WO2024092126A1 - Compositions et méthodes pour immunothérapies améliorées - Google Patents

Compositions et méthodes pour immunothérapies améliorées Download PDF

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WO2024092126A1
WO2024092126A1 PCT/US2023/077916 US2023077916W WO2024092126A1 WO 2024092126 A1 WO2024092126 A1 WO 2024092126A1 US 2023077916 W US2023077916 W US 2023077916W WO 2024092126 A1 WO2024092126 A1 WO 2024092126A1
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domain
cell
composition
mhc
seq
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PCT/US2023/077916
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English (en)
Inventor
Michael Yi
Boi QUACH
Michael Bethune
Matthew SIEGEL
Nhung Nguyen
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Cargo Therapeutics, Inc.
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Publication of WO2024092126A1 publication Critical patent/WO2024092126A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector

Definitions

  • 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.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • WSGR Docket No.61078-716.601 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.
  • MHC major histocompatibility complex
  • 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.
  • MHC major histocompatibility complex
  • 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 .
  • 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.
  • the first extracellular domain of the MHC is a domain that binds to B2M.
  • the first domain is B2M or an MHC-binding fragment or variant thereof.
  • the second domain is CD8 or an MHC-binding fragment or variant thereof, or LILRB1 variant thereof.
  • 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%, or WSGR Docket No.61078-716.601 comprises an amino acid sequence set forth in SEQ ID NO.
  • the second domain or third domain binds to a second extracellular domain of the MHC that is different than the first extracellular domain.
  • the second extracellular domain of the MHC embodiments the MHC is an MHC class I.
  • the MHC is encoded by an HLA- A02:01 gene.
  • 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.
  • the cell-surface receptor is CD8, or wherein the cell-surface receptor is not TCRalpha/beta or TCR delta/gamma.
  • 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.
  • CAR-T chimeric antigen receptor T
  • 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).
  • 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.
  • the second domain is linked to the third domain by a third linker sequence.
  • the first domain is linked to the second domain and the first domain is linked to the third domain.
  • the second domain is linked to the first domain and the second domain is linked to the third domain.
  • the first, second, and or third linker sequence is at least 5 amino acids in length.
  • the first, second, and or third linker sequence is at most 30 amino acids in length.
  • the linker sequence is from 5 to 30 amino acids in length.
  • the third domain is a domain that binds to an immune checkpoint protein.
  • the third domain is a PDL1 or PDL2 domain.
  • 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 is WSGR 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.
  • compositions comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding the recombinant fusion protein of the composition described herein.
  • 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.
  • the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • 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.
  • the antigen binding domain is an anti-CD19 binding domain.
  • 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.
  • VL variable light chain domain
  • LCDR1 light chain CDR1
  • LCDR2 and LCDR3 of RASQDISKYLN
  • SRLHSGV and GNTLPYTFG SRLHSGV and GNTLPYTFG
  • VH variable heavy chain domain
  • the antigen binding domain is an anti-CD22 binding domain.
  • 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.
  • VL variable light chain domain having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively
  • HCDR1 heavy chain CDR1
  • HCDR2 and HCDR3 of GDSVSSNSAA TYYRSKWYN
  • AREVTGDLEDAFDI AREVTGDLEDAFDI
  • 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-H
  • 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.
  • the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.
  • the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.
  • 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.
  • the cell is a lymphocyte.
  • the cell is a T cell.
  • the cell is an allogeneic cell.
  • the cell comprises a disruption of NLRC5, RFX5, RFXANK, RFXAP, a viral immunoevasin, ICAM1, CD80, CD58, OX40L, and any combination thereof.
  • 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.
  • the cell is an allogeneic cell.
  • the cell is a population of cells.
  • the population of cells comprises at least 1x10 ⁇ 5 cells.
  • the pharmaceutical composition comprises a population of CAR-T cells.
  • the disease or condition is cancer, and or an auto- immune disease.
  • the cancer is lymphoma or leukemia.
  • the cancer is a solid tumor cancer.
  • 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 chrom
  • UCS
  • FIG. 1 coreceptor by a host T cell, thereby reducing TCR-pMHC avidity and recruitment of Lck to host TCR complex.
  • 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.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.
  • 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.
  • 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.
  • 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.
  • FIG.6B shows the flow cytometry results of B2M expression of the various constructs of anti- MHCI-B2M fusions in B2M KO T cells.
  • FIGS.7A-7C show the % survival of the TRAC KO and RFX5 KO graft T cells.
  • 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.
  • 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 CTFR low events counted by flow cytometry.
  • CTFR Cell Trace Far Red
  • compositions and methods comprising recombinant polypeptides and/or recombinant nucleic acids encoding the recombinant polypeptides, comprising recombinant Beta-2 microglobulin (B2M) fusion proteins.
  • B2M Beta-2 microglobulin
  • 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.
  • MHC major histocompatibility complex
  • the recombinant B2M fusion protein may further comprise a third domain that binds to an extracellular receptor on a second cell.
  • the recombinant B2M fusion protein may be expressed in a cell.
  • the cell may further comprise a chimeric antigen receptor (CAR).
  • the B2M fusion protein and/or CAR may comprise recombinant polypeptides and/or recombinant nucleic acids encoding the recombinant polypeptides.
  • the B2M fusion protein and/or CAR may be expressed in a cell.
  • the compositions and methods disclosed herein my further comprise the disruption of a gene in the recombinant cells described herein.
  • 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.
  • 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.
  • 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.
  • a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
  • polynucleotides 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.
  • 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.
  • loci locus 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.
  • 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.
  • 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.
  • thiol containing nucleotides 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.
  • loci locus
  • locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering
  • 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 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.
  • 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.
  • 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.
  • 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.
  • a “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
  • 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.
  • 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.
  • 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.
  • non-naturally occurring 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.
  • 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.
  • the terms “gene” or “gene fragment” are used interchangeably herein.
  • 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.
  • 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.
  • 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.
  • “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences.
  • 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.
  • binding domain refers to a molecule, such as a protein, or polypeptide sequence, which specifically binds to a target.
  • 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 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.
  • 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.
  • terapéuticaally 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.
  • 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).
  • “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “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.
  • 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.
  • 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.
  • the first domain may be B2M or an MHC-binding fragment or variant thereof.
  • 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.
  • the first domain comprises an amino acid sequence MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 1).
  • 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.
  • 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 comprise WSGR 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.
  • 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.
  • 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.
  • 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.
  • the first domain of the B2M fusion protein may be a binding domain 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • a KA of less than or equal to about 10 ⁇ 5 M-1.
  • 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.
  • 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.
  • the first domain of the B2M fusion protein may be a binding domain 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • the first domain of the B2M fusion protein WSGR Docket No.61078-716.601 M-1, 10 ⁇ 10 M-1, 10 ⁇ 11 M-1, 10 ⁇ 12 M-1, or 10 ⁇ 13 M-1.
  • the first domain of the B2M nM 500 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • 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.
  • KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • 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.
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • KA KA of less than or equal to about 10 ⁇ 5 M-1.
  • 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.
  • the first domain of the B2M nM 500 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • 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.
  • KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • 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.
  • the first domain of the B2M fusion protein may be a domain that binds to HLA-A.
  • the first domain of the B2M fusion protein binds to MHCI HLA-A with low affinity.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, WSGR Docket No.61078-716.601 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • the first domain of the B2M fusion protein binds to MHCI HLA-A with a high affinity.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • 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.
  • the first domain may be expressed in an allogeneic and/or grafted T-cell.
  • the first domain may inhibit killing of the grafted cells in which the first domain is WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first domain may be modified N-terminally or C-terminally.
  • the first domain may be operatively linked to a second domain by a linker.
  • 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.
  • the first domain may WSGR 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.
  • the first domain may be linked to three or more additional domains by additional linkers.
  • 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.
  • 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.
  • 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.
  • the first domain may anchor the recombinant B2M fusion protein to an MHC of a first cell.
  • the MHC may be a class 1 MHC molecule.
  • the class 1 MHC may be encoded by an HLA-A*02:01 gene.
  • 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.
  • 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.
  • 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.
  • the first and second domains are operatively linked by a linker domain.
  • 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.
  • the second domain may be a domain that bind to an immune checkpoint protein.
  • the second domain may be a PDL1 or PDL2 domain.
  • the second domain may be a domain that binds to PD1 or PD2.
  • WSGR Docket No.61078-716.601 [0070]
  • the second domain comprises CD8 or an MHC binding fragment or variant thereof.
  • the second domain can comprise CD8alpha or an MHC binding fragment or variant thereof.
  • the second domain comprises an amino acid sequence MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPR GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 2).
  • the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2.
  • 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.
  • the second domain is a CD8alpha domain or an MHC-binding fragment or variant thereof.
  • 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.
  • the second domain comprises two or more CD8alpha domains or an MHC-binding fragments or variants thereof.
  • the second domain comprises two or more CD8alpha domains or an MHC-binding fragments or variants thereof that are linked together.
  • the second domain is linked to the first domain via a linker.
  • 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.
  • the second domain can comprise CD8beta or an MHC binding fragment or variant thereof.
  • the second domain comprises an amino acid sequence MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQA PSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPEL TFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSL GVAIHLCCRRRRARLRFMKQFYK (SEQ ID NO: 2B).
  • the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 2B.
  • 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.
  • the second domain is a CD8beta domain or an MHC-binding fragment or variant thereof.
  • 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.
  • the second domain comprises two or more CD8beta domains or WSGR Docket No.61078-716.601 an MHC-binding fragments or variants thereof.
  • 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.
  • 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.
  • 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.
  • the second domain is linked to the C terminus of the first domain.
  • the second domain is linked to the N terminus of the first domain.
  • the second domain is linked to a linker at the C terminus of the first domain.
  • the second domain is linked to a linker at N terminus of the first domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the recombinant protein comprises, from N to C terminus: (i) CD8alpha or an MHC WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the second domain comprises LILRB1 or MHC binding fragment or variant thereof.
  • the second domain comprises an amino acid sequence MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYR EKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKP WSGR Docket No.61078-716.601 TLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVS PSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDA GYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAP SDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRS TYQSQK
  • 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.
  • an antibody e.g. W6/32
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the second domain comprises CD160 or an MHC binding fragment or variant thereof.
  • the second domain comprises an amino acid sequence MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEEAEGFVVFLCKD RSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGTYQCCARSQKSGIRLQGH FFSILFTETGNYTVTGLKQRQHLEFSHNEGTLSSGFLQEKVWVMLVTSLVALQAL (SEQ ID NO: 5).
  • the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 5.
  • 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.
  • the second domain comprises an amino acid sequence MRYMILGLLALAAVCSAAKKVEFKEPACNVTFKSEANECTTLIKCTTEHEKLIIRHKDKIGK WSGR Docket No.61078-716.601 YAVYAIWQPGDTNDYNVTVFQGENRKTFMYKFPFYEMCDITMYMSKQYKLWPPQKCLEN TGTFCSTALLITALALVCTLLYLKYKSRRSFIDEKKMP (SEQ ID NO: 6).
  • the second domain comprises a portion of the amino acid sequence according to SEQ ID NO: 6.
  • 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.
  • 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.
  • 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.
  • the second domain comprises an amino acid sequence MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMED KNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGAD YKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNS KREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILL CLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 7).
  • 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.
  • the second domain comprises an amino acid sequence MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVE WSGR Docket No.61078-716.601 NDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRK INTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGR NFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYS SKDTTKRPVTTTKREVNSAI (SEQ ID NO: 8).
  • 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.
  • the domain that binds to CTLA-4 comprises an amino acid sequence MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINT TVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWA ITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV(SEQ ID NO: 9).
  • 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.
  • the domain that binds to CTLA-4 comprises an amino acid sequence MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQE NLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRI HQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQ KSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVL PTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFK SSKTSSCDKSDTCF (SEQ ID NO: 10).
  • 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 some WSGR 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.
  • the second domain that binds to LAG3 comprises an amino acid sequence MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEK TKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPV DPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 11).
  • the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 11.
  • 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.
  • the second domain comprises Galectin-3 or a LAG-3 binding fragment or variant thereof.
  • the second domain that binds to LAG3 comprises an amino acid sequence MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAP PGAYPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLP LPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWG REERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSA SYTMI (SEQ ID NO: 12).
  • 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.
  • the second domain that binds to LAG3 comprises an amino acid sequence MDTTRYSKWGGSSEEVPGGPWGRWVHWSRRPLFLALAVLVTTVLWAVILSILLSKASTER AALLDGHDLLRTNASKQTAALGALKEEVGDCHSCCSGTQAQLQTTRAELGEAQAKLMEQE SALRELRERVTQGLAEAGRGREDVRTELFRALEAVRLQNNSCEPCPTSWLSFEGSCYFFSVP KTTWAAAQDHCADASAHLVIVGGLDEQGFLTRNTRGRGYWLGLRAVRHLGKVQGYQWV DGVSLSFSHWNQGEPNDAWGRENCVMMLHTGLWNDAPCDSEKDGWICEKRHNC (SEQ ID NO: 13).
  • the second domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 13.
  • the second domain WSGR 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.
  • the second domain comprises fibrinogen-like protein 1 domain or a LAG-3 binding fragment or variant thereof.
  • the second domain that binds to LAG3 comprises an amino acid sequence MAKVFSFILVTTALTMGREISALEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQ FLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVI QRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNS RYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHD NYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVV MKIRPNDFIPNVI (SEQ ID NO: 14).
  • 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.
  • PtdSer galectin 9, phosphatidylserine
  • HMGB1 high mobility group protein B1
  • the domain that binds to TIM3 may be a galectin 9, phosphatidylserine (PtdSer), CEACAM1 or high mobility group protein B1 (HMGB1) domain.
  • the second domain comprises galectin 9 or a TIM3 binding fragment or variant thereof.
  • the second domain that binds to TIM3 comprises an amino acid sequence MAFSGCQAPYLSPAVPFSGTIQGGLQDGFQITVNGAVLSCSGTRFAVDFQTGFSGNDIAFHF NPRFEDGGYVVCNTRQKGTWGPEERKMHMPFQKGMPFDLCFLVQSSDFKVMVNGSLFVQ YFHRVPFHRVDTISVNGSVQLSYISFQNPRAVPVQPAFSTVPFSQPVCFPPRPRGRRQKPPSV RPANPAPITQTVIHTVQSASGQMFSQTPAIPPMMYPHPAYPMPFITTIPGGLYPSKSIILSGTVL PSAQRFHINLCSGSHIAFHMNPRFDENAVVRNTQINNSWGSEERSLPRKMPFVRGQSFSVWI LCEAHCLKVAVDGQHVFEYYHRLRNLPTINKLEVGGDIQLTHVQT (SEQ ID NO: 15).
  • 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.
  • PtdSer phosphatidylserine
  • the second domain that binds to TIM3 WSGR Docket No.61078-716.601 comprises an amino acid sequence MASCVGSRTLSKDDVNYKMHFRMINEQQVEDITIDFFYRPHTITLLSFTIVSLMYFAFTRDDS VPEDNIWRGILSVIFFFLIISVLAFPNGPFTRPHPALWRMVFGLSVLYFLVFLLFLNFEQVKS LMYWLDPNLRYATREADVMEYAVNCHVITWERIISHFDIFAFGHFWGWAMKALLIRSYGL CWTISITWELTELFFMHLLPNFAECWWDQVILDILLCNGGGIWLGMVVCRFLEMRTYHWAS FKDIHTTTGKIKRAVLQFTPASWTYVRWFDPKSSFQRVAGVYLFMIIWQLTELNTFFLKHIF VFQASHPLSWGRILFIGGITAPTVRQYYAYLTDTQCKRVGTQCWVFGVIGFLEAIVCIKFGQ DLFSKTQILYVV
  • 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.
  • the second domain that binds to TIM3 comprises an amino acid sequence MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQ LFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTL QVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQS LPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDT YYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCN RTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERM KLSQGNTTLSINPVKREDAG
  • 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.
  • HMGB1 high mobility group protein B1
  • the second domain that binds to TIM3 comprises an amino acid sequence MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSI GDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEK WSGR Docket No.61078-716.601 SKKKKEEEEDEEDEEDEEEEEEEDEEDEDEEEDDDDE (SEQ ID NO: 18).
  • 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.
  • the second domain that binds to TIGIT comprises an amino acid sequence MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVT HVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEG NYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWH SDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVY YPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPV DKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRNAIIFLVLGILVFLILLGIGIYFYW SKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSS
  • 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.
  • the second domain that binds to TIGIT comprises an amino acid sequence MARAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPPVPG LYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDA TLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCI SKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFE EPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAV AQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGGIIGGIIAAIIA TAVAATGILICRQQRKEQTLQGAEEDEDLEGPPSYKPP
  • 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.
  • the second domain that binds to TIGIT comprises an amino acid sequence MARTLRPSPLCPGGGKAQLSSASLLGAGLLLQPPTPPPLLLLLFPLLLFSRLCGALAGPIIVEP HVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKN YSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETV AAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIISQYKLFPTRFARGRRITCVVKHPA LEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPD GLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADI EDLATEPKKLPFPLSTLATIKDDTIATIIASVVGGA
  • 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 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • 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.
  • 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.
  • the second domain of the B2M fusion protein WSGR 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.
  • KA of greater than or equal to about 200 nM, 300 nM, 400 nM, 500 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M.
  • KA of greater than or equal to about 10 ⁇ 5 M-1.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, 75 ⁇ M, 100 ⁇ M, 125 ⁇ M, 140 ⁇ M or 150 ⁇ M or greater than 150 ⁇ M.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the second cell may be a host cell.
  • the first cell may be an allogeneic and/or grafted cell.
  • the cell surface receptor of the second cell may be a CD8 receptor.
  • the cell-surface receptor is not TCRalpha/beta or TCR delta/gamma.
  • 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.
  • 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.
  • 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.
  • 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 domain WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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 domain WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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 any WSGR Docket No.61078-716.601 additional domains.
  • 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
  • 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.
  • 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.
  • the recombinant B2M fusion protein may comprise a third domain that binds to an immune checkpoint protein.
  • 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.
  • 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.
  • the third domain may be a domain that bind to an immune checkpoint protein.
  • the third domain may be a PDL1 or PDL2 domain.
  • the third domain may be a domain that binds to PD1 or PD2.
  • the third domain comprises an amino acid sequence of SEQ ID NO: 7.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 7.
  • 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.
  • the third domain may be a domain that binds to CTLA-4.
  • 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.
  • 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.
  • 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.
  • 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.
  • the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 12.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 12.
  • 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.
  • the third domain that binds to LAG3 comprises an amino acid sequence SEQ ID NO: 13.
  • 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.
  • the third domain may be a domain that binds to TIM3.
  • the domain that binds to TIM3 may be a galectin 9, phosphatidylserine (PtdSer), CEACAM1 or high mobility group protein B1 (HMGB1) domain.
  • the third WSGR Docket No.61078-716.601 domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 15.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 15.
  • 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.
  • 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.
  • 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.
  • the third domain that binds to TIM3 comprises an amino acid sequence SEQ ID NO: 18.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 18.
  • 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.
  • the third domain may be a domain that binds to TIGIT.
  • the domain that binds to TIGIT may be a CD155, CD112, or CD113 domain.
  • the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 19.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 19.
  • 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.
  • the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 20.
  • the third domain comprises an amino acid sequence with at least about 80% sequence identity to SEQ ID NO: 20.
  • 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.
  • the third domain that binds to TIGIT comprises an amino acid sequence SEQ ID NO: 21.
  • the third domain comprises an amino acid sequence with at WSGR Docket No.61078-716.601 least about 80% sequence identity to SEQ ID NO: 21.
  • 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.
  • the third domain may be a domain that binds to a T-cell receptor.
  • the third domain may be an intracellular signaling domain.
  • the stalk domain comprises an amino acid sequence MAAPAVSGLSRQVRCFSTSVVRPFAKLVRPPVQVYGIEGRYATALYSAASKQNKLEQVEKE LLRVAQILKEPKVAASVLNPYVKRSIKVKSLNDITAKERFSPLTTNLINLLAENGRLSNTQGV VSAFSTMMSVHRGEVPCTVTSASPLEEATLSELKTVLKSFLSQGQVLKLEAKTDPSILGGMI VRIGEKYVDMSVKTKIQKLGRAMREIV(SEQ ID NO: 22).
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, and/or 50 ⁇ M.
  • 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.
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, or greater than 50 ⁇ M.
  • 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.
  • 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]
  • the third domain of the B2M fusion protein binds to an immune checkpoint protein with a high affinity.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, and/or 50 ⁇ M.
  • 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.
  • 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.
  • 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, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, 50 ⁇ M, or greater than 50 ⁇ M.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the third domain may inhibit killing of the grafted cells in which it is expressed by at least about 30% compared to grafted cells in WSGR 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.
  • 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.
  • 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.
  • the third domain may be linked to three or more additional domains by additional linkers.
  • 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.
  • 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.
  • 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.
  • the additional domain is an intracellular signaling domain.
  • 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.
  • 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.
  • the cross-linking ties MHC/B2M complexes together irrespective to the peptide presented by the complex.
  • the cross-linking inhibits or prevents cognate TCRs from clustering MHCI/B2M complexes presenting a same peptide into a functional cell-to-cell synapse.
  • the first domain may be B2M or an MHC- binding fragment or variant thereof.
  • 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.
  • the first domain of a plurality of recombinant fusion proteins may be cross-linked.
  • 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.
  • the first domain may be cross-linked to a cell membrane.
  • the cell membrane may be the cell membrane of the cell in which the first domain is expressed.
  • the second domain may be cross-linked to a cell membrane.
  • the cell membrane may be the cell membrane of the cell in which the first domain is expressed.
  • the third domain may be cross-linked to a cell membrane.
  • the cell membrane may be the cell membrane of the cell in which the first domain is expressed.
  • the additional domain may be cross-linked to a cell membrane.
  • the cell membrane may be the cell membrane of the cell in which the first domain is expressed.
  • cross-linking the first domain may comprise cytoskeletal anchoring modifications.
  • the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin.
  • the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain.
  • the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.
  • cross-linking the second domain may comprise cytoskeletal anchoring modifications.
  • 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.
  • the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.
  • cross-linking the additional domain may comprise cytoskeletal anchoring modifications.
  • the cytoskeletal anchoring modifications may comprise anchoring the first domain to a talin.
  • the cytoskeletal anchoring modifications may comprise anchoring the first domain to a ITGB3 cytoplasmic domain.
  • the cytoskeletal anchoring modification may comprise anchoring the first domain to a CD44 cytoplasmic domain.
  • 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.
  • a cross linker can create/form a strong chemical bond (for example, a covalent bond) between biopolymer chains, such as nucleic acids and proteins.
  • 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.
  • 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-
  • 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.
  • 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.
  • 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 a WSGR 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.
  • 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.
  • 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.
  • the cross-linked first domain may be expressed in an allogeneic and/or grafted T-cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 about WSGR 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.
  • 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.
  • 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]
  • a recombinant B2M fusion protein may comprise a linker domain.
  • a recombinant B2M fusion protein may comprise a plurality of linker domains.
  • the first domain may be modified N-terminally or C-terminally.
  • the first domain may be operatively linked to a second domain by a linker.
  • 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.
  • 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.
  • 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.
  • the third domain may be linked to three or more additional domains by additional linkers.
  • additional domain may be operatively linked to a first domain by a linker.
  • 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.
  • the additional domain may be linked to a first domain, and/or a second domain, and/or a third domain by a third linker.
  • the additional domain may be linked to three or more additional domains by additional linkers.
  • 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.
  • the linker there are no particular limitations to the length and/or amino acid composition of the linker.
  • 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.
  • 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.
  • 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.
  • 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]
  • 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.
  • a cell expressing a B2M fusion protein further comprises the CAR.
  • 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.
  • a recombinant nucleic acid encoding a recombinant polypeptide comprising a B2M WSGR 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.
  • the antigen is an antigen expressed by a tumor cell.
  • the antigen binding domain comprises a scFv, a nanobody, a ligand, or a receptor.
  • 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.
  • 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 (2016) 21(5):455-64 (CD19 nanobody); F.
  • 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).
  • the antigen binding domain specifically binds to CD19.
  • the antigen binding domain is an anti-CD19 binding domain.
  • 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.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to WSGR Docket No.61078-716.601 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.
  • 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.
  • the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 amino WSGR Docket No.61078-716.601 acid sequence with at least 96% identity of SEQ ID NO: 33.
  • 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.
  • the antigen binding domain is an anti-CD22 binding domain.
  • 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.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFS GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEI.
  • 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.
  • VH variable heavy chain domain
  • 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.
  • VH variable heavy chain domain
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK.
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFS WSGR Docket No.61078-716.601 GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKGGGGSQVQLQQSGPGLVK PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINP DTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS.
  • 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.
  • the antigen binding domain comprises an scFv with 100% sequence identity QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK.
  • the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 34.
  • 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.
  • 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.
  • 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 amino WSGR 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.
  • 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.
  • 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, B
  • an antigen that is selected
  • the antigen binding domain of a CAR provided herein is operatively linked to a transmembrane domain by a hinge domain.
  • the antigen binding domain of a CAR provided herein is directly linked to a transmembrane domain by a hinge domain.
  • the hinge domain of a CAR provided herein is from CD28.
  • the hinge domain of a CAR provided herein has the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.
  • the hinge domain of a CAR or provided herein is from CD8.
  • the hinge domain of a CAR provided herein has the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY.
  • 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.
  • 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.
  • the hinge domain includes a CD8a or CD28 hinge region.
  • the hinge is a PD-1 hinge or CD152 hinge.
  • 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.
  • 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.
  • 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.
  • the hinge domain is linked downstream of the antigen-binding domain of a CAR and upstream of the transmembrane domain of a CAR.
  • suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4.
  • 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.
  • the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain.
  • the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain.
  • 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.
  • the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and the WSGR Docket No.61078-716.601 extracellular hinge domain.
  • the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain.
  • the length and/or amino acid composition of the linker there are no particular limitations to the length and/or amino acid composition of the linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.
  • 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.
  • a transmembrane domain such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof.
  • 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.
  • a short oligo- or polypeptide linker 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.
  • 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.
  • a CAR may further include a spacer domain between the antigen- binding portion and the transmembrane domain, e.g., a CD8a hinge.
  • the CAR comprises a transmembrane domain from CD28.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intracellular signaling domain consists of an amino acid sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRP PPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB (CD137).
  • 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.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2.
  • 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.
  • the CAR comprises an intracellular domain comprising a truncated CD3epsilon intracellular domain.
  • TCR T cell receptor
  • 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.
  • 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.
  • the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.
  • 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”).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • a CAR includes a CD3zeta primary signaling domain and one or more costimulatory signaling domains.
  • a CAR includes a 4-1BB costimulatory signaling domain.
  • the intracellular primary signaling and WSGR Docket No.61078-716.601 costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.
  • a CAR lacks a CD2 intracellular signaling domain.
  • 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.
  • 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.
  • 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.
  • the CAR comprises two or more intracellular signaling domains.
  • 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.
  • 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.
  • 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.
  • 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 intracellular WSGR Docket No.61078-716.601 signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.
  • 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.
  • 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,
  • 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 a4b
  • 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.
  • the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an WSGR Docket No.61078-716.601 intracellular domain comprising a CD28 zeta intracellular signaling domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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]
  • 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 plasma WSGR 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.
  • the protein localization tag is an ER localization tag.
  • the ER localization tag comprises an amino acid sequence of SEQ ID NO: 23.
  • the ER localization tag consists of an amino acid sequence of SEQ ID NO: 23.
  • the ER localization tag comprises an amino acid sequence of SEQ ID NO: 24.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ER localization tag WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the ER localization tag consists of an amino acid sequence of SEQ ID NO: 40.
  • 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.
  • 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.
  • 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).
  • the lysosome localization tag consists of the amino acid sequence KFERQ (SEQ ID NO: 39).
  • 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.
  • a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids.
  • 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.
  • 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.
  • 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.
  • metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases.
  • the protease cleavage site is TEV protease cleavage site.
  • Disruption of Genes WSGR Docket No.61078-716.601 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.
  • the gene may be a gene that encodes a T-cell receptor.
  • 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.
  • the disruption of the gene may include administering a gene editing platform to a recombinant cell of the disclosure.
  • the disruption of the gene may include knock-down (KD), knock-out (KO), and/or knock-in and or overexpression of the gene.
  • 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.
  • 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.
  • gene editing technology may include RNA interference (RNAi), including small-interfering RNAs (siRNAs).
  • RNAi RNA interference
  • siRNAs small-interfering RNAs
  • gene editing technologies may include microRNAs (miRNAs).
  • CRISPR/cas Gene Editing Systems 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.
  • the CRISPR sequence sometimes called a CRISPR locus, comprises alternating repeats and spacers.
  • the 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.
  • 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.
  • the Cse (Cas subtype, E. coli) proteins form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains.
  • Cascade a functional complex
  • Cascade processes CRISPR transcripts into spacer-repeat units that Cascade retains.
  • 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.
  • Cas9 is a nuclease with two active cutting sites, one for each strand of the double helix.
  • the Cas9 is derived from a S. pyogenes Cas9.
  • 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.
  • the RNA can guide the Cas protein to a target promoter, sterically blocking RNA polymerases.
  • TALEN Gene Editing System refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene.
  • 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.
  • TALEs Transcription activator-like effects
  • 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.
  • 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.
  • a TALE protein is fused to a nuclease (N), e.g., a wild-type or mutated FokI endonuclease.
  • N nuclease
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ZFN Zinc Finger Nuclease Gene Editing System WSGR Docket No.61078-716.601
  • ZFN Zinc Finger Nuclease Gene Editing System WSGR Docket No.61078-716.601
  • ZFN Zinc Finger Nuclease or “Zinc Finger Nuclease” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • a targeting RNA is fewer than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the targeting RNA comprises a sequence of 10 nucleic acids.
  • the targeting RNA comprises a sequence of 11 nucleic acids.
  • the targeting RNA comprises a sequence of 12 nucleic acids.
  • the targeting RNA comprises a sequence of 13 nucleic acids.
  • the targeting RNA comprises a sequence of 14 nucleic acids.
  • the targeting RNA comprises a sequence of 15 nucleic acids.
  • the targeting RNA comprises a sequence of 16 nucleic acids.
  • 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 19 WSGR 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.
  • the targeting RNA comprises a sequence of 25 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 26 nucleic acids.
  • 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 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.
  • the components of an gene editing system as described herein 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.
  • 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.
  • a targeting RNA may be selected to target any target DNA sequence.
  • 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.
  • 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.
  • a targeting RNA is selected to reduce the degree of secondary structure within the targeting RNA. Secondary structure may be determined by any suitable polynucleotide WSGR Docket No.61078-716.601 folding algorithm. Some programs are based on calculating the minimal Gibbs free energy.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the Guide RNA refers to an RNA aptamer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA molecules that bind to specific targets of interest 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).
  • SELEX Fitzwater et al., Methods Enzymol., vol.267, pp.275-301 (1996), which is incorporated herein in its entirety.
  • 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 [0162]
  • the gene editing systems of the disclosure may include a system for RNA interference.
  • 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.
  • RNAi may comprise siRNA.
  • 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.
  • 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 effective WSGR Docket No.61078-716.601 intramolecular hybridization of the stem elements. Typically, the length to be spanned is at least about 10-24 atoms.
  • RNAi may include shRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • the oligonucleotide is a mimic of a naturally occurring miRNA, or an analog or homolog thereof.
  • 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.
  • 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.
  • a gene editing system, and/or gene expression modulator of the present disclosure may be provided to a cell.
  • the gene editing system, and/or gene expression modulator may be provided to a recombinant cell of the disclosure.
  • 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.
  • 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).
  • the gene may be a gene that encodes an MHC.
  • the gene may be a gene that encodes an extracellular domain of a T-cell receptor or MHC.
  • the gene may be a gene that encodes an intracellular receptor of a T-cell receptor or MHC.
  • the gene may be selected from the group consisting of TCRalpha, TCRbeta, CD80, CD58, OX40L, and any combination thereof.
  • shRNA [0249]
  • T-cell receptor expression may be decreased using small-hairpin RNAs chains (e.g., CD3 zeta) T cells.
  • targeting shRNAs have been designed for key components of the TCR complex.
  • an shRNA targeting CD3 epsilon can comprise the sequence ctggaggcttgctgaaggctgtatgctgAACGCCAACTGATAAGAGGCAgttttggccactgactgacTGCCTCTTC AGTTGGCGTTcaggacacaaggcctgttactagcactcacatggaacaaatggccca.
  • 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).
  • shRNAs small-hairpin RNAs
  • an shRNA targeting B2M can comprise the sequence: ctggaggcttgctgaaggctgtatgctgAATCTTTGGAGTACGCTGGATgttttggccactgactgacATCCAGCGCT CCAAAGATTcaggacacaaggcctgttactagcactcacatggaacaaatggccca.
  • Dominant Negative Inhibition [0251]
  • over-expression of a dominant-negative inhibitor protein may be capable of interrupting T cell receptor expression and/or function.
  • a minigene that incorporates part, or all, of a polynucleotide encoding for one of the TCR components is prepared, but is modi-fied so WSGR 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).
  • TCR- alpha, TCR-beta, CD3-gamma, CD3-delta, CD3-epsilon, or CD3-zeta is prepared, but is modi-fied so WSGR 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
  • 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.
  • inhibitory signal motif e.g., a cytoplasmic domain from a KIR protein
  • phosphatases e.g., SHP1 and SHP2.
  • minigenes may also encode a portion of a protein that serves as a means to identify the over-expressed minigene.
  • 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.
  • over-expression of a dominant-negative inhibitor protein may be capable of interrupting MHC expression and/or function.
  • a minigene that incorporates be used.
  • 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.
  • DN dominant negative protein
  • 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%.
  • 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, the WSGR 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%.
  • 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%.
  • 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%.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about WSGR 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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, the WSGR 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%.
  • 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%.
  • 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%.
  • 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.
  • the composition may comprise any combination of the recombinant polypeptides and recombinant polynucleic acids disclosed herein.
  • WSGR Docket No.61078-716.601 Cells [0259]
  • 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.
  • the recombinant fusion protein may be a B2M fusion protein.
  • the cell may express a B2M fusion protein and/or chimeric antigen receptor disclosed herein.
  • the recombinant B2M fusion protein and/or CAR are expressed on the surface of the cell.
  • the MHC molecule may be a class 1 MHC molecule.
  • the extracellular binding domain 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.
  • the intracellular signaling domain of the cell surface receptor participates in transducing the signal from the binding into the interior of the cell.
  • 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.
  • a vector construct of the present disclosure 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.
  • the cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell.
  • the cell is autologous or allogeneic.
  • 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.
  • Cells may be transduced/transfected with a polynucleic acid encoding the recombinant B2M fusion protein and/or CAR.
  • a cell may be transduced with a bicistronic nucleic acid encoding a B2M fusion protein and a CAR.
  • a cell may be transduced with a nucleic acid encoding a B2M fusion protein and an additional nucleic acid encoding a CAR.
  • 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.
  • 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]
  • allogeneic T-cells may be isolated and cultured ex vivo.
  • the allogeneic T-cells may comprise a CAR of the disclosure.
  • a B2M fusion protein may be administered to the cultured CAR-T cell.
  • a B2M fusion protein may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient.
  • the cultured, B2M/CAR-T cell may be administered to a patient.
  • autologous T-cells may be isolated from a subject, and cultured ex vivo.
  • the autologous T-cells may comprise a CAR of the disclosure.
  • a B2M fusion protein may be administered to the cultured CAR-T cell.
  • a B2M fusion protein may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient.
  • the cultured, B2M/CAR-T cell may be administered to a patient.
  • cells of the disclosure may comprise a CAR, and/or a B2M fusion protein, and/or a disruption of a gene.
  • 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.
  • the cell comprising disruption of a gene may be a T-cell.
  • the cell comprising disruption of a gene may further comprise a CAR.
  • 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.
  • the recombinant cell is an animal cell.
  • the animal cell may be a mammalian cell.
  • the animal cell may be a mouse cell.
  • the animal cell may be a human cell.
  • 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.
  • 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.
  • TIL tumor-infiltrating lymphocyte
  • the immune system cell may be selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells.
  • 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.
  • 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.
  • the nucleic acid molecule may be introduced into a host cell by a transduction procedure, transfection procedure, electroporation procedure, or a biolistic procedure.
  • cell cultures including at WSGR 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.
  • Cells of the present disclosure may be autologous/autogeneic (“self”) or non-autologous (“non- self,” e.g., allogeneic, syngeneic or xenogeneic).
  • autologous refers to cells derived from the same individual to which they are subsequently administered.
  • Allogeneic refers to cells of the same species that differ genetically from the cell in comparison.
  • T cells refers to cells of a different individual that are genetically identical to the cell in comparison.
  • the cells are T cells obtained from a mammal.
  • the mammal is a primate.
  • the primate is a human.
  • 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.
  • 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.
  • an isolated or purified population of T cells may be used.
  • TCTL and TH lymphocytes are purified from PBMCs.
  • 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 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+.
  • MCS magnetic-activated cell sorting
  • TN are CD45RA+ CD62L+ CD95-
  • TSCM are CD45RA+ CD62L+ CD95+
  • TCM are CD45RO+ CD62L+ CD95+
  • TEM are CD45RO+ CD62L- CD95+.
  • An exemplary approach for such sorting may be described in Wang et al. (2016) Blood 127(24):2980- 90.
  • 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
  • a specific subpopulation of T cells may be further isolated by positive or negative selection techniques.
  • compositions 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 wherein WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell is a mammalian cell.
  • the cell can be a human cell.
  • the cell can be a blood cell.
  • the blood cell is a lymphocyte.
  • 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 1x10 5 cells. In some embodiments, the population of cells comprises at least about 1x10 6 cells. In some embodiments, the population of cells comprises at least about 1x10 7 cells. In some embodiments, the population of cells comprises at least about 1x10 8 cells.
  • the population of cells comprises at least about 1x10 9 cells. In some embodiments, the population of cells comprises from about 1x10 5 cells to about 1x10 9 cells. In some embodiments, the population of cells comprises from about 1x10 5 cells to about 1x10 8 cells. In some embodiments, the population of cells comprises from about 1x10 5 cells to about 1x10 7 cells. In some embodiments, the population of cells comprises from about 1x10 5 cells to about 1x10 6 cells.
  • 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.
  • the present disclosure describes a pharmaceutical composition comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and a pharmaceutically acceptable excipient or carrier.
  • 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.
  • terapéuticaally 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 of WSGR 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.
  • 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).
  • a pharmaceutical composition of the present disclosure includes from 1x10 5 to 5x10 10 of the cells of the present disclosure, or greater than 5x10 10 cells of the present disclosure.
  • 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.
  • 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.
  • parenteral e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.
  • 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 ge
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20 ) and polysorbate 80 (sold under the trademark Tween 80 ).
  • Suitable polyethylene- polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188 .
  • 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.
  • 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.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • Methods of Treatment WSGR Docket No.61078-716.601 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).
  • agents e.g., an antibody or an antigen binding fragment thereof, or a molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • the disease, disorder, or condition is cancer.
  • the cancer is lymphoma or leukemia.
  • 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).
  • 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.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • B-cell prolymphocytic leukemia precursor B lymphoblastic leukemia
  • hairy cell leukemia hairy cell leukemia
  • DLBCL diffuse large B- cell lymphoma
  • follicular lymphoma marginal
  • 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, an
  • 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.
  • 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.
  • a systemic route of administration such as an intraperitoneal or intravenous route.
  • the terms “individual”, “subject,” “host” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • 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.
  • 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.
  • 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.
  • the cell population being administered can be allogeneic blood cells, hematopoietic stem cells, hematopoietic progenitor cells, embryonic WSGR 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.
  • administerered 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 ⁇ 10 4 cells 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.
  • the route is intravenous.
  • administration by injection or infusion can be made.
  • 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.
  • the efficacy of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician.
  • 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.
  • administration of a pharmaceutical composition may comprise administering a B2M fusion protein to a subject.
  • administration of a pharmaceutical composition may comprise administering a B2M fusion protein to a subject, and administering a WSGR Docket No.61078-716.601 population of CAR-T cells to the subject.
  • 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.
  • 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.
  • the polynucleotide may be introduced into a cell via any other delivery system.
  • the polynucleotide may be introduced into a cell using a cell transformation method.
  • the polynucleotide may be introduced into a cell using a cell transduction method.
  • 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.
  • 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.
  • 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.
  • chemical transfection methods are techniques that catalyze DNA cross-membrane transport.
  • chemical methods may use Ca2+phosphate, polycations or dendrimers including for WSGR 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.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell.
  • 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.
  • 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.
  • 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.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above.
  • the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell.
  • the recombinant cell or recombinant vector may comprise a selectable biomarker.
  • the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.
  • the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker.
  • the selectable biomarker may be an antibiotic cassette.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • mScarlet fluorescent protein gene mScarlet fluorescent protein gene
  • red fluorescent protein (RFP) gene red fluorescent protein
  • iRFP infrared fluorescent protein
  • CFP yellow fluorescent protein
  • YFP yellow fluorescent protein
  • Cy5.5 fluorescent protein gene Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art.
  • antibiotic selectable resistance marker gene examples 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.
  • 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.
  • 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.
  • endonuclease polypeptide(s) may be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles.
  • 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.
  • 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.
  • LNP lipid nanoparticle
  • 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.
  • 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 as WSGR 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.
  • LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.
  • 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.
  • the lipids may be combined in any number of molar ratios to produce a LNP.
  • the polynucleotide(s) may be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may each be administered separately to a cell or a patient.
  • the site- directed polypeptide such as a CRISPR/cas9 polypetide
  • 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).
  • RNP ribonucleoprotein particle
  • RNA is capable of forming specific interactions with RNA or DNA.
  • RNPs ribonucleoprotein particles
  • the endonuclease in the RNP may be modified or unmodified.
  • the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerous modifications are known in the art and may be used.
  • the endonuclease and sgRNA may be generally combined in a 1:1 molar ratio.
  • Adeno-Associated Virus 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,.
  • 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.
  • rAAV 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.
  • a method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production.
  • 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.
  • Suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.
  • adenovirus or baculovirus rather than plasmids
  • rAAV production is 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.
  • 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 2 WSGR Docket No.61078-716.601 [0316]
  • 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.
  • 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.
  • Cas9 mRNA is formulated in a lipid nanoparticle, while sgRNA and donor DNA are delivered in an AAV vector.
  • Cas9 mRNA and sgRNA are co- formulated in a lipid nanoparticle, while donor DNA is delivered in an AAV vector.
  • 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.
  • 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.
  • MVE multivesicular-endosomes
  • 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.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may be introduced into a cell using biological methods.
  • 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.
  • the polynucleotide may be introduced into a cell via any other delivery system.
  • the polynucleotide may be introduced into a cell using a cell transformation method.
  • the polynucleotide may be introduced into a cell using a cell transduction method.
  • 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.
  • 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.
  • 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.
  • chemical transfection methods are techniques that catalyze DNA cross-membrane transport.
  • 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.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell.
  • the polynucleotide integrating into the cell may be single stranded.
  • the polynucleotide integrating WSGR Docket No.61078-716.601 may be double stranded DNA.
  • the polynucleotide may be short nucleotide sequences.
  • the polynucleotide may be long nucleotide sequences.
  • the integration into the genome of the cell may be transient integration in the cell.
  • the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell.
  • the polynucleotide may integrate into the cell genome within a random locus.
  • the polynucleotide may integrate into the cell genome within a directed or targeted locus.
  • the polynucleotide may replicate when the cell genome replicates.
  • 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.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above.
  • the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell.
  • the recombinant cell or recombinant vector may comprise a selectable biomarker.
  • the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.
  • the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker.
  • the selectable biomarker may be an antibiotic cassette.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • mScarlet fluorescent protein gene mScarlet fluorescent protein gene
  • red fluorescent protein (RFP) gene red fluorescent protein
  • iRFP infrared fluorescent protein
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • Cy5.5 fluorescent protein gene Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art.
  • kits 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.
  • 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.
  • 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.
  • the expression vector when the expression vector encodes a recombinant polypeptide that does not comprise the protease (trans configuration), the expression vector further encodes the protease.
  • the expression vector is configured to express the recombinant polypeptide and the protease from the same promoter.
  • 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.
  • 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.
  • 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.
  • the instructions of the kits may be recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • 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.
  • 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.
  • 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.
  • the means for obtaining the instructions is recorded on a suitable substrate.
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • MHC major histocompatibility complex
  • 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 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.
  • the first extracellular domain of the MHC is a domain that binds to B2M. 10.
  • composition of any one of embodiments 1-9, wherein the first domain is B2M or an MHC- binding fragment or variant thereof.
  • 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.
  • 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.
  • an MHC-binding fragment or variant thereof is an MHC-binding fragment or variant thereof.
  • VL chain domain 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.
  • VH variable heavy chain domain 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.
  • 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.
  • 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.
  • 33. The composition of embodiment 32, wherein the first domain is linked to the third domain by a second linker sequence.
  • 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.
  • composition of embodiment 45 wherein the additional domain comprises a domain from WSGR 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.
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • the antigen binding domain is an anti-CD19 binding domain.
  • 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.
  • VL variable light chain domain
  • LCDR1 light chain CDR1
  • LCDR2 and LCDR3 of RASQDISKYLN
  • SRLHSGV and GNTLPYTFG SRLHSGV and GNTLPYTFG
  • VH variable heavy chain domain having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG
  • VL variable light chain domain having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of WSGR 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.
  • 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, B
  • CD137 CD137
  • CD28 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, SLP
  • composition of any one of embodiments 55-61, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.
  • 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 dominant negative (DN) protein is selected from the group consisting of DN-RFX5, DN-RFXANK, DN-RFXAP, a DN-viral immunoevas
  • composition of any one of embodiments 64-69, wherein the cell is an allogeneic cell.
  • the composition of any one of embodiments 64-70, wherein the cell is a population of cells.
  • the composition of embodiment 71, wherein the population of cells comprises at least 1x10 ⁇ 5 cells.
  • a pharmaceutical composition comprising the composition of any one of embodiments 1-72 and a pharmaceutically acceptable excipient diluent, excipient or carrier.
  • 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 embodiment 73 to the subject.
  • the pharmaceutical composition comprises a population of CAR-T cells.
  • the method of embodiment 74 or 75, wherein the disease or condition is cancer, and or an auto- immune disease.
  • the cancer is lymphoma or leukemia.
  • the cancer is a solid tumor cancer.
  • 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 WSGR Docket No.61078-716.601 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),
  • UCS uterine car
  • B2M fusions were human codon optimized, synthesized and cloned into a MSGV1 retroviral vector by Gene Art (Life Technologies). B2M was fused N-terminally or C- terminally with a second or third domain via a linker. Retroviral vector production, T cell transduction and T cell gene editing [0335] Retroviral supernatant was produced via transient transfection of 293 GP producer cells.293 GP cells were seeded onto poly-D-lysine (R&D Systems) coated plates the day prior to transfection.
  • plasmids encoding the genes of interest were co- transfected with Lipofectamine 2000 (Life Technologies). Media was replaced 24h after transfection. WSGR Docket No.61078-716.601 transduction.
  • Primary human T cells were isolated from fresh or frozen PBMCs obtained from healthy donor leukopaks from Stem Cell Technologies using negative MACS selection (Miltenyi). Isolated T cells are cryopreserved in Bambanker (GC Lymphotec Inc.).
  • T cells were activated with TransACT (Miltenyi) and 100 IU/mL IL-2 (Miltenyi) for 48 h and cultured in RPMI-1640 + 10% fetal bovine serum + non-essential amino acids (R10). T cells were transduced with retroviral vector on days 2 or 3 post activation.
  • TransACT TransACT
  • IL-2 Miltenyi
  • R10 non-essential amino acids
  • Table 5 Exemplary CAR Constructs WSGR Docket No.61078-716.601
  • Example 3 Additional modifications to cells expressing B2M fusion proteins constructs
  • Cells expressing B2M fusion proteins constructs will be subject to additional modifications, such as ablation of the endogenous T-cell receptor, e.g., endogenous TRAC, ablation of endogenous B2M, ablation of endogenous RFXANK, ablation of endogenous RFXAP, ablation of endogenous WSGR Docket No.61078-716.601 thereof.
  • ablation of the endogenous T-cell receptor e.g., endogenous TRAC
  • ablation of endogenous B2M ablation of endogenous RFXANK
  • ablation of endogenous RFXAP ablation of endogenous WSGR Docket No.61078-716.601 thereof.
  • T cells were activated with TransACT and 100 IU/mL IL-2 for 48-72 hr. T cells were gene edited using 1:1 molar ratio of sgRNA:Cas9 on days 2 or 3 post activation via CRISPR/Cas9 using the Neon electroporator (Life Technologies). T cells were gene edited with CRISPR/cas9 and assessed for KO by flow cytometry 5 days post-editing.
  • Exemplary sgRNA sequences to be used are shown in Table 6.
  • sgRNA sequences WSGR Docket No.61078-716.601 [0342] DN-RFX5, and/or DN-RFXANK constructs can be expressed in cells expressing B2M fusion proteins constructs. [0343] Table 7. Table 7 WSGR Docket No.61078-716.601 WSGR Docket No.61078-716.601 WSGR Docket No.61078-716.601 WSGR Docket No.61078-716.601 WSGR Docket No.61078-716.601 WSGR Docket No.61078-716.601 Example 4 – Functional Testing of Graft T cells 1. T cell Mixed Lymphocyte Reaction WSGR Docket No.61078-716.601 [0344] (Stem Cell Technologies).
  • %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells.
  • Graft T cells transduced with CD8a-B2M fusions were co-cultured with primed, alloreactive host T cells at a 1:1 ratio for 48 hours, in the T cell mixed lymphocyte reaction described herein. % graft survival was measured by flow cytometry.
  • FIG. 3 shows that after 48h, % graft T cells were higher in the co-culture with graft T cells transduced with CD8-B2M fusions than in the co-culture with control graft T cells. The result suggests that the CD8-B2M fusions inhibits alloreactive T cell killing by targeting CD8 coreceptor binding.
  • Graft T cells transduced with PD-L1-linker-B2M or PD-L2-linker-B2M fusions were co- cultured with primed, alloreactive host T cells at a 1:1 ratio for 48 hours, in the T cell mixed lymphocyte reaction described herein. % graft survival was measured by flow cytometry.
  • FIG. 4 shows that after 48h, % graft T cells were higher in the co-culture with graft T cells transduced with PD-L1-linker-B2M or with PD-L2-linker-B2M than in the co-culture with control graft T cells.
  • NK cell Mixed Lymphocyte Reaction (Stem Cell Technologies). Purity was confirmed via flow cytometry. Graft T cells were mixed at a 1:1 E:T ratio with activated, allogeneic host NK cells in R10 + 1000 IU/mL IL-2 and co-incubated for 48 2. %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells.
  • PBMC Mixed Lymphocyte Reaction [0348] (Stem Cell Technologies). Purity was confirmed via flow cytometry.
  • Graft T cells were mixed at a 3:1 E:T ratio with allogeneic host PBMC cells in R10 + 20 IU/mL IL-2 and co-incubated for up to 14 days 2.
  • Host CD4+/CD8+ proliferation was determined by flow cytometry, gating on CD4+ or CD8+, TCR+ T cells at various time points.
  • TRAC KO edited graft T cells were pre-incubated with an anti-MHCI (clone TP25.99) primed allogeneic CD8+ T cells were added at a 1:1 E:T ratio for 48 hr in a mixed lymphocyte reaction described herein. Survival of graft T cells was assessed by flow cytometry. As shown in FIG.5, % graft survival were higher when the graft T cells were pre-incubated with the anti-MHCI scFv than the control.
  • Example 6 Cellular incorporation of Anti-MHCI scFv constructs
  • An anti-MHCI scFv (clone TP25.99) was incorporated into cells as a fusion to B2M, a fusion to a transmembrane domain or secreted as a soluble scFv, as depicted in FIG.6A.
  • Different constructs of anti-MHCI/B2M fusion proteins were expressed in the B2M knocked-out cells, and the B2M expression of the different constructs were tested by flow cytometry to determine the optimal configuration.
  • the constructs that provided the optimal configurations are N- Term vL-vH (G4S)3 (SEQ ID NO: 240), N-Term vH-vL (G4S)3 (SEQ ID NO: 242), N-Term vH- vL(G4S)6 (SEQ ID NO: 243), C-Term vH-vL(G4S)6 (SEQ ID NO: 247).
  • Graft T cells with TRAC KO were engineered to express a B2M/anti-MHCI scFv fusion, an anti-MHCI scFv as a fusion to a transmembrane domain, or a secreted soluble anti-MHCI scFv.
  • the engineered graft T cells with or without RFX5 KO were mixed at a 1:1 E:T ratio with primed, allogeneic CD8+ host T cells or host T cells in R10 + 20 IU/mL IL-2 and co-incubated for 48 h at 37 2.
  • %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells.
  • FIG.7A shows that expressing the optimal configurations of the B2M/anti-MHCI scFv fusion in the graft T cells inhibited alloreactive CD8+ T killing and increased the percentage of graft T cell survival to a similar level as expressing a soluble anti-MHCI scFv (FIG. 7C).
  • FIG. 7B shows that expressing anti-MHCI scFv as a fusion to a transmembrane domain resulted in lower % of graft survival.
  • TRAC KO edited graft T cells were engineered to have B2M KO, B2M/CIITA knockout and CD47 overexpression, RFX5 and CD58 knockout, or RFX/CD58 knockout and expressing the WSGR Docket No.61078-716.601 cells were mixed with allogeneic PBMCs at a 3:1 E:T ratio for 14 days in a mixed lymphocyte reaction described herein. Survival of graft T cells were assessed by flow cytometry on Day 4, Day 7, Day 9, Day 12, and Day 14. As shown in FIG.8A, the graft T cells with RFX/CD58 knockout and Day 14.
  • FIG.8B demonstrated that overexpressing CD8a-B2M fusion proteins in the graft T cells elicited minimal NK cell proliferation.
  • FIG.8C showed that overexpressing CD8a-B2M fusion proteins in the graft T cells reduced host CD8+ T cell proliferation.

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Abstract

La présente demande concerne des protéines de fusion recombinantes, des fusions de récepteurs antigéniques chimériques modifiés, des cellules modifiées pour exprimer un ou plusieurs récepteurs de protéines de fusion/antigéniques chimériques recombinants, des protéines de fusion B2M, et leurs utilisations pour le traitement de maladies, d'affections et/ou de troubles, et leurs utilisations pour la fabrication de populations de cellules.
PCT/US2023/077916 2022-10-27 2023-10-26 Compositions et méthodes pour immunothérapies améliorées WO2024092126A1 (fr)

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Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1995013365A1 (fr) 1993-11-09 1995-05-18 Targeted Genetics Corporation Production de titres eleves de vecteurs d'aav recombinants
WO1995013392A1 (fr) 1993-11-09 1995-05-18 Medical College Of Ohio Lignees cellulaires stables aptes a exprimer le gene de replication du virus adeno-associe
WO1996017947A1 (fr) 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants
WO1997006243A1 (fr) 1995-08-10 1997-02-20 Pasteur Merieux Serums Et Vaccins Procede de purification de virus par chromatographie
WO1997008298A1 (fr) 1995-08-30 1997-03-06 Genzyme Corporation Purification d'adenovirus et de virus adeno-associe (aav) par voie chromatographique
WO1997009441A2 (fr) 1995-09-08 1997-03-13 Genzyme Corporation Vecteurs aav ameliores pour la therapie genique
WO1997021825A1 (fr) 1995-12-15 1997-06-19 Systemix, Inc. Procede de production de lignees de cellules d'encapsidation retrovirales generant un surnageant retroviral a efficacite de transduction elevee
US5786211A (en) 1994-06-06 1998-07-28 Children's Hospital, Inc. Adeno-associated virus materials and methods
US5871982A (en) 1994-10-28 1999-02-16 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
WO1999011764A2 (fr) 1997-09-05 1999-03-11 Targeted Genetics Corporation Procedes de generation de preparations de vecteurs de aav recombinants dont le titre est eleve et qui sont exemptes de virus assistant
US6258595B1 (en) 1999-03-18 2001-07-10 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
WO2001083692A2 (fr) 2000-04-28 2001-11-08 The Trustees Of The University Of Pennsylvania Vecteurs aav recombinants dotes de capsides aav5 et vecteurs aav5 pseudotypes dans des capsides heterologues
US20030170238A1 (en) 2002-03-07 2003-09-11 Gruenberg Micheal L. Re-activated T-cells for adoptive immunotherapy
WO2013169802A1 (fr) 2012-05-07 2013-11-14 Sangamo Biosciences, Inc. Procédés et compositions pour l'intégration médiée par nucléase de transgènes
WO2015048577A2 (fr) 2013-09-27 2015-04-02 Editas Medicine, Inc. Compositions et méthodes relatives aux répétitions palindromiques groupées, courtes et régulièrement espacées
US9613872B2 (en) 2014-09-29 2017-04-04 Kabushiki Kaisha Toshiba Method of manufacturing semiconductor device
US9614423B2 (en) 2012-04-07 2017-04-04 Traugott Weller Method for producing rotating electrical machines
US9620777B2 (en) 2013-09-30 2017-04-11 Tdk Corporation Positive electrode and lithium ion secondary battery using thereof
US9818600B2 (en) 2014-03-21 2017-11-14 Hitachi Kokusai Electric, Inc. Substrate processing apparatus and method of manufacturing semiconductor device
US20180362975A1 (en) * 2015-12-04 2018-12-20 Novartis Ag Compositions and methods for immunooncology
US20190290686A1 (en) * 2017-12-23 2019-09-26 Rubius Therapeutics, Inc. Artificial antigen presenting cells and methods of use
WO2021205172A1 (fr) * 2020-04-08 2021-10-14 Autolus Limited Cellule

Patent Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1995013365A1 (fr) 1993-11-09 1995-05-18 Targeted Genetics Corporation Production de titres eleves de vecteurs d'aav recombinants
WO1995013392A1 (fr) 1993-11-09 1995-05-18 Medical College Of Ohio Lignees cellulaires stables aptes a exprimer le gene de replication du virus adeno-associe
US5658776A (en) 1993-11-09 1997-08-19 Targeted Genetics Corporation Generation of high titers of recombinant AAV vectors
US5786211A (en) 1994-06-06 1998-07-28 Children's Hospital, Inc. Adeno-associated virus materials and methods
US5871982A (en) 1994-10-28 1999-02-16 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
WO1996017947A1 (fr) 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants
WO1997006243A1 (fr) 1995-08-10 1997-02-20 Pasteur Merieux Serums Et Vaccins Procede de purification de virus par chromatographie
WO1997008298A1 (fr) 1995-08-30 1997-03-06 Genzyme Corporation Purification d'adenovirus et de virus adeno-associe (aav) par voie chromatographique
WO1997009441A2 (fr) 1995-09-08 1997-03-13 Genzyme Corporation Vecteurs aav ameliores pour la therapie genique
WO1997021825A1 (fr) 1995-12-15 1997-06-19 Systemix, Inc. Procede de production de lignees de cellules d'encapsidation retrovirales generant un surnageant retroviral a efficacite de transduction elevee
WO1999011764A2 (fr) 1997-09-05 1999-03-11 Targeted Genetics Corporation Procedes de generation de preparations de vecteurs de aav recombinants dont le titre est eleve et qui sont exemptes de virus assistant
US6258595B1 (en) 1999-03-18 2001-07-10 The Trustees Of The University Of Pennsylvania Compositions and methods for helper-free production of recombinant adeno-associated viruses
WO2001083692A2 (fr) 2000-04-28 2001-11-08 The Trustees Of The University Of Pennsylvania Vecteurs aav recombinants dotes de capsides aav5 et vecteurs aav5 pseudotypes dans des capsides heterologues
US20030170238A1 (en) 2002-03-07 2003-09-11 Gruenberg Micheal L. Re-activated T-cells for adoptive immunotherapy
US9614423B2 (en) 2012-04-07 2017-04-04 Traugott Weller Method for producing rotating electrical machines
WO2013169802A1 (fr) 2012-05-07 2013-11-14 Sangamo Biosciences, Inc. Procédés et compositions pour l'intégration médiée par nucléase de transgènes
WO2015048577A2 (fr) 2013-09-27 2015-04-02 Editas Medicine, Inc. Compositions et méthodes relatives aux répétitions palindromiques groupées, courtes et régulièrement espacées
US9620777B2 (en) 2013-09-30 2017-04-11 Tdk Corporation Positive electrode and lithium ion secondary battery using thereof
US9818600B2 (en) 2014-03-21 2017-11-14 Hitachi Kokusai Electric, Inc. Substrate processing apparatus and method of manufacturing semiconductor device
US9613872B2 (en) 2014-09-29 2017-04-04 Kabushiki Kaisha Toshiba Method of manufacturing semiconductor device
US20180362975A1 (en) * 2015-12-04 2018-12-20 Novartis Ag Compositions and methods for immunooncology
US20190290686A1 (en) * 2017-12-23 2019-09-26 Rubius Therapeutics, Inc. Artificial antigen presenting cells and methods of use
WO2021205172A1 (fr) * 2020-04-08 2021-10-14 Autolus Limited Cellule

Non-Patent Citations (69)

* Cited by examiner, † Cited by third party
Title
A. R. GRUBER ET AL., CELL, vol. 106, no. 1, 2008, pages 23 - 24
ARNOULD S, J MOL BIOL., vol. 355, 2006, pages 443 - 58
BARRANGOU ET AL., SCIENCE, vol. 315, 2007, pages 1709 - 1712
BITINAITE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 10570 - 5
CARROLL ET AL., GENETICS SOCIETY OF AMERICA, vol. 188, 2011, pages 773 - 782
CARTER, CURRENT OPINIONS IN BIOTECHNOLOGY, 1992, pages 1533 - 539
CERMAK ET AL., NUCL. ACIDS RES., vol. 39, 2011, pages e82
CHEN ET AL., PROTEIN ENG DES SEL, vol. 22, 2009, pages 249 - 56
CHEVALIER ET AL., MOL. CELL, vol. 10, 2002, pages 895 - 905
CLARK ET AL., GENE THERAPY, vol. 3, 1996, pages 1124 - 1132
D.L. PORTER ET AL., N ENGL J MED, vol. 365, no. 8, 2011, pages 725 - 33
DOYON ET AL., J AM CHEM SOC, vol. 128, 2006, pages 2477 - 84
DOYON ET AL., NATURE METHODS, vol. 8, 2010, pages 74 - 79
E. ROMAO ET AL., CURR PHARM DES, vol. 22, no. 43, 2016, pages 6500 - 18
E.L. SMITH ET AL., MOL THER, vol. 26, no. 6, 2018, pages 1447 - 56
EPINAT ET AL., NUCLEIC ACIDS RES, vol. 31, 2003, pages 2952 - 62
F. LE GALL ET AL., FEBS LETT, vol. 453, no. 1-2, 1999, pages 164 - 68
F. RAHBARIZADEH ET AL., ADV DRUG DELIV REV, vol. 141, 2019, pages 41 - 46
FITZWATER ET AL., METHODS ENZYMOL., vol. 267, 1996, pages 275 - 301
GRETEN T F ET AL: "Peptide-@b2-microglobulin-MHC fusion molecules bind antigen-specific T cells and can be used for multivalent MHC-Ig complexes", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 271, no. 1-2, 20 December 2002 (2002-12-20), pages 125 - 135, XP004393932, ISSN: 0022-1759, DOI: 10.1016/S0022-1759(02)00346-0 *
GRISSA ET AL., BMC BIOINFORMATICS, vol. 8, 2007, pages 172
GUO ET AL., J. MOL. BIOL., vol. 200, 2010, pages 96
HAFT ET AL., PLOS COMPUT. BIOL., vol. 1, 2005, pages e60
HERMONAT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6466
HOCKEMEYER ET AL., NATURE BIOTECH., vol. 29, 2011, pages 731 - 734
HOCKEMEYER, D., NAT. BIOTECHNOL., vol. 29, 2011, pages 731 - 734
HORVATH ET AL., SCIENCE, vol. 327, 2010, pages 167 - 170
HUERTAS, P., NAT. STRUCT. MOL. BIOL., vol. 17, 2010, pages 11 - 16
KIM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 1156 - 1160
KUNIN ET AL., GENOME BIOL., vol. 8, 2007, pages R61
LAUGHLIN ET AL., GENE, vol. 23, 1983, pages 65 - 73
LEBKOWSKI ET AL., MOL. CELL. BIOL., vol. 7, 1988, pages 349
LIU, BIOINFORMATICS, vol. 24, 2008, pages 1850 - 1857
M. THEMELI ET AL., NAT BIOTECHNOL, vol. 31, no. 10, 2013, pages 928 - 33
M.A. GHETIE ET AL., BLOOD, vol. 83, no. 5, 1994, pages 1329 - 36
M.A. GHETIE ET AL., CLIN CANCER RES, vol. 5, no. 12, 1999, pages 3920 - 27
MAKAROVA ET AL., BIOLOGY DIRECT, vol. 1, 2006, pages 7
MARRAGINI ET AL., SCIENCE, vol. 321, 2008, pages 1843 - 1845
MCLAUGHLIN ET AL., J. VIROL., vol. 62, 1988, pages 1963
MOJICA ET AL., J. MOL. EVOL., vol. 60, 2005, pages 174 - 182
MOSCOU ET AL., SCIENCE, vol. 326, 2009, pages 3501 - 12
MUZYCZKA, CURR. TOPICS IN MICROBIAL. AND IMMUNOL., vol. 158, 1992, pages 97 - 129
P A CARRG M CHURCH, NATURE BIOTECHNOLOGY, vol. 27, no. 12, 2009, pages 1151 - 62
PAUL ET AL., HUMAN GENE THERAPY, vol. 4, 1993, pages 609 - 615
PEERLIEBERMAN, GENE THERAPY, vol. 18, 2011, pages 1127 - 1133
PERRIN ET AL., VACCINE, vol. 13, 1995, pages 1244 - 1250
POURCEL ET AL., MICROBIOL., vol. 151, 2005, pages 2551 - 2561
RATSCHIN ET AL., MOL. CELL. BIOL., vol. 4, 1984, pages 2072
ROSEN ET AL., NUCLEIC ACIDS RES, 2006
S.A. ROSENBERG, NAT REV CLIN ONCOL, vol. 8, no. 10, 2011, pages 577 - 85
S.M. KIPRIYANOV ET AL., INT J CANCER, vol. 77, no. 5, 1998, pages 763 - 72
S.R. BANIHASHEMI ET AL., IRAN J BASIC MED SCI, vol. 21, no. 5, 2018, pages 455 - 64
SAMULSKI ET AL., J. VIROL., vol. 63, 1989, pages 3822 - 3828
SAMULSKI ET AL., PROC. NATL. ACAD. S6. USA, vol. 79, 1982, pages 2077 - 2081
SELIGMAN ET AL., NUCLEIC ACIDS RES, vol. 30, 2002, pages 3870 - 9
SENAPATHYCARTER, J. BIOL. CHEM., vol. 259, 1984, pages 4661 - 4666
SERA, BIOCHEMISTRY, vol. 41, 2002, pages 7074 - 7081
SILVA ET AL., CURRENT GENE THERAPY, vol. 11, 2011, pages 11 - 27
SILVA ET AL., J MOL BIOL, vol. 361, 2006, pages 744 - 54
SMITH, NUCLEIC ACIDS RES., vol. 363, no. 2, 2006, pages 283 - 94
STERN ET AL., TRENDS. GENET., vol. 28, 2010, pages 335 - 340
SUSSMAN ET AL., J MOL BIOL, vol. 342, 2004, pages 31 - 41
SZCZEPEK, NATURE BIOTECH., vol. 25, 2007, pages 786 - 793
T. TSUKAHARA ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 438, no. 1, 2013, pages 84 - 89
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 3251
WANG ET AL., BLOOD, vol. 127, no. 24, 2016, pages 2980 - 90
WANG T ET AL., SCIENCE, vol. 343, 2013, pages 833 - 836
WOOD ET AL., SCIENCE, vol. 333, 2011, pages 307
ZUKERSTIEGLER, NUCLEIC ACIDS RES., vol. 9, 1981, pages 133 - 148

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