acids disclosed herein.WSGR Docket No.61078-716.601 Cells

[0259] In some aspects, the present disclosure describes cells comprising the recombinant fusion proteins disclosed herein, cells comprising the chimeric antigen receptors disclosed herein, cells comprising the recombinant polynucleic acids disclosed herein, cells comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and / or cells comprising the recombinant fusion proteins and chimeric antigen receptors disclosed herein. In some embodiments, the recombinant fusion protein may be a B2M fusion protein. In some embodiments, the cell may express a B2M fusion protein and / or chimeric antigen receptor disclosed herein. In some embodiments, the recombinant B2M fusion protein and / or CAR are expressed on the surface of the cell. In some embodiments, when the recombinant B2M fusion protein is expressed on the surface of the cell, it may be bound to an MHC molecule. In some embodiments, the MHC molecule may be a class 1 MHC molecule. In some embodiments, in the case of a cell surface receptor (e.g., a CAR, TCR, etc.) - the extracellular binding domain is displayed on the cell surface, the transmembrane portion passes through the cell membrane, and the one or more intracellular signaling domains are disposed adjacent to the intracellular side of the cell membrane. Upon binding of the extracellular binding domain to the target ligand / antigen, the intracellular signaling domain of the cell surface receptor participates in transducing the signal from the binding into the interior of the cell.

[0260] In some embodiments, cells may be genetically engineered (e.g transduced, transformed, or transfected) with, for example, a vector construct of the present disclosure that may be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or may be an expression vector for the expression of the polypeptides of interest. Cells may be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. In some embodiments, the cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell. In some embodiments, the cell is autologous or allogeneic. In some embodiments, the cell is a T cell, a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.

[0261] Cells may be transduced / transfected with a polynucleic acid encoding the recombinant B2M fusion protein and / or CAR. In some embodiments, a cell may be transduced with a bicistronic nucleic acid encoding a B2M fusion protein and a CAR. In some, a cell may be transduced with a nucleic acid encoding a B2M fusion protein and an additional nucleic acid encoding a CAR. In some embodiments, the cell is further transduced with an additional nucleic acid encoding one or more additional therapeutic agents such as, for example, but not limited to, an antibody, an antibody fragment thereof, or a protein therapeutic.WSGR Docket No.61078-716.601

[0262] In some aspects, allogeneic T-cells may be isolated and cultured ex vivo. In some embodiments, the allogeneic T-cells may comprise a CAR of the disclosure. In some embodiments, a B2M fusion protein may be administered to the cultured CAR-T cell. In some embodiments, a B2M fusion protein may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient. In some embodiments, the cultured, B2M / CAR-T cell may be administered to a patient.

[0263] In some aspects, autologous T-cells may be isolated from a subject, and cultured ex vivo. In some embodiments, the autologous T-cells may comprise a CAR of the disclosure. In some embodiments, a B2M fusion protein may be administered to the cultured CAR-T cell. In some embodiments, a B2M fusion protein may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient. In some embodiments, the cultured, B2M / CAR-T cell may be administered to a patient.

[0264] In some aspects, cells of the disclosure may comprise a CAR, and / or a B2M fusion protein, and / or a disruption of a gene. In some embodiments, a cell may be modified comprising disruption of a gene, for example, gene knock-out, knock-down, mutation, knock-in, and / or overexpression, by administering a gene editing system and / or gene modulator of the disclosure. In some embodiments, the cell comprising disruption of a gene may be a T-cell. In some embodiments, the cell comprising disruption of a gene may further comprise a CAR. In some embodiments, the cell comprising the disruption of a gene and a CAR may further comprise a B2M fusion protein or sequence encoding a B2M fusion protein.

[0265] In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell may be a mammalian cell. In some embodiments, the animal cell may be a mouse cell. In some embodiments, the animal cell may be a human cell. In some embodiments, the recombinant cell may be an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor-infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell. In some embodiments, the immune system cell may be selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells. In some embodiments, the immune system cell may be a T lymphocyte. The immune cell may also be a precursor cell, i.e., a cell that may be capable of differentiating into an immune cell.

[0266] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. In some embodiments, the nucleic acid molecule may be introduced into a host cell by a transduction procedure, transfection procedure, electroporation procedure, or a biolistic procedure. Accordingly, cell cultures including atWSGR Docket No.61078-716.601 least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0267] Cells of the present disclosure may be autologous / autogeneic (“self”) or non-autologous (“non- self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells derived from the same individual to which they are subsequently administered. “Allogeneic” as used herein refers to cells of the same species that differ genetically from the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison. In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0268] T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL separation.

[0269] In some aspects, an isolated or purified population of T cells may be used. In some embodiments, TCTL and TH lymphocytes are purified from PBMCs. In some embodiments, the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and / or genetic modification. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L- CD95+. An exemplary approach for such sorting may be described in Wang et al. (2016) Blood 127(24):2980- 90.

[0270] A specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, may be further isolated by positive or negative selection techniques.

[0271] Further disclosed herein includes a composition comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a B2M fusion protein and / or a CAR of the disclosure, wherein the cell expresses the B2M fusion protein and / or CAR of the disclosure, wherein the cell expressing the B2M fusion protein and / or CAR of the disclosure is a grafted cell, and whereinWSGR Docket No.61078-716.601 the grafted cell is administered to a host, and wherein the grafted cell count when measured after administration to the host is at least about 5% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 10% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 20% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 30% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 40% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 50% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 60% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 70% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 80% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 90% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 100% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 150% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 200% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 300% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure. InWSGR Docket No.61078-716.601 some embodiments, the grafted cell count when measured after administration to the host is greater than 300% greater than a grafted cell count of grafted cells that do not express the B2M fusion protein and / or CAR of the disclosure.

[0272] In some embodiments, the cell is a mammalian cell. The cell can be a human cell. The cell can be a blood cell. In some embodiments, the blood cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells is a population of blood cells. The blood cells can be lymphocytes. The lymphocytes can be T cells. In some embodiments, the population of cells is a homogeneous mixture of cells of the same cell type. In some embodiments, the population of cells is a heterogeneous mixture of cells of different cell types. In some embodiments, the population of cells comprises at least about 1x105cells. In some embodiments, the population of cells comprises at least about 1x106cells. In some embodiments, the population of cells comprises at least about 1x107cells. In some embodiments, the population of cells comprises at least about 1x108cells. In some embodiments, the population of cells comprises at least about 1x109cells. In some embodiments, the population of cells comprises from about 1x105cells to about 1x109cells. In some embodiments, the population of cells comprises from about 1x105cells to about 1x108cells. In some embodiments, the population of cells comprises from about 1x105cells to about 1x107cells. In some embodiments, the population of cells comprises from about 1x105cells to about 1x106cells. Pharmaceutical Compositions

[0273] In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the B2M fusion proteins and / or CARs disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the cells disclosed herein, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of the cells disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability ofWSGR Docket No.61078-716.601 the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a pharmaceutical composition of the present disclosure includes from 1x105to 5x1010of the cells of the present disclosure, or greater than 5x1010cells of the present disclosure.

[0274] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents.

[0275] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0276] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0277] Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine,WSGR Docket No.61078-716.601 and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0278] An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and / or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0279] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0280] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20 ) and polysorbate 80 (sold under the trademark Tween 80 ). Examples of suitable polyethylene- polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188 . Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij . Example concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0281] In some aspects, the pharmaceutical composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v). Methods of TreatmentWSGR Docket No.61078-716.601

[0282] Further disclosed here include methods for treating a disease, disorder, or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. The pharmaceutical composition can be administered alone or in combination with other agents (e.g., an antibody or an antigen binding fragment thereof, or a molecule). In some embodiments, a vaccine, an oncoloytic viruse, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and / or a bispecific antibody can be combined with the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells). Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anti-cancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and other antineoplastics.

[0283] In some embodiments, the disease, disorder, or condition is a cancer, an inflammatory disease, a neuronal disorder, HIV / AIDS, diabetes, a cardiovascular disease, an infectious disease, or an autoimmune disease. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the disease, disorder, or condition is a hyperproliferative disorder. Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms, and may express native proteins inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal).

[0284] B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B- cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, MALT lymphoma, Waldenstrom’s macroglobulinemia, and / or other disorders characterized by the overgrowth of B-lineage cells.WSGR Docket No.61078-716.601

[0285] Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma / oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma / anaplastic oligodendroglioma / glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkin disease / lymphoma; kidney cancer; melanoma; multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated / small cell / atypical lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia / small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and mycosis fungoides / Sëzary Syndrome; non-melanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intra- abdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hürthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and / or uterine sarcoma.

[0286] Methods for administering immune cells for therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapyWSGR Docket No.61078-716.601 methods are described in US 2003 / 0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli et al., Nat Biotechnol (2013) 31(10):928-33; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(l):84-89. Administration to Subjects

[0287] The terms “administering,” “introducing” and “transplanting” are used interchangeably in the context of the placement of the recombinant polypeptides, nucleic acids, and / or gene editing molecules, and / or recombinant cells of the disclosure into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. The recombinant polypeptides, nucleic acids, and / or gene editing molecules, or recombinant cells of the disclosure can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the patient, i.e., long-term engraftment. For example, in some aspects described herein, an effective amount of the recombinant polypeptides, nucleic acids, and / or gene editing molecules, or recombinant cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0288] The terms “individual”, “subject,” “host” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being.

[0289] The term “donor” is used to refer to an individual that is not the patient. In some embodiments, the donor is an individual who does not have or is not suspected of having the medical condition to be treated. In some embodiments, multiple donors, e.g., two or more donors, can be used. In some embodiments, each donor used is an individual who does not have or is not suspected of having the medical condition to be treated.

[0290] When provided therapeutically, the recombinant polypeptides, nucleic acids, and / or gene editing molecules, or recombinant cells of the disclosure may be provided at (or after) the onset of a symptom or indication of a medical condition, e.g., upon the onset of disease. In some aspects the recombinant cells being administered according to the compositions and methods described herein comprises allogeneic T cells. In some aspects the recombinant cells being administered according to the compositions and methods described herein comprises allogeneic T cells. obtained from one or more donors. In some embodiments, the cell population being administered can be allogeneic blood cells, hematopoietic stem cells, hematopoietic progenitor cells, embryonicWSGR Docket No.61078-716.601 stem cells, or induced embryonic stem cells. “Allogeneic” refers to a cell, cell population, or biological samples comprising cells, obtained from one or more different donors of the same species, where the genes at one or more loci are not identical to the recipient.

[0291] “Administered” refers to the delivery of a recombinant cell composition of the disclosure into a subject by a method or route that results in at least partial localization of the cell composition at a desired site. A cell composition can be administered by any appropriate route that results in effective treatment in the subject, i.e. administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, i.e. at least 1×104cells are delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made.

[0292] The cells are administered systemically. The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” refer to the administration of a population of recombinant cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.

[0293] The efficacy of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” if any one or all of the signs or symptoms of, as but one example, tumor size is reduced (e.g., reduced by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0294] In some aspects, administration of a pharmaceutical composition may comprise administering a B2M fusion protein to a subject. In some embodiments, administration of a pharmaceutical composition may comprise administering a B2M fusion protein to a subject, and administering aWSGR Docket No.61078-716.601 population of CAR-T cells to the subject. In some embodiments, the B2M fusion protein and CAR-T cells may be administered at the same time. In some embodiments, the B2M fusion protein may be administered before the CAR-T cells. In some embodiments, the B2M fusion protein may be administered after the CAR-T cells. Methods of Delivery

[0295] In some embodiments, the recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may be introduced into a cell using biological methods. For example, biological methods may employ delivery methods such as vectors or synthetic liposomes, non-limiting examples described above; other examples may include non-viral biological agents, such as bacteria, bacteriophage, virus-like particles, erythrocyte ghosts, exosomes among others known in the art. In some embodiments, the polynucleotide may be introduced into a cell via any other delivery system. In some embodiments, the polynucleotide may be introduced into a cell using a cell transformation method. In some embodiments, the polynucleotide may be introduced into a cell using a cell transduction method. In some embodiments, the polynucleotide may be introduced into a cell by any transfection method wherein the transfection leads to uptake of any artificial introduction of foreign cargo e.g. nucleic acid into a cell. In some embodiments, the polynucleotide comprising the sequences encoding the fusion protein may be introduced into a cell using physical methods in which physical energy is applied for intracellular delivery. Such physical methods use application of force to generate transient pores in the cell membrane. Some non-limiting examples of physical methods of delivering the polynucleotide or a cell bearing polynucleotide include, microfluidic electroporation, nanochannel electroporation, nanostraw electroporation, laser-induced photoporation, optical transfection, mechanoporation, ballistic gene delivery, cell squeezing, microinjection, nanofountain probe electroporation, particle bombardment, field-induced membrane disruption, sonoporation, optoporation, magnetoporation, constriction channel based intracellular delivery, thermoporation and any other electroporation-based cell delivery technique or device. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cell intracellular environment such as for example, the cytoplasm or nucleus of the cell. The methods of delivery may depend on if the introduction may be for a single-cell intracellular delivery or not. art. In some embodiments, the polynucleotide may be introduced into a cell using chemical methods, such as chemical vector-based non-viral cargo delivery which may require modifying cell-penetrating peptides or proteins or endosomal escape to transfect cargo molecules into the cytoplasm directly. The chemical transfection methods are techniques that catalyze DNA cross-membrane transport. In some embodiments, chemical methods may use Ca2+phosphate, polycations or dendrimers including forWSGR Docket No.61078-716.601 example, without limitations, such methods as, use of cationic polymers e.g. diethylaminoethyl- dextran (DEAE-dextran). Chemical methods of cell delivery may apply cell transfection with cationic lipids (non-viral vectors), also known as lipofection or lipid-mediated / liposome transfection are used in cargo or gene transfection.

[0296] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell. In some embodiments, the polynucleotide integrating into the cell may be single stranded. In some embodiments, the polynucleotide integrating may be double stranded DNA. In some embodiments, the polynucleotide may be short nucleotide sequences. In some embodiments, the polynucleotide may be long nucleotide sequences. In some embodiments, the integration into the genome of the cell may be transient integration in the cell. In some embodiments, the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell. In some embodiments, the polynucleotide may integrate into the cell genome within a random locus. In some embodiments, the polynucleotide may integrate into the cell genome within a directed or targeted locus. In instances where the polynucleotide may be integrated into the cell genome, the polynucleotide may replicate when the cell genome replicates.

[0297] In some embodiments, the cells bearing the polynucleotide comprising the sequences encoding the fusion protein having integrated or transduced or transformed into the cell may be characterized using various methods.

[0298] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above. In some embodiments, the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell. In some embodiments, the recombinant cell or recombinant vector may comprise a selectable biomarker. In some embodiments, the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.

[0299] In some embodiments, the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker. In some embodiments, the selectable biomarker may be an antibiotic cassette. In some embodiments, the selectable marker may be a vector or molecule that produces a morphological change, wherein the morphological change denotes integration of the recombinant polynucleotide or cell or vector bearing the polynucleotide. In some embodiments, the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a transgene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker.WSGR Docket No.61078-716.601 Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, mScarlet fluorescent protein gene, red fluorescent protein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry / texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art. Non-limiting examples of antibiotic selectable resistance marker gene include, kanamycin gene, ampicillin gene, streptomycin gene, neomycin gene, puromycin gene gentamycin gene, erythromycin gene, Blasticidin S gene, hygromycin B gene among many others known in the art. In some embodiments, the polynucleotide integrating may be small interfering RNA or miRNA wherein the siRNA or miRNA may be short hairpin transcripts, or the short hairpins may be made from a selectable DNA vector.

[0300] Recombinant polypeptides and / or recombinant nucleic acids, gene editing molecules, guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotide(s) (RNA or DNA) of the present disclosure can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, endonuclease polypeptide(s) may be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In some embodiments, the DNA endonuclease may be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.

[0301] The recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may be delivered by non-viral delivery vehicles including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA- conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vehicles for siRNA that are also useful for delivery of other polynucleotides). Lipid Nanoparticles

[0302] The recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may be delivered to a cell or a patient by a lipid nanoparticle (LNP).

[0303] A LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0304] LNPs may be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, may be included in LNPs asWSGR Docket No.61078-716.601 ‘helper lipids’ to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.

[0305] LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0306] Any lipid or combination of lipids that are known in the art may be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0307] The lipids may be combined in any number of molar ratios to produce a LNP. In addition, the polynucleotide(s) may be combined with lipid(s) in a wide range of molar ratios to produce a LNP.

[0308] The recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may each be administered separately to a cell or a patient. On the other hand, the site- directed polypeptide, such as a CRISPR / cas9 polypetide, may be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA. The pre-complexed material may then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

[0309] RNA is capable of forming specific interactions with RNA or DNA. While this property is exploited in many biological processes, it also comes with the risk of promiscuous interactions in a nucleic acid-rich cellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which the RNA is pre-complexed with an endonuclease. Another benefit of the RNP is protection of the RNA from degradation.

[0310] The endonuclease in the RNP may be modified or unmodified. Likewise, the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerous modifications are known in the art and may be used.

[0311] The endonuclease and sgRNA may be generally combined in a 1:1 molar ratio. Alternatively, the endonuclease, crRNA and tracrRNA may be generally combined in a 1:1:1 molar ratio. However, a wide range of molar ratios may be used to produce a RNP. Adeno-Associated Virus

[0312] A recombinant adeno-associated virus (AAV) vector may be used for delivery of the recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure,.WSGR Docket No.61078-716.601 Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived, and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01 / 83692. See Table 1. TABLE 1

[0313] A method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983,WSGR Docket No.61078-716.601 Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661- 4666). The packaging cell line is then infected with a helper virus, such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0314] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol.5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos.5,786,211; 5,871,982; and 6,258,595.

[0315] AAV vector serotypes can be matched to target cell types. For example, the following exemplary cell types may be transduced by the indicated AAV serotypes among others. See Table 2. TABLE 2WSGR Docket No.61078-716.601

[0316] In addition to adeno-associated viral vectors, other viral vectors can be used. Such viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplex virus.

[0317] In some embodiments, Cas9 mRNA, sgRNA targeting one or two loci in target gene, and donor DNA is each separately formulated into lipid nanoparticles, or are all co-formulated into one lipid nanoparticle, or co-formulated into two or more lipid nanoparticles.

[0318] In some embodiments, Cas9 mRNA is formulated in a lipid nanoparticle, while sgRNA and donor DNA are delivered in an AAV vector. In some embodiments, Cas9 mRNA and sgRNA are co- formulated in a lipid nanoparticle, while donor DNA is delivered in an AAV vector.

[0319] Options are available to deliver the Cas9 nuclease as a DNA plasmid, as mRNA or as a protein. The guide RNA can be expressed from the same DNA, or can also be delivered as an RNA. The RNA can be chemically modified to alter or improve its half-life, or decrease the likelihood or degree of immune response. The endonuclease protein can be complexed with the gRNA prior to delivery. Viral vectors allow efficient delivery; split versions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can donors for HDR. A range of non-viral delivery methods also exist that can deliver each of these components, or non-viral and viral methods can be employed in tandem. For example, nano- particles can be used to deliver the protein and guide RNA, while AAV can be used to deliver a donor DNA. Exosomes

[0320] The recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may be delivered via exosomes. Exosomes, a type of microvesicle bound by phospholipid bilayer, can be used to deliver nucleic acids to specific tissue. Many different types of cells within the body naturally secrete exosomes. Exosomes form within the cytoplasm when endosomes invaginate and form multivesicular-endosomes (MVE). When the MVE fuses with the cellular membrane, the exosomes are secreted in the extracellular space. Ranging between 30-120 nm in diameter, exosomes can shuttle various molecules from one cell to another in a form of cell-to-cell communication. Cells that naturally produce exosomes, such as mast cells, can be genetically altered to produce exosomes with surface proteins that target specific tissues, alternatively exosomes can be isolated from the bloodstream. Specific nucleic acids can be placed within the engineered exosomes with electroporation. When introduced systemically, the exosomes can deliver the nucleic acids to the specific target tissue.

[0321] In some embodiments, the recombinant polypeptides, polynucleotides, and / or gene editing molecules of the disclosure, may be introduced into a cell using biological methods. For example,WSGR Docket No.61078-716.601 biological methods may employ delivery methods such as vectors or synthetic liposomes, non-limiting examples described above; other examples may include non-viral biological agents, such as bacteria, bacteriophage, virus-like particles, erythrocyte ghosts, exosomes among others known in the art. In some embodiments, the polynucleotide may be introduced into a cell via any other delivery system. In some embodiments, the polynucleotide may be introduced into a cell using a cell transformation method. In some embodiments, the polynucleotide may be introduced into a cell using a cell transduction method. In some embodiments, the polynucleotide may be introduced into a cell by any transfection method wherein the transfection leads to uptake of any artificial introduction of foreign cargo e.g., nucleic acid into a cell. In some embodiments, the polynucleotide comprising the sequences encoding the fusion protein may be introduced into a cell using physical methods in which physical energy is applied for intracellular delivery. Such physical methods use application of force to generate transient pores in the cell membrane. Some non-limiting examples of physical methods of delivering the polynucleotide or a cell bearing polynucleotide include, microfluidic electroporation, nanochannel electroporation, nanostraw electroporation, laser-induced photoporation, optical transfection, mechanoporation, ballistic gene delivery, cell squeezing, microinjection, nanofountain probe electroporation, particle bombardment, field-induced membrane disruption, sonoporation, optoporation, magnetoporation, constriction channel based intracellular delivery, thermoporation and any other electroporation-based cell delivery technique or device. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cell intracellular environment such as for example, the cytoplasm or nucleus of the cell. The methods of delivery may depend on if the introduction may be for a single-cell intracellular delivery or not. art. In some embodiments, the polynucleotide may be introduced into a cell using chemical methods, such as chemical vector-based non-viral cargo delivery which may require modifying cell-penetrating peptides or proteins or endosomal escape to transfect cargo molecules into the cytoplasm directly. The chemical transfection methods are techniques that catalyze DNA cross-membrane transport. In some embodiments, chemical methods may use Ca2+phosphate, polycations or dendrimers including for example, without limitations, such methods as, use of cationic polymers e.g. diethylaminoethyl- dextran (DEAE-dextran). Chemical methods of cell delivery may apply cell transfection with cationic lipids (non-viral vectors), also known as lipofection or lipid-mediated / liposome transfection are used in cargo or gene transfection.

[0322] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell. In some embodiments, the polynucleotide integrating into the cell may be single stranded. In some embodiments, the polynucleotide integratingWSGR Docket No.61078-716.601 may be double stranded DNA. In some embodiments, the polynucleotide may be short nucleotide sequences. In some embodiments, the polynucleotide may be long nucleotide sequences. In some embodiments, the integration into the genome of the cell may be transient integration in the cell. In some embodiments, the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell. In some embodiments, the polynucleotide may integrate into the cell genome within a random locus. In some embodiments, the polynucleotide may integrate into the cell genome within a directed or targeted locus. In instances where the polynucleotide may be integrated into the cell genome, the polynucleotide may replicate when the cell genome replicates.

[0323] In some embodiments, the cells bearing the polynucleotide comprising the sequences encoding the fusion protein having integrated or transduced or transformed into the cell may be characterized using various methods.

[0324] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above. In some embodiments, the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell. In some embodiments, the recombinant cell or recombinant vector may comprise a selectable biomarker. In some embodiments, the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.

[0325] In some embodiments, the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker. In some embodiments, the selectable biomarker may be an antibiotic cassette. In some embodiments, the selectable marker may be a vector or molecule that produces a morphological change, wherein the morphological change denotes integration of the recombinant polynucleotide or cell or vector bearing the polynucleotide. In some embodiments, the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a transgene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker. Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, mScarlet fluorescent protein gene, red fluorescent protein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry / texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art. Non-limiting examples of antibiotic selectable resistance marker gene include, kanamycin gene, ampicillin gene, streptomycin gene, neomycin gene, puromycin gene gentamycin gene,WSGR Docket No.61078-716.601 erythromycin gene, Blasticidin S gene, hygromycin B gene among many others known in the art. In some embodiments, the polynucleotide integrating may be small interfering RNA or miRNA wherein the siRNA or miRNA may be short hairpin transcripts, or the short hairpins may be made from a selectable DNA vector. Kits

[0326] Also provided by the present disclosure are kits. In certain embodiments, provided are kits that include any of the recombinant nucleic acids, recombinant polypeptides, and / or expression vectors of the present disclosure, and instructions for introducing the recombinant nucleic acid, recombinant polypeptides, and / or expression vector into a cell. According to some embodiments, when the expression vector encodes a recombinant polypeptide that does not comprise the protease (trans configuration), the expression vector further encodes the protease. In certain embodiments, the expression vector is configured to express the recombinant polypeptide and the protease from the same promoter. For example, the expression vector may be a bicistronic expression vector for expression of separate recombinant polypeptides and protease molecules under the same promoter in the cell.

[0327] The kits of the present disclosure may further include any other reagents useful for regulatable signaling of the cell surface receptor, such as transfection / transduction reagents useful for introducing the nucleic acid or expression vector into cells of interest, e.g., immune cells (e.g., T cells) or other cells of interest.

[0328] Components of the kits may be present in separate containers, or multiple components may be present in a single container. A suitable container includes a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.

[0329] The instructions of the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0330] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features whichWSGR Docket No.61078-716.601 may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. List of Numbered Embodiments 1. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) binds to a second extracellular domain of the MHC that is different than the first extracellular domain or (ii) binds to an immune checkpoint protein, wherein the first and second domains are operatively linked by a linker domain. 2. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain or (ii) activates a cell-surface receptor that is an immune checkpoint protein, wherein the cell-surface receptor is expressed by a second cell. 3. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) binds to a second extracellular domain of the MHC that is different than the first extracellular domain or (ii) binds to an immune checkpoint protein, wherein the first and second domains are operatively linked by a linker domain. 4. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain or (ii) activates a cell-surface receptor that is an immune checkpoint protein, wherein the cell-surface receptor is expressed by a second cell. 5. The composition of any one of embodiments 1-4, wherein the second domain binds to a second extracellular domain of the MHC that is different than the first extracellular domain and / or inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain.WSGR Docket No.61078-716.601 6. The composition of embodiment 5, wherein the recombinant fusion protein further comprises (c) a third domain that binds to an immune checkpoint protein and / or activates a cell-surface receptor that is an immune checkpoint protein; wherein the first, second and third domains are operatively linked. 7. The composition of any one of embodiments 1-4, wherein the second domain binds to an immune checkpoint protein and / or activates a cell-surface receptor that is an immune checkpoint protein. 8. The composition of embodiment 7, wherein the recombinant fusion protein further comprises (c) a third domain that binds to a second extracellular domain of the MHC that is different than the first extracellular domain and / or inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain; wherein the first, second and third domains are operatively linked. 9. The composition of any one of embodiments 1-8, wherein the first extracellular domain of the MHC is a domain that binds to B2M. 10. The composition of any one of embodiments 1-9, wherein the first domain is B2M or an MHC- binding fragment or variant thereof. 11. The composition of any one of embodiments 1-10, wherein the second or third domain is CD8 or an MHC-binding fragment or variant thereof, or LILRB1 or an MHC-binding fragment or variant MHC-binding fragment or variant thereof. 12. The composition of embodiment 11, wherein the second or third domain is CD160 or an MHC- binding fragment or variant thereof, wherein the CD160 or MHC-binding fragment or variant thereof is an E319K variant. 13. an MHC-binding fragment or variant thereof. 14. 15. chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QSVTNN (SEQ ID NO: 293), FAS (SEQ ID NO: 294) and HQDYSSPLT (SEQ ID NO: 295), respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GYTFTSNW (SEQ ID NO: 290), IAPGSGNT (SEQ ID NO: 291) and ARLLRGALDY (SEQ ID NO: 292), respectively.WSGR Docket No.61078-716.601 16. sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity SEQ ID NO.252 or 253. 17. sequence set forth in SEQ ID NO.252 or 253. 18. The composition of any one of embodiments 2, 4 and 9-11, wherein the second domain or third binds to a second extracellular domain of the MHC that is different than the first extracellular domain. 19. The composition of any one of embodiments 1, 3, 5-7 and 12, wherein the second extracellular domain of the MHC is a CD8 binding domain or an LILRB1 binding domain, or an anti-MHC-I 20. The composition of any one of embodiments 1, 3, 5-7, and 12-17, wherein the second 21. The composition of any one of embodiments 1-20, wherein the MHC is an MHC class I. 22. The composition of any one of embodiments 1-21, wherein the MHC is encoded by an HLA- A02:01 gene. 23. The composition of any one of embodiments 1, 3 and 5-22, wherein the second or third domain inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell. 24. The composition of any one of embodiments 2, 4 and 5-23, wherein the cell-surface receptor is CD8, or wherein the cell-surface receptor is not TCR alpha / beta or TCR delta / gamma. 25. The composition of any one of embodiments 1-24, wherein the first cell is an allogeneic cell or an autologous cell. 26. The composition of any one of embodiments 1-24, wherein the first cell is a grafted cell, or a host cell. 27. The composition of any one of embodiments 2, 4 and 5-26, wherein the second cell is a host cell. 28. The composition of any one of embodiments 2, 4 and 5-27, wherein the second cell is a T-cell. 29. The composition of any one of embodiments 2, 4 and 5-28, wherein the second cell is a CD8+T- cell. 30. The composition of any one of embodiments 1-29, wherein the first domain is linked to the second domain. 31. The composition of embodiment 30, wherein the first domain is linked to the second domain by a first linker sequence.WSGR Docket No.61078-716.601 32. The composition of any one of embodiments 6-31, wherein the first domain is linked to the third domain. 33. The composition of embodiment 32, wherein the first domain is linked to the third domain by a second linker sequence. 34. The composition of any one of embodiments 1-33, wherein the second domain is linked to the third domain. 35. The composition of embodiment 34, wherein the second domain is linked to the third domain by a third linker sequence. 36. The composition of any one of embodiments 6-29, wherein the first domain is linked to the second domain and the first domain is linked to the third domain. 37. The composition of any one of embodiments 6-29, wherein the second domain is linked to the first domain and the second domain is linked to the third domain. 38. The composition of any one of embodiments 30-35, wherein the first, second, and / or third linker sequence is at least 5 amino acids in length. 39. The composition of any one of embodiments 30-35 and 38, wherein the first, second, and or third linker sequence is at most 30 amino acids in length. 40. The composition of any one of embodiments 30-35 and 38, wherein the linker sequence is from 5 to 30 amino acids in length. 41. The composition of any one of embodiments 6-40, wherein the second or third domain is a domain that binds to an immune checkpoint protein. 42. The composition of any one of embodiments 6-41, wherein the second or third domain is a PDL1 or PDL2 domain. 43. The composition of any one of embodiments 6-42, wherein the second or third domain is a domain that binds to PD1 or PD2. 44. The composition of any one of embodiments 6-40, wherein the second or third domain is a domain that binds to CTLA-4, LAG-3, TIM-3, TIGIT OR VISTA. 45. The composition of any one of embodiments 6-40, wherein the recombinant fusion protein further comprises an additional domain that binds to a T cell receptor. 46. The composition of embodiment 45, wherein the additional domain is an intracellular signaling domain. 47. The composition of embodiment 45, wherein the additional domain comprises a domain fromWSGR Docket No.61078-716.601 48. The composition of embodiment 47, wherein the additional domain comprises an intracellular 49. The composition of embodiment 47 or 48, wherein the additional domain comprises a stalk domain. 50. The composition of any one of embodiments 1-49, wherein the first or second or third or additional domain is crosslinked to the MHC, a Talin, a ITGB3 cytoplasmic domain, and / or a CD44 cytoplasmic domain. 51. The composition of embodiment 50, wherein the crosslinking prevents MHC clustering. 52. A composition comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding the recombinant fusion protein of the composition of any one of embodiments 1-49. 53. The composition of any one of embodiments 1-52, wherein the recombinant fusion protein comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence any one of SEQ ID NOs.42-56, 62-111, and 240- 258. 54. The composition of embodiment 50, wherein the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR). 55. The composition of embodiment 54, wherein the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. 56. The composition of embodiment 55, wherein the antigen binding domain is an anti-CD19 binding domain. 57. The composition of embodiment 56, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. 58. The composition of embodiment 55, wherein the antigen binding domain is an anti-CD22 binding domain. 59. The composition of embodiment 58, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 ofWSGR Docket No.61078-716.601 QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. 60. The composition of embodiment 55, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY- ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART- 1, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2. 61. The composition of any one of embodiments 50-60, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. 62. The composition of any one of embodiments 55-61, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. 63. The composition of any one of embodiments 55-62, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28. 64. A composition comprising a cell, wherein the cell comprises the composition of any one of embodiments 1-63. 65. The composition of embodiment 64, wherein the cell is a lymphocyte. 66. The composition of embodiment 64, wherein the cell is a T cell.WSGR Docket No.61078-716.601 67. The composition of any one of embodiments 64-66, wherein the cell is an allogeneic cell. 68. The composition of any one of embodiments 62-67, wherein the cell comprises a disruption of a CIITA, NLRC5, RFX5, RFXANK, RFXAP, a viral immunoevasin, ICAM1, CD80, CD58, OX40L, SUGT1, TAP1, T...

Claims

WSGR Docket No.61078-716.601 CLAIMS WHAT IS CLAIMED IS:

1. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) binds to a second extracellular domain of the MHC that is different than the first extracellular domain or (ii) binds to an immune checkpoint protein, wherein the first and second domains are operatively linked by a linker domain.

2. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that binds to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain or (ii) activates a cell- surface receptor that is an immune checkpoint protein, wherein the cell-surface receptor is expressed by a second cell.

3. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a first extracellular domain of a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) binds to a second extracellular domain of the MHC that is different than the first extracellular domain or (ii) binds to an immune checkpoint protein, wherein the first and second domains are operatively linked by a linker domain.

4. A composition comprising a recombinant fusion protein, the recombinant fusion protein comprising: (a) a first domain that anchors the recombinant fusion protein to a major histocompatibility complex (MHC) of a first cell, and (b) a second domain that (i) inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain or (ii) activates a cell-surface receptor that is an immune checkpoint protein,WSGR Docket No.61078-716.601 wherein the cell-surface receptor is expressed by a second cell.

5. The composition of any one of claims 1-4, wherein the second domain binds to a second extracellular domain of the MHC that is different than the first extracellular domain and / or inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain.

6. The composition of claim 5, wherein the recombinant fusion protein further comprises (c) a third domain that binds to an immune checkpoint protein and / or activates a cell-surface receptor that is an immune checkpoint protein; wherein the first, second and third domains are operatively linked.

7. The composition of any one of claims 1-4, wherein the second domain binds to an immune checkpoint protein and / or activates a cell-surface receptor that is an immune checkpoint protein.

8. The composition of claim 7, wherein the recombinant fusion protein further comprises (c) a third domain that binds to a second extracellular domain of the MHC that is different than the first extracellular domain and / or inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain; wherein the first, second and third domains are operatively linked.

9. The composition of any one of claims 1-8, wherein the first extracellular domain of the MHC is a domain that binds to B2M.

10. The composition of any one of claims 1-9, wherein the first domain is B2M or an MHC- binding fragment or variant thereof.

11. The composition of any one of claims 1-10, wherein the second or third domain is CD8 or an MHC-binding fragment or variant thereof, or LILRB1 or an MHC-binding fragment or or an MHC-binding fragment or variant thereof.

12. The composition of claim 11, wherein the second or third domain is CD160 or an MHC- binding fragment or variant thereof, wherein the CD160 or MHC-binding fragment or variant thereof is an E319K variant.

13. MHC-binding fragment or variant thereof. 14.WSGR Docket No.61078-716.601 15. chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QSVTNN (SEQ ID NO: 293), FAS (SEQ ID NO: 294) and HQDYSSPLT (SEQ ID NO: 295), respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GYTFTSNW (SEQ ID NO: 290), IAPGSGNT (SEQ ID NO: 291) and ARLLRGALDY (SEQ ID NO: 292), respectively.

16. sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity SEQ ID NO.252 or 253.

17. sequence set forth in SEQ ID NO.252 or 253.

18. The composition of any one of claims 2, 4 and 9-11, wherein the second domain or third binds to a second extracellular domain of the MHC that is different than the first extracellular domain.

19. The composition of any one of claims 1, 3, 5-7 and 12, wherein the second extracellular domain of the MHC is a CD8 binding domain or an LILRB1 binding domain, or an anti- 20. The composition of any one of claims 1, 3, 5-7, and 12-17, wherein the second extracellular 21. The composition of any one of claims 1-20, wherein the MHC is an MHC class I.

22. The composition of any one of claims 1-21, wherein the MHC is encoded by an HLA-A02:01 gene.

23. The composition of any one of claims 1, 3 and 5-22, wherein the second or third domain inhibits binding of a cell-surface receptor to the MHC when bound to the MHC of the first cell via the first domain, wherein the cell-surface receptor is expressed by a second cell.

24. The composition of any one of claims 2, 4 and 5-23, wherein the cell-surface receptor is CD8, or wherein the cell-surface receptor is not TCR alpha / beta or TCR delta / gamma.

25. The composition of any one of claims 1-24, wherein the first cell is an allogeneic cell or an autologous cell.

26. The composition of any one of claims 1-24, wherein the first cell is a grafted cell, or a host cell.

27. The composition of any one of claims 2, 4 and 5-26, wherein the second cell is a host cell.

28. The composition of any one of claims 2, 4 and 5-27, wherein the second cell is a T-cell.WSGR Docket No.61078-716.601 29. The composition of any one of claims 2, 4 and 5-28, wherein the second cell is a CD8+ T- cell.

30. The composition of any one of claims 1-29, wherein the first domain is linked to the second domain.

31. The composition of claim 30, wherein the first domain is linked to the second domain by a first linker sequence.

32. The composition of any one of claims 6-31, wherein the first domain is linked to the third domain.

33. The composition of claim 32, wherein the first domain is linked to the third domain by a second linker sequence.

34. The composition of any one of claims 1-33, wherein the second domain is linked to the third domain.

35. The composition of claim 34, wherein the second domain is linked to the third domain by a third linker sequence.

36. The composition of any one of claims 6-29, wherein the first domain is linked to the second domain and the first domain is linked to the third domain.

37. The composition of any one of claims 6-29, wherein the second domain is linked to the first domain and the second domain is linked to the third domain.

38. The composition of any one of claims 30-35, wherein the first, second, and / or third linker sequence is at least 5 amino acids in length.

39. The composition of any one of claims 30-35 and 38, wherein the first, second, and or third linker sequence is at most 30 amino acids in length.

40. The composition of any one of claims 30-35 and 38, wherein the linker sequence is from 5 to 30 amino acids in length.

41. The composition of any one of claims 6-40, wherein the second or third domain is a domain that binds to an immune checkpoint protein.

42. The composition of any one of claims 6-41, wherein the second or third domain is a PDL1 or PDL2 domain.

43. The composition of any one of claims 6-42, wherein the second or third domain is a domain that binds to PD1 or PD2.

44. The composition of any one of claims 6-41, wherein the second or third domain is a domain that binds to CTLA-4, LAG-3, TIM-3, TIGIT OR VISTA.WSGR Docket No.61078-716.601 45. The composition of any one of claims 6-40, wherein the recombinant fusion protein further comprises an additional domain that binds to a T cell receptor.

46. The composition of claim 45, wherein the additional domain is an intracellular signaling domain.

47.

48. The composition of claim 47, wherein the additional domain comprises an intracellular 49. The composition of claim 47 or 48, wherein the additional domain comprises a stalk domain.

50. The composition of any one of claims 1-49, wherein the first or second or third or additional domain is crosslinked to the MHC, a Talin, a ITGB3 cytoplasmic domain, and / or a CD44 cytoplasmic domain.

51. The composition of claim 50, wherein the crosslinking prevents MHC clustering.

52. A composition comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding the recombinant fusion protein of the composition of any one of claims 1-49.

53. The composition of any one of claims 1-52, wherein the recombinant fusion protein comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence any one of SEQ ID NOs.42-56, 62-111, and 240-258.

54. The composition of claim 50, wherein the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR).

55. The composition of claim 54, wherein the CAR comprises (a) an extracellular domain comprising an antigen binding domain;(b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain.

56. The composition of claim 55, wherein the antigen binding domain is an anti-CD19 binding domain.

57. The composition of claim 56, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively.WSGR Docket No.61078-716.601 58. The composition of claim 55, wherein the antigen binding domain is an anti-CD22 binding domain.

59. The composition of claim 58, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively.

60. The composition of claim 55, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate- carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2.

61. The composition of any one of claims 55-60, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70.

62. The composition of any one of claims 55-61, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.WSGR Docket No.61078-716.601 63. The composition of any one of claims 55-62, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.

64. A composition comprising a cell, wherein the cell comprises the composition of any one of claims 1-63.

65. The composition of claim 64, wherein the cell is a lymphocyte.

66. The composition of claim 64, wherein the cell is a T cell.

67. The composition of any one of claims 64-66, wherein the cell is an allogeneic cell.

68. The composition of any one of claims 62-67, wherein the cell comprises a disruption of a CIITA, NLRC5, RFX5, RFXANK, RFXAP, a viral immunoevasin, ICAM1, CD80, CD58, OX40L, SUGT1, TAP1, TAP2, TAPBP, HLA-A, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD74, an immunoevasin, US11, K3, ICP47 and any combination thereof.

69. The composition of any one of claims 64-67, wherein the cell express a dominant negative protein, wherein the dominant negative (DN) protein is selected from the group consisting of NLRC5, DN-RFX5, DN-RFXANK, DN-RFXAP, a DN-viral immunoevasin, ICAM1, DN- CD80, DN-CD58, DN-CD2, DN-OX40L, DN-SUGT1, DN-TAP1, DN-TAP2, DN-TAPBP, DN-HLA-A, DN-HLA-C, DN-HLA-DR, DN-HLA-DP, DN-HLA-DQ, DN-CD74, a DN- immunoevasin, DN-US11, DN-K3, DN-ICP47 and any combination thereof.

70. The composition of any one of claims 64-69, wherein the cell is an allogeneic cell.

71. The composition of any one of claims 64-70, wherein the cell is a population of cells.

72. The composition of claim 71, wherein the population of cells comprises at least 1x10^5 cells.

73. A pharmaceutical composition comprising the composition of any one of claims 1-72 and a pharmaceutically acceptable excipient diluent, excipient or carrier.

74. A method of treating a disease or condition in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 73 to the subject.

75. The method of claim 74, wherein the pharmaceutical composition comprises a population of CAR-T cells.

76. The method of claim 74 or 75, wherein the disease or condition is cancer, and or an auto- immune disease.

77. The method of claim 76, wherein the cancer is lymphoma or leukemia.

78. The method of claim 76, wherein the cancer is a solid tumor cancer.WSGR Docket No.61078-716.601 79. The method of claim 76, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and / or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and / or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

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