WO2024123760A1 - Multiplex cell selection compositions and uses thereof - Google Patents

Abstract

Multiplex cell selection compositions and methods of uses thereof.

Description

MULTIPLEX CELL SELECTION COMPOSITIONS AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/430,113, filed December 5, 2022, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] T cells genetically engineered to express chimeric antigen receptors (CARs) are mainstays of adoptive cell therapy for the treatment of diseases like cancer. In the face of immunosuppressive tumor microenvironments that limit the therapeutic potential of such immunotherapies, bulk production and enrichment of T cells expressing correct and complete receptors are critical aspects of providing sufficient, effective therapeutic product to target cancer cells. Efficient and consistent ex vivo expansion of CAR-T cells prior to patient infusion can enhance the effectiveness of the immunotherapy.

[0003] Expression of CARs with correct selectivity and potency can be challenging. Screening for T cell populations that express one or more different CARs simultaneously and obtaining a final pool of recombinant cells with high yield and high purity are similarly difficult tasks. There remains a need to minimize the number of T cells inadequately or incorrectly expressing CARs and improve the efficiency with which T cells expressing correct and complete CARs can be selected.

SUMMARY

[0004] Provided herein is a composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a receptor complex and a second sequence encoding a second polypeptide comprising a second portion of the receptor complex, wherein the first polypeptide comprises: (a) a first extracellular domain comprising a selection tag, and (b) a first transmembrane domain from a first receptor, wherein the second polypeptide comprises: (a) a second transmembrane domain from a second receptor; wherein the first receptor and the second receptor are different receptors; and wherein (i) the selection tag is not a cytokine binding domain from a cytokine receptor, and/or (ii) wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor; and wherein cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide.

[0005] In some embodiments, the selection tag is a peptide sequence. In some embodiments, the selection tag is a portion of a full-length polypeptide. In some embodiments, the selection tag of the first polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide.

[0006] In some embodiments, the selection tag comprises a purification tag or a fluorescent tag.

[0007] In some embodiments, the selection tag comprises a peptide tag. In some embodiments, the peptide tag comprises a peptide sequence derived from a full-length protein. In some embodiments, the selection tag comprises a purification tag selected from the group consisting of a histidine-glycine tag, poly-arginine tag, poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein tag, streptavidin tag, streptavidin binding peptide (SPB) tag, a tag consisting of the amino acid sequence WSHPQFEK, a tag consisting of the ammo acid sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, CD34 tag, CD133 tag, CD304 tag, chloramphenicol acetyl transferase (CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, epidermal growth factor receptor (EGF) tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag, Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profmity eXact tag, Protein C tag, SI -tag, S-tag, biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, a Vesicular Stomatitis Virus (VSV) glycoprotein (G protein) epitope tag, or a V5 tag (which is derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus of the simian virus 5 (SV5) family).

[0008] In some embodiments, the selection tag comprises multiple copies of a purification tag sequence. [0009] In some embodiments, the selection tag comprises a fluorescent tag detectable by flow cytometry. [0010] In some embodiments, the fluorescent tag detectable by flow cytometry is selected from the group consisting of mNeonGreen, mtagBFP2, tagRFP, mCherry, tagRFP658, mTurquois, mBeRFP, mEGFP, mOrange2, mGrape2.

[0011] In some embodiments, the selection tag comprises a truncated receptor.

[0012] In some embodiments, the truncated receptor comprises a truncated receptor selected from the group consisting of truncated epidermal growth factor receptor (EGFRt), truncated CD34 (CD34t), and truncated human epidermal growth factor receptor 2 (HER2t).

[0013] In some embodiments, the first transmembrane domain is a Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) transmembrane domain. [0014] In some embodiments, the first transmembrane domain is a transmembrane domain from a protein selected from the group consisting of Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), TREM2, IREM2, KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5A), CD300 Molecule Like Family Member B (CD300LB), Sialic Acid Binding Ig-Like Lectin 14 (SIGLEC14), SIGLEC15, and SIGLEC16.

[0015] In some embodiments, the second transmembrane domain is a DNAX Activating Protein of 12 kDa (DAP12) transmembrane domain.

[0016] In some embodiments, the first polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the second polypeptide.

[0017] In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the second polypeptide by the cell.

[0018] In some embodiments, retention of the first polypeptide in the endoplasmic reticulum (ER) is increased in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide.

[0019] In some embodiments, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain.

[0020] In some embodiments, the second polypeptide does not comprise a full-length intracellular domain. [0021] In some embodiments, the second polypeptide does not comprise an intracellular domain.

[0022] In some embodiments, the second polypeptide consists of the second transmembrane domain.

[0023] In some embodiments, the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor.

[0024] In some embodiments, the selection tag is not a cytokine binding domain from a cytokine receptor. [0025] In some embodiments, the selection tag is not a cytokine binding domain from a cytokine receptor, and the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor.

[0026] In some embodiments, the second polypeptide comprises an intracellular domain that is at most about 65 amino acids in length.

[0027] In some embodiments, the second polypeptide comprises a second selection tag, wherein the second selection tag is the same as the selection tag of the first polypeptide.

[0028] In some embodiments, the second polypeptide does not comprise a selection tag.

[0029] In some embodiments, the second polypeptide does not comprise an extracellular domain. [0030] In some embodiments, the second polypeptide comprises an extracellular domain that is at most about 350 amino acids in length.

[0031] In some embodiments, the first receptor is TREM1 and the second receptor is DAP12.

[0032] In some embodiments, the recombinant polynucleic acid further comprises a third sequence encoding a third polypeptide.

[0033] In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide are each different molecules.

[0034] In some embodiments, the third polypeptide comprises a third transmembrane domain from a third receptor.

[0035] In some embodiments, the second receptor and the third receptor are the same.

[0036] In some embodiments, the first receptor is TREM1, the second receptor is DAP 12 and the third receptor is DAP12.

[0037] In some embodiments, the third receptor and the second receptor are different receptors.

[0038] In some embodiments, the third receptor and the first receptor are different receptors.

[0039] In some embodiments, the first receptor is IgM and the second receptor is CD79a.

[0040] In some embodiments, the third receptor is CD790.

[0041] In some embodiments, the first receptor is IgM and the second receptor is CD790.

[0042] In some embodiments, the third receptor is CD79a.

[0043] In some embodiments, the first receptor is CD79a and the second receptor is CD79 .

[0044] In some embodiments, the third receptor is IgM.

[0045] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, or any combination thereof, lacks an antigen binding domain.

[0046] In some embodiments, the IgM lacks one or more fragment antigen binding (Fab) domains.

[0047] In some embodiments, the third polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain.

[0048] In some embodiments, the third polypeptide does not comprise a full-length intracellular domain.

[0049] In some embodiments, the third polypeptide does not comprise an intracellular domain.

[0050] In some embodiments, the third polypeptide consists of the third transmembrane domain.

[0051] In some embodiments, the third polypeptide comprises an intracellular domain that is at most about 65 amino acids in length.

[0052] In some embodiments, the third polypeptide comprises a third selection tag, wherein the third selection tag is the same as the selection tag of the first polypeptide and/or the second polypeptide.

[0053] In some embodiments, the third polypeptide does not comprise a selection tag.

[0054] In some embodiments, the third polypeptide does not comprise an extracellular domain. [0055] In some embodiments, the third polypeptide comprises an extracellular domain that is at most about 350 amino acids in length.

[0056] In some embodiments, cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide, the second polypeptide, and the third polypeptide.

[0057] In some embodiments, the first polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the third polypeptide.

[0058] In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the third polypeptide by the cell.

[0059] In some embodiments, retention of the first polypeptide in the endoplasmic reticulum (ER) is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide, the second polypeptide, and the third polypeptide.

[0060] In some embodiments, the first polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

[0061] In some embodiments, the second polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

[0062] In some embodiments, the third polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

[0063] In some embodiments, the first polypeptide and the second polypeptide form a complex.

[0064] In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide form a complex.

[0065] In some embodiments, the complex is a non-functional receptor complex or is not capable of signaling to a cell in which it is expressed.

[0066] In some embodiments, the first polypeptide and the second polypeptide form a dimer.

[0067] In some embodiments, the first polypeptide and the second polypeptide form a trimer. In some embodiments, the first polypeptide and the second polypeptide form a trimer containing two molecules of the first polypeptide and one molecule of the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide form a trimer containing two molecules of the second polypeptide and one molecule of the first polypeptide.

[0068] In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide form a trimer. [0069] In some embodiments, the first transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0070] In some embodiments, the second transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0071] In some embodiments, the third transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0072] In some embodiments, the first polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69.

[0073] In some embodiments, the second polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69.

[0074] In some embodiments, the third polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69.

[0075] In some embodiments, the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the first polypeptide and a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide.

[0076] In some embodiments, the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest.

[0077] In some embodiments, the sequence encoding the first gene of interest encodes a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as a single polypeptide molecule.

[0078] In some embodiments, the sequence encoding the first polypeptide is linked to the sequence encoding the first protein of interest by a sequence encoding a linker.

[0079] In some embodiments, the linker comprises a protease cleavage site or a 2A self-cleaving peptide. [0080] In some embodiments, the sequence encoding a first gene of interest encodes a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as separate polypeptide molecules.

[0081] In some embodiments, the sequence encoding the first polypeptide and the sequence encoding the first gene of interest are separated or linked by an IRES or have different promoters.

[0082] In some embodiments, the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest.

[0083] In some embodiments, the sequence encoding a second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as a single polypeptide molecule.

[0084] In some embodiments, the nucleotide sequence encoding the second polypeptide is linked to the nucleotide sequence encoding the second protein of interest by a sequence encoding a linker. [0085] In some embodiments, the linker comprises a protease cleavage site or a 2A self-cleaving peptide. [0086] In some embodiments, the sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules.

[0087] In some embodiments, the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an internal ribosome entry site (IRES) or have different promoters.

[0088] In some embodiments, the recombinant polynucleic acid comprises a third recombinant polynucleic acid comprising a sequence encoding the third polypeptide.

[0089] In some embodiments, the third recombinant polynucleic acid further comprises a sequence encoding a third gene of interest.

[0090] In some embodiments, the sequence encoding the third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as a single polypeptide molecule.

[0091] In some embodiments, the sequence encoding the third polypeptide is linked to the sequence encoding the third protein of interest by a sequence encoding a linker.

[0092] In some embodiments, the linker comprises a protease cleavage site or a 2A self-cleaving peptide. [0093] In some embodiments, the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as separate polypeptide molecules.

[0094] In some embodiments, the sequence encoding the third polypeptide and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters.

[0095] In some embodiments, the first protein of interest, the second protein of interest, and/or the third protein of interest is a chimeric antigen receptor (CAR).

[0096] In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR).

[0097] In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain.

[0098] In some embodiments, the antigen binding domain is an anti-CD19 binding domain.

[0099] In some embodiments, the antigen binding domain is a single chain variable fragment (“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. [0100] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 75 or 76.

[0101] In some embodiments, the anti-CD19 binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1, LCDR2 and LCDR3 sequences in Tables 12-14; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 and HCDR3 sequences in Tables 9-11.

[0102] In some embodiments, the scFv comprises a heavy chain variable region (VH) with at least about 80% sequence identity to a sequence selected from the group consisting of the VH sequences in Table 8. [0103] In some embodiments, the scFv comprises a light chain variable region (VL) with at least about 80% sequence identity to a sequence selected from the group consisting of the VL sequences in Table 8. [0104] In some embodiments, the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 7.

[0105] In some embodiments, the antigen binding domain is an anti-CD20 binding domain.

[0106] In some embodiments, the anti-CD20 binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1, LCDR2 and LCDR3 sequences in Tables 20-22; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 and HCDR3 sequences in Tables 17-19.

[0107] In some embodiments, the scFv comprises a heavy chain variable region (VH) with at least about 80% sequence identity to a sequence selected from the group consisting of the VH sequences in Table 16. [0108] In some embodiments, the scFv comprises a light chain variable region (VL) with at least about 80% sequence identity to a sequence selected from the group consisting of the VL sequences in Table 16. [0109] In some embodiments, the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 15.

[0110] In some embodiments, the antigen binding domain is an anti-CD22 binding domain.

[OHl] In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively.

[0112] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 77.

[0113] In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothehn, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, R0R1, CD2 and GD2.

[0114] In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain from CD2.

[0115] In some embodiments, the intracellular domain of the CAR comprises at least one intracellular signaling domain selected from the group consisting of CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD35, CD3E, CD35, CD22, CD79a, CD79p, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. [0116] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from CD8 (CD8TM) or CD28 (CD28TM).

[0117] In some embodiments, the extracellular domain of the CAR comprises a hinge domain from CD8 (CD8H) or CD28 (CD28H). In some embodiments, the CAR comprises a CD8H and CD8TM. In some embodiments, the CAR comprises a CD28H and CD28 TM. In some embodiments, the CAR comprises a CD8H and CD28 TM. In some embodiments, the CAR comprises a CD28H and CD8TM.

[0118] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 24.

[0119] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 78-80.

[0120] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 25.

[0121] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 26. [0122] In some embodiments, the CAR comprises an anti-CD19 scFv, a hinge domain from CD8a, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD28 and an intracellular signaling domain from CD3zeta.

[0123] In some embodiments, the CAR comprises an anti-CD20 scFv, a hinge domain from CD28, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta.

[0124] In some embodiments, the CAR comprises an anti-CD22 scFv, a hinge domain from CD8a, a transmembrane domain from CD8a, a cytoplasmic domain comprising a costimulatory domain from 4- 1BB and an intracellular signaling domain from CD3zeta.

[0125] Provided herein is a composition comprising a cell comprising any of the compositions described herein, wherein the cell expresses the first polypeptide and the second polypeptide.

[0126] In some embodiments, the cell further expresses the third polypeptide.

[0127] In some embodiments, the cell is an engineered cell.

[0128] In some embodiments, the cell is a lymphocyte.

[0129] In some embodiments, the cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, or a suppressor T cell. In some embodiments, the T cell is a CD8+ cell. In some embodiments, the T cell is a CD4+ cell. In some embodiments, the T cell is a CD8+CD4+ cell. In some embodiments, the T cell is a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, or a naive T cell.

[0130] In some embodiments, the cell is a B cell. In some embodiments, the B cell is a naive mature B cell, a plasmablast, a plasma cell, or a memory B cell. In some embodiments, the B cell is a CD19+ cell.

[0131] In some embodiments, the cell is a population of cells. In some embodiments, the population of cells is a population of T cells. In some embodiments, the population of T cells is a population of cytotoxic T cells, effector T cells, helper T cells, natural killer T cells, or suppressor T cells. In some embodiments, the population of T cells is a population of CD8+ cells. In some embodiments, the population of T cells is a population of CD4+ cells. In some embodiments, the population of T cells is a population of CD8+CD4+ cells. In some embodiments, the population of T cells is a population of regulatory T cells, memory T cells, stem cell memory T cells, central memory T cells, effector memory T cells, or a naive T cells. In some embodiments, the population of cells is a population of B cells. In some embodiments, the population of B cells is a population of naive mature B cells, plasmablasts, plasma cells, or memory B cells. In some embodiments, the population of B cells is a population of CD19+ cells.

[0132] In some embodiments, the population of cells comprises at least lxl0^A5 cells.

[0133] In some embodiments, at least 50% of the cells in the population of cells express the first protein of interest and the second protein of interest. [0134] In some embodiments, at least 50% of the cells in the population of cells express the first protein of interest, the second protein of interest, and the third protein of interest.

[0135] In some embodiments, at least 50% of the cells in the population of cells express at least two CARs. [0136] In some embodiments, at least 50% of the cells in the population of cells express at least three CARs.

[0137] Provided herein is a pharmaceutical composition comprising any of the compositions described herein.

[0138] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of any of the pharmaceutical compositions described herein.

[0139] In some embodiments, the cancer is lymphoma or leukemia.

[0140] In some embodiments, the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0141] Provided herein is a method of multiplex cell selection, the method comprising (a) culturing a cell described herein; (b) selecting cells from (a) based on cell surface expression of the selection tag, thereby obtaining a population of selected cells.

[0142] In some embodiments, the selecting comprises purifying or isolating cells.

[0143] In some embodiments, the selection tag is a fluorescent tag and the selecting comprises isolating cells by flow cytometry.

[0144] In some embodiments, the selection tag is a purification tag and the selecting comprises isolating the cells by contacting the cells with an agent comprising a binding moiety that binds to the selection tag and removing the cells that do not bind to the agent comprising the binding moiety. [0145] In some embodiments, the agent comprises a binding moiety further comprising a bead or solid surface attached to the binding moiety that binds to the selection tag. In some embodiments, the bead is magnetic.

[0146] In some embodiments, the agent comprises further comprises a magnetic bead that is attached to the moiety that binds to the selection tag.

[0147] In some embodiments, the binding moiety comprises an antibody capable of binding to the selection tag.

[0148] In some embodiments, the method comprises introducing the recombinant nucleic acid into the cell prior to culturing the cell, the engineered cell, the engineered T cell, or the engineered B cell.

[0149] In some embodiments, at least 50% of the cells in the population of selected cells express the first polypeptide and the second polypeptide.

[0150] In some embodiments, at least 50% of the cells in the population of selected cells express the first protein of interest and the second protein of interest.

[0151] In some embodiments, at least 50% of the cells in the population of selected cells express the first polypeptide, the second polypeptide, and the third polypeptide.

[0152] In some embodiments, at least 50% of the cells in the population of selected cells express the first protein of interest, the second protein of interest, and the third protein of interest.

[0153] Provided herein is a method of multiplex cell selection, comprising the steps of: (a) introducing any recombinant polynucleic acid as described herein into a population of cells; (b) culturing the population of cells from (a); and (c) selecting cells from the population of cells from (b) based on cell surface expression of the selection tag, thereby obtaining a population of selected cells.

[0154] In some embodiments, the population of selected cells is a population of chimeric antigen receptor (CAR)-expressing immune effector cells.

[0155] In some embodiments, at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least two different CARs.

[0156] In some embodiments, at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least three different CARs.

INCORPORATION BY REFERENCE

[0157] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS [0158] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0159] FIG. 1A depicts an exemplary diagram of a receptor complex containing an EGFRt selection tag fused to the transmembrane domain of TREM1 that is in association with two DAP12 proteins at the cell membrane.

[0160] FIG. IB shows the percentage of cells with cell-surface expression of the EGFRt tag. In this experiment, cells were transduced with the TREM1 -EGFRt tag construct only.

[0161] FIG. 1C shows the percentage of cells with cell-surface expression of the EGFRt tag. In this experiment, cells were transduced with the TREM1 -EGFRt tag construct and a DAP12 construct containing the DAP12 extracellular (EC) domain and transmembrane (TM) domain.

[0162] FIG. 2A depicts exemplary polynucleotide constructs encoding EGFRt-TREMl and DAP12, each fused to a different fluorescent protein.

[0163] FIG. 2B shows the percentage of cells with cell-surface expression of the EGFRt tag or the fluorescent proteins. In this experiment, cells were transduced with the constructs shown in FIG. 2A, and then purified using a biotinylated anti-EGFR antibody followed by anti-biotin MACS beads. The flow cytometry plot on the left shows the percentage of cells with cell-surface expression of the blue fluorescent protein (BFP) and NeonGreen (GFP) prior to purification. The flow cytometry plot in the middle shows the percentage of cells with cell-surface expression of BFP and GFP in the flow-through that were not captured by MACS beads. The flow cytometry plot on the right shows the percentage of cells with cellsurface expression of BFP and GFP in eluted cell population captured by MACS.

[0164] FIG. 2C shows the recovery yield of cells expressing EGFRt-TREMl and DAP12 that were recovered from the cells prior to MACS purification and by elution after MACS purification as shown in FIG. 2B.

[0165] FIG. 3A depicts an exemplary diagram of a receptor complex containing an IgM transmembrane domain that is in association with CD79a and CD790 at the cell-surface.

[0166] FIG.3B shows the percentage of cells with cell-surface expression of membrane-bound IgM (IgM containing an IgM transmembrane domain), CD79a, and CD790. In this experiment, cells were transduced with either the IgM construct only depicted in FIG. 3A; both the CD79a and CD790 constructs depicted in FIG. 3A; or all three of the IgM, CD79a, and CD790 constructs depicted in FIG. 3A. Membrane IgM, CD79a, and CD79 only show surface expression when all components are present simultaneously in the cell. [0167] FIG. 4A shows the percentage of cells with cell-surface expression of the EGFRt tag after MACS purification. The EGFRt tag was included on the IgM construct (right panels), the CD79a construct (left panels), or the CD79 construct (middle panels.

[0168] FIG. 4B shows the percentage of cells expressing all three constructs (IgM, CD79a, and CD790). In this experiment, the EGFRt tag was included on one of the IgM, CD79a, or CD79J3 constructs. Samples shown include cells prior to MACS purification using beads that bind to the EGFRt tag, flow-through cells not captured by MACS, and eluted cells captured by MACS.

[0169] FIG. 4C shows the recovery yield of cells expressing all three constructs (IgM, CD79a, and CD790) when EGFRt is fused to either CD79a, CD790, or IgM.

[0170] FIG. 5A depicts exemplary polynucleotide constructs encoding EGFRt-TREMl and DAP12, each linked to a different gene of interest by a P2A sequence.

[0171] FIG. 5B depicts exemplary polynucleotide constructs encoding TREM1, EGFRt-DAP12, and DAP12, each linked to a different gene of interest by a P2A sequence.

[0172] FIG. 5C depicts exemplary polynucleotide constructs encoding IgM, EGFRt-CD79a, and CD790, each linked to a different gene of interest by a P2A sequence.

[0173] FIG. 6A depicts two exemplary polynucleotide constructs. One polynucleotide construct (Vector 2878: SEQ ID NO: 94; Vector 2879: SEQ ID NO: 95; or Vector 2880: SEQ ID NO: 96) encodes Tag- TREM1 (tag used was either a truncated CD34 (CD34t) tag or a truncated EGFR (EGFRt) tag), an anti- CD19 CAR, and an anti-CD20 CAR (left). The other polynucleotide construct (Vector 2883: SEQ ID NO: 97) encodes DAP12 and an anti-CD22 CAR (right).

[0174] FIG. 6B shows the percentage of cells with cell surface expression of the CD22 CAR and an EGFRt or CD34t tag after transduction with the indicated vectors as described in FIG. 6A and before (top panels) and after (bottom panels) MACS beads purification.

[0175] FIG. 6C shows the percentage of cells with cell surface expression of the CD22 CAR and the CD19 CAR after transduction with the indicated vectors as described in FIG. 6A and before (top panels) and after (bottom panels) MACS beads purification.

[0176] FIG. 7A shows the percentage of cells with cell surface expression of the CD34t tag and the CD22 CAR prior to MACS separation (top panels), in the flow through (middle panels), and after MACS selection (bottom panels). The cells were transduced with Vector 2883 and Vector 3050, 3051, 3056, or 3057. See Table 6 for construct details.

[0177] FIG. 7B shows the percentage of cells with cell surface expression of the CD34t tag and the CD22 CAR prior to MACS separation (top panels), in the flow through (middle panels), or after MACS selection (bottom panels). The cells were transduced with Vector 2884 and Vector 3050, 3051, 3056, or 3057. See Table 6 for construct details. [0178] FIG. 8A shows the percentage of cells with cell surface expression of the CD34t tag and the CD22 CAR prior to MACS separation (top panels), in the flow through (middle panels), or after MACS selection (bottom panels). The cells were transduced with Vector 2884 and Vector 3137, 3138, or 3139. See Table 6 for construct details.

[0179] FIG.8B shows the percentage of cells with cell surface expression of the CD34t tag and the CD22 CAR prior to MACS separation (top panels), in the flow through (middle panels), or after MACS selection (bottom panels). The cells were transduced with Vector 2884 and Vector 3140, 3141, or 3142. See Table 6 for construct details.

[0180] FIG. 9A shows the percentage of cells expressing the CD34t tag and the CD22 CAR prior to MACS purification. The cells were transduced with a vector expressing CD34t-TREMl and the CD20 CAR and a vector expressing DAP12, the CD 19 CAR, and the CD22 CAR.

[0181] FIG. 9B shows the percentage of cells expressing the CD34t tag and the CD22 CAR after MACS purification. The cells were transduced with a vector expressing CD34t-TREMl and the CD20 CAR and a vector expressing DAP12, the CD 19 CAR, and the CD22 CAR.

[0182] FIG. 9C shows the percentage of cells expressing the CD19 CAR and the CD22 CAR after MACS purification. The cells were transduced with a vector expressing CD34t-TREMl and the CD20 CAR and a vector expressing DAP12, the CD 19 CAR, and the CD22 CAR.

[0183] FIG. 10A depicts three exemplary polynucleotide constructs: one encoding a CD22 CAR and a truncated CD79a construct linked by a P2A sequence (Vector 3588: SEQ ID NO: 107), one encoding a CD19 CAR and a truncated CD79b construct linked by a P2A sequence (Vector 3589: SEQ ID NO: 108), and one encoding a CD20 CAR and a CD34t-mIgM linked by a P2A sequence (Vector 3590: SEQ ID NO: 109). See Table 6 for construct details.

[0184] FIG. 10B shows the percentage of cells expressing the CD34t tag and the CD22 CAR prior to MACS purification using the CD34t tag. The cells were transduced with a vector expressing the CD22 CAR and a truncated CD79a construct (Vector 3588: SEQ ID NO: 107), a vector expressing the CD19 CAR and a truncated CD79b construct (Vector 3589: SEQ ID NO: 108), and a vector expressing the CD20 CAR and mlgM with a CD34t tag (Vector 3590: SEQ ID NO: 109).

[0185] FIG. 10C shows the percentage of cells expressing the CD34t tag and the CD22 CAR after MACS purification. The cells were transduced with a vector expressing the CD22 CAR and a truncated CD79a construct (Vector 3588: SEQ ID NO: 107), a vector expressing the CD 19 CAR and a truncated CD79b construct (Vector 3589: SEQ ID NO: 108), and a vector expressing the CD20 CAR and mlgM with a CD34t tag (Vector 3590: SEQ ID NO: 109).

[0186] FIG. 10D shows the percentage of cells expressing the CD 19 CAR and the CD22 CAR after MACS purification. The cells were transduced with a vector expressing the CD22 CAR and a truncated CD79a construct (Vector 3588: SEQ ID NO: 107), a vector expressing the CD 19 CAR and a truncated CD79b construct (Vector 3589: SEQ ID NO: 108), and a vector expressing the CD20 CAR and mlgM with a CD34t tag (Vector 3590: SEQ ID NO: 109).

[0187] FIG. 10E shows the percentage of cells expressing the CD20 CAR and the CD22 CAR prior to MACS purification. The cells were transduced with a vector expressing the CD22 CAR and a truncated CD79a construct (Vector 3588: SEQ ID NO: 107), a vector expressing the CD19 CAR and a truncated CD79b construct (Vector 3589: SEQ ID NO: 108), and a vector expressing the CD20 CAR and mlgM with a CD34t tag (Vector 3590: SEQ ID NO: 109).

DETAILED DESCRIPTION OF THE INVENTION

[0188] Disclosed herein include compositions and methods for selective expansion of a cell population, such as a CAR-T cell population. The compositions and methods described herein can be useful, for example, to generate cell populations in which a high percentage of the cells each express multiple different proteins of interest, such as one or more CARs, for example, two, three, or more CARs.

[0189] The present disclosure provides a recombinant polynucleic acid encoding a polypeptide comprising a first portion of a receptor complex and a second polynucleic acid encoding a polypeptide comprising a second portion of a receptor complex.

[0190] Also provided herein is a receptor complex comprising a first polypeptide comprising a first portion of a receptor complex and a second polypeptide comprising a second portion of the receptor complex.

[0191] Also provided herein is a cell comprising a receptor complex comprising a first polypeptide comprising a first portion of a receptor complex and a second sequence encoding a second polypeptide comprising a second portion of the receptor complex.

[0192] In some embodiments, the first polypeptide comprises: (a) a first extracellular domain comprising a selection tag, and (b) a first transmembrane domain from a first receptor; and the second polypeptide comprises: (a) a second transmembrane domain from a second receptor, wherein the first receptor and second receptors are different receptors, and wherein (i) the selection tag is not a cytokine binding domain from a cytokine receptor, and/or (ii) wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor, and wherein cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide. In some embodiments, the second polypeptide comprises a second selection tag, wherein the second selection tag is the same as the selection tag of the first polypeptide. In some embodiments, the second polypeptide does not comprise a selection tag. In some embodiments, the second polypeptide does not comprise an extracellular domain. In some embodiments, the recombinant polynucleic acid further comprises a third sequence encoding a third polypeptide that comprises a third transmembrane domain from a third receptor, wherein the first receptor and third receptor are different receptors, and wherein (i) the selection tag is not a cytokine binding domain from a cytokine receptor, and/or (ii) wherein the third polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor, and wherein cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding either the second polypeptide or the third polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide, the second polypeptide, and the third polypeptide.

Definitions

[0193] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0194] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0195] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0196] ‘ ‘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 nucleic acid or amino acid residues in the polynucleotide or polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0197] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0198] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong. Although any recombinant polypeptides, methods and other aspects similar or equivalent to those described herein can also be used in the practice or testing of the recombinant polypeptides, methods and other aspects, representative illustrative recombinant polypeptides, methods and other aspects are now described.

[0199] The terms “nucleic acid,” “polynucleic acid,” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, and/or synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid can comprise one or more bases and/or linkages that do not occur naturally in DNA or RNA, such as phosphoramidite linkages, 2’-modified ribose or deoxyribose, morpholino phosphoramidites, peptide-nucleic acid links, locked nucleic acid links, xanthine, 7-methylguanine, inosine, dihydrouracil, 5- methylcytosine, 5- hydroxymethylcytosine, and others. See, e.g., C.I.E. Smith et al., Ann Rev Pharmacol Toxicol (2019) 59:605-30, incorporated herein by reference. A nucleic acid can be double-stranded or single-stranded (for example, a sense strand or an antisense strand). A nucleic acid may contain unconventional or modified nucleotides. The terms “polynucleotide sequence,” “polynucleic acid sequence,” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a nucleic acid molecule.

[0200] As used herein, the term “CAR-T cell” means a T cell or population of T cells modified through molecular biological methods to contain a nucleic acid encoding a chimeric antigen receptor (CAR) or to express a CAR, such as on the T cell surface. The CAR is a recombinant polypeptide having an antigen binding domain with binding specificity to a desired target which is operably connected to an intracellular signaling domain comprising one or more T cell activation domains. By bypassing MHC-class I and class II restriction, CAR engineered T cells of both CD8+ and CD4+ subsets can be recruited for redirected target cell recognition. Exemplary CARs include, but are not limited to, fusions of an immunoglobulin binding functionality (e.g. , as a single-chain variable fragment (scFv) derived from a monoclonal antibody) to a transmembrane domain or to a hinge domain and a transmembrane domain, such as a CD8 or CD28 transmembrane domain, a CD8 hinge and CD8 transmembrane domain, a CD28 hinge and CD28 transmembrane domain, a CD8 hinge and CD28 transmembrane domain, or a CD28 hinge and CD8 transmembrane domain, and a CD28 intracellular domain, a 4-1BB/CD137 intracellular domain, a CD2 intracellular domain, an 0X40 (CD134) intracellular domain, a CD7 intracellular domain, a CD27 intracellular domain, a CD30 intracellular domain, a CD40 intracellular domain, a CD54 (ICAM) intracellular domain, a CD83 intracellular domain, or a CD278 (ICOS) intracellular domain, and a CD3- zeta (CD3Q intracellular domain. Such molecules result in the transmission of a CD3^ T cell activation signal in response to the immunoglobulin binding functionality portion of the CAR binding to its cognate antigen.

[0201] As used herein, the term “antibody” is used to refer to any antibody or antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab', Fab, single domain antibodies (DABs), Fv, scFv (single chain Fv). Techniques for preparing and using various antibody -based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (see, e.g. , Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The term “antibody” as used herein includes, but is not limited to, an antibody fragment or a variant molecule that retains the ability to bind its cognate antigen (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment, a heavy chain fragment and a binding domain-immunoglobulin fusion). The antibody can be a monoclonal or polyclonal, antibody and from any naturally occurring antibody isotype. The antibody can also be a human, humanized, chimeric, or CDR-grafted antibody. In other embodiments, the antibody has a heavy chain constant region chosen from, for example, IgGl, IgG2, IgG3, or IgG4. In another embodiment, the antibody has a light chain chosen from, for example, kappa or lambda. In one embodiment, the constant region is altered, e.g., mutated relative to a wild-type antibody constant region (e.g., a wild-type human IgGl, IgG2, IgG3, or IgG4 constant region), to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0202] As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0203] 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.

[0204] As used herein, the term “binding agent” means a molecule that can associate with any other molecule by way of covalent bonds, hydrogen bonds, ionic bonds, Van der Waals forces, London forces, or any combination of the forces. Binding agents include, but are not limited to, proteins and fragments thereof, peptidomimetic compounds, antibodies and fragments thereof, nucleic acids, toxins, and small molecules. [0205] As used herein, a “therapeutically effective amount” or “therapeutically effective dose” 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. In cases where the disease or disorder is a cancer, a therapeutically effective amount or dose of an agent means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” encompasses, but is not limited to, 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 can refer to 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” may depend on the purpose of the treatment and may be ascertainable by one skilled in the art using known techniques.

[0206] As used herein, the term “treat,” “treating,” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0207] It is appreciated that certain features of the recombinant polypeptides, and/or recombinant nucleic acids encoding the recombinant polypeptides, methods, and other aspects, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the recombinant polypeptides, and/or recombinant nucleic acids encoding the recombinant polypeptides, methods, and other aspects, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present recombinant polypeptides, methods and other aspects and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

Receptor complexes for cell selection [0208] Provided herein are compositions comprising a sequence encoding at least a first and a second polypeptide of a receptor complex. Also provided herein are exogenous receptor complexes comprising at least a first and a second polypeptide. Also provided herein are cells expressing at least a first and a second polypeptide of a receptor complex. In some embodiments, a receptor complex comprises at least a first polypeptide, a second polypeptide, and a third polypeptide. In some embodiments, a receptor complex comprises at least a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. Expression at the cell surface of one or more of the polypeptides that make up a receptor complex can be dependent on the presence (e.g., expression) of one or more of the other polypeptides that make up the receptor complex. For example, expression at the cell surface of one or more of the polypeptides that make up a receptor complex can be dependent on the presence (e.g., expression) of each of the polypeptides that make up a receptor complex.

[0209] At least one of the polypeptides that make up a given receptor complex described herein can comprise a selection tag. Thus, expression at the cell surface of a polypeptide containing a selection tag that is a portion of (e.g., a subunit of) a receptor complex can be dependent on the presence (e.g., expression) of one or more of each of the other polypeptides that make up the receptor complex. In some embodiments, a selection tag (e.g., a purification tag) is expressed at the cell surface only when each of the polypeptides that make up the receptor complex are present (e.g., expressed) in a given cell.

[0210] The compositions and methods described herein can be used to select cells (e.g. , via a selection tag) that express each of the polypeptides that make up a receptor complex from a population of cells.

[0211] The compositions and methods described herein can also be used to select cells that express multiple proteins of interest from a population of cells.

[0212] For example, each protein of interest can be expressed as a protein molecule containing at least one of the polypeptides that makes up a receptor complex. For example, each protein of interest can be expressed as a protein molecule containing at least one of the polypeptides that makes up a receptor complex and a given protein of interest, wherein the polypeptide that makes up a receptor complex and a given protein of interest are linked via a protease cleavage site.

[0213] For example, each protein of interest can be expressed from the same nucleic acid molecule that encodes at least one of the polypeptides that makes up a receptor complex. For example, each protein of interest can be expressed from the same nucleic acid molecule that encodes at least one of the polypeptides that makes up a receptor complex, wherein the sequence encoding the polypeptide that makes up a receptor complex and the sequence encoding a given protein of interest are separated or linked by an internal ribosome entry site (“IRES”) or have different promoters.

[0214] For example, a first CAR can be expressed as a first protein containing the first CAR and a first polypeptide that makes up a receptor complex, wherein the first polypeptide that makes up the receptor complex and the first CAR are linked via a protease cleavage site; and a second CAR can be expressed as a second protein molecule containing the second CAR and a second polypeptide that makes up the receptor complex, wherein the second polypeptide that makes up the receptor complex and the second CAR are linked via a protease cleavage site. For example, the first or second polypeptide that makes up the receptor complex can contain a selection tag and cells expressing the first protein and the second protein can be selected from a population of cells via the selection tag of the receptor complex that is expressed at the cell surface. Cells selected in such a manner will express both the first CAR and the second CAR.

[0215] Each of the polypeptides that make up a receptor complex can comprise a transmembrane domain. In some embodiments, the transmembrane domains of the polypeptides that make up a receptor complex interact with each other. At least one of the polypeptides that makes up a receptor complex can comprise an extracellular domain, e.g., an extracellular domain comprising a selection tag. In some embodiments, one or more of the polypeptides that make up a receptor complex can comprise an intracellular domain. In some embodiments, none of the polypeptides that make up a receptor complex comprises an intracellular domain.

Polypeptides (subunits) of receptor complexes

Extracellular domain

[0216] The extracellular domain of one or more polypeptides of a receptor complex can serve to present a selection tag. In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex comprises an extracellular domain that comprises a selection tag. In some embodiments, an extracellular domain of a polypeptide of a receptor complex does not comprise a selection tag. In some embodiments, an extracellular domain of a polypeptide of a receptor complex only comprises a selection tag. In some embodiments, an extracellular domain of a polypeptide of a receptor complex does not comprise an extracellular domain of a receptor.

[0217] In some embodiments, an extracellular domain of a polypeptide of a receptor complex comprises a truncated extracellular domain of a receptor. In some embodiments, an extracellular domain of a polypeptide of a receptor complex only comprises a truncated extracellular domain. For example, an extracellular domain of a polypeptide of a receptor complex can comprise a truncated extracellular domain of a receptor. For example, an extracellular domain of a polypeptide of a receptor complex can comprise a truncated extracellular domain of TREM1.

[0218] In some embodiments, an extracellular domain of a polypeptide of a receptor complex comprises a full-length extracellular domain of a receptor. For example, an extracellular domain of a polypeptide of a receptor complex can comprise a full-length extracellular domain of an extracellular domain of DAP12. In some embodiments, an extracellular domain of a polypeptide of a receptor complex comprises a truncated extracellular domain and a selection tag. In some embodiments, an extracellular domain of a polypeptide of a receptor complex comprises a full-length extracellular domain and a selection tag. In some embodiments, the extracellular domain of a polypeptide of a receptor complex may be fewer than 350 (or altematively, 100, 110, 120, 125, 130, 150, 185, 200, 225, 250, 275, 300, 320, 340) amino acids long. In some embodiments, the extracellular domain of a polypeptide of a receptor complex may be fewer than 30 (or alternatively 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 19, 20, 25, 27) amino acids long. In some embodiments, the extracellular domain of a polypeptide of a receptor complex may be longer than 350 (or alternatively, 400, 500, 600, 650, 675, 700, 750, 775, 800) amino acids long. In some embodiments, the extracellular domain of a polypeptide of a receptor complex may be longer than 30 (or alternatively 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70) amino acids long. In some embodiments, the extracellular domain of a polypeptide of a receptor complex is from about 30 to about 250 amino acids in length. In some embodiments, the extracellular domain of a polypeptide of a receptor complex is from about 250 to about 700 amino acids in length. In some embodiments, the selection tag of an extracellular domain of the first, second, third, and/or fourth polypeptides is at most about 700 (or alternatively, 675, 650, 600, 500, 400, 340, 338, 312, 300, 275, 250, 240, 230, 220, 200, 180, 150, 130, 120, 110, 100, 90, 80, 77, 70, 60, 50, 40) amino acids in length. In some embodiments, the selection tag of an extracellular domain of the first, second, third, and/or fourth polypeptides is at most about 20 (or alternatively, 19, 18, 17, 16, 15, 10, 5, 4, 3, 2, 1) amino acids in length.

[0219] In some embodiments, the extracellular domain of a polypeptide of a receptor complex comprises an amino acid sequence of any of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a polypeptide of a receptor complex consists or consists essentially of an amino acid sequence of any of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a polypeptide of the receptor complex comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a polypeptide of the receptor complex consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-17.

[0220] In some embodiments, the extracellular domain of a first polypeptide of a receptor complex may comprise any one of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a second polypeptide of the receptor complex may comprise any one of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a third polypeptide of the receptor complex may comprise any one of SEQ ID NOs: 1-17. In some embodiments, the extracellular domain of a fourth polypeptide of the receptor complex may comprise any one of SEQ ID NOs: 1-17.

[0221] In some embodiments, at least one, two, three, or four polypeptides in a receptor complex can comprise a selection tag. In some embodiments, at least two, three, or four polypeptides in a receptor complex can comprise a different selection tag. In some embodiments, at least two, three, or four polypeptides in a receptor complex can comprise the same selection tag. [0222] In some embodiments, only the first polypeptide of a receptor complex comprises a selection tag. In some embodiments, only the second polypeptide of a receptor complex comprises a selection tag. In some embodiments, only the third polypeptide of a receptor complex comprises a selection tag. In some embodiments, only the fourth polypeptide of a receptor complex comprise a selection tag.

[0223] In some embodiments, the first polypeptide and the second polypeptide each comprise a selection tag. In some embodiments, the first, second, and third polypeptides each comprise a selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise a selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise a selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise a selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise a selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise a selection tag.

[0224] In some embodiments, the first polypeptide and the second polypeptide each comprise a different selection tag. In some embodiments, the first, second, and third polypeptides each comprise a different selection tag. In some embodiments, the first, second, third, and fourth polypeptides each comprise a different selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise a different selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise a different selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise a different selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise a different selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise a different selection tag.

[0225] In some embodiments, the first polypeptide and the second polypeptide each comprise the same selection tag. In some embodiments, the first, second, and third polypeptides each comprise the same selection tag. In some embodiments, the first, second, third, and fourth polypeptides each comprise the same selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise the same selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise the same selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise the same selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise the same selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise the same selection tag.

[0226] In some embodiments, at least one, two, three, or four polypeptides in a receptor complex comprise an extracellular domain that comprises a selection tag. In some embodiments, at least two, three, or four polypeptides in a receptor complex each comprise an extracellular domain that comprises a different selection tag. In some embodiments, at least two, three, or four polypeptides in a receptor complex each comprise an extracellular domain that comprises the same selection tag. [0227] In some embodiments, only the first polypeptide comprises an extracellular domain that comprises a selection tag. In some embodiments, only the second polypeptide comprises an extracellular domain that comprises a selection tag. In some embodiments, only the third polypeptide comprises an extracellular domain that comprises a selection tag. In some embodiments, only the fourth polypeptide comprises an extracellular domain that comprises a selection tag.

[0228] In some embodiments, the first polypeptide and the second polypeptide each comprise a selection tag. In some embodiments, the first, second, and third polypeptides each comprise a selection tag. In some embodiments, the first, second, third, and fourth polypeptides each comprise a selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise a selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise a selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise a selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise a selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise a selection tag.

[0229] In some embodiments, the first polypeptide and the second polypeptide each comprise a different selection tag. In some embodiments, the first, second, and third polypeptides each comprise a different selection tag. In some embodiments, the first, second, third, and fourth polypeptides each comprise a different selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise a different selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise a different selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise a different selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise a different selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise a different selection tag.

[0230] In some embodiments, the first polypeptide and the second polypeptide each comprise the same selection tag. In some embodiments, the first, second, and third polypeptides each comprise the same selection tag. In some embodiments, the first, second, third, and fourth polypeptides each comprise the same selection tag. In some embodiments, the first polypeptide and the third polypeptide each comprise the same selection tag. In some embodiments, the first polypeptide and the fourth polypeptide each comprise the same selection tag. In some embodiments, the second polypeptide and the third polypeptide each comprise the same selection tag. In some embodiments, the second polypeptide and the fourth polypeptide each comprise the same selection tag. In some embodiments, the third polypeptide and the fourth polypeptide each comprise the same selection tag.

[0231] In some embodiments, an extracellular domain of a polypeptide of the receptor complex comprises a fluorescent tag. In some embodiments, the fluorescent tag is not a selection tag. In some embodiments, an extracellular domain of a polypeptide of the receptor complex does not comprise a fluorescent tag. In some embodiments, an extracellular domain of a polypeptide of the receptor complex comprises a selection tag and a fluorescent tag. In some embodiments, an extracellular domain of a polypeptide of the receptor complex comprises a selection tag, a fluorescent tag, and an extracellular domain of a polypeptide of a receptor complex. In some embodiments, the extracellular domain of the receptor complex comprises a selection tag, a fluorescent tag, and a truncated extracellular domain of a transmembrane protein.

[0232] In some embodiments, the selection tag comprises a purification tag. In some embodiments, the selection tag comprises multiple repeats of a purification tag sequence. In some embodiments, the selection tag comprises multiple repeats of the same purification tag sequence. In some embodiments, the selection tag may comprise two or more different purification tags. In some embodiments, an extracellular domain of a polypeptide of the receptor complex comprises multiple repeats of a purification tag and a fluorescent tag. In some embodiments, an extracellular domain of a polypeptide of the receptor complex comprises multiple repeats of a purification tag, a fluorescent tag, and an extracellular domain of a polypeptide of a receptor complex. In some embodiments, the extracellular domain of the receptor complex comprises multiple repeats of a purification tag, a fluorescent tag, and a truncated extracellular domain of a transmembrane protein.

[0233] Examples of selection tags may include, but are not limited to, a histidine-glycine tag, poly-arginine tag, poly-aspartate tag, poly-cysteine tag, poly-phenylalanine tag, c-myc tag, Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, a tag consisting of the amino acid sequence WSHPQFEK, a tag consisting of the ammo acid sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, CD34 tag, CD133 tag, CD304 tag, chloramphenicol acetyl transferase (CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, epidermal growth factor receptor (EGF) tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag, Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profmity eXact tag, Protein C tag, SI -tag, S-tag, biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, a VSV-G epitope tag derived from the Vescular Stomatis viral glycoprotein, and a V5 tag derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus of simian virus 5 (SV5) or any other suitable selection tag known in the art. In some embodiments, the selection tag is not a cytokine binding domain from a cytokine receptor. [0234] In some embodiments, the selection tag comprises a truncated receptor. In some embodiments, the truncated receptor can be truncated epidermal growth factor receptor (EGFRt), truncated CD34 (CD34t), or truncated human epidermal growth factor receptor 2 (HER2t). In some embodiments, the selection tag comprises an amino acid sequence of any one of SEQ ID NOs: 70-74. In some embodiments, the selection tag consists or consists essentially of an amino acid sequence of any one of SEQ ID NOs: 70-74. In some embodiments, the selection tag comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 70-74. In some embodiments, the selection tag consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 70- 74. Examples of fluorescent tags include, but are not limited to, eGFP, GFP, mNeonGreen, mtagBFP2, BFP, tagRFP, mCherry, tagRFP658, mTurquois, mBeRFP, mEGFP, mOrange2, mOrange, mGrape2, mTurquoise2, mCerulean3, EGFP, mWasabi, Superfolder GFP, mClover3, Venus, Citrine, mKOK, tdTomato, TagRFP-T, mRuby3, mScarlet, FusionRed, mStable, mKate2, mMaroonl, mCardinal, T- Sapphire, mCyRFPl, LSSmOrange, mBeRFP, CreiLOV, UnaG, miRFP670, TDsmURFP, iRFP670, mIFP, 1FP2.0, 1RFP720, FlAsH/peptidea, ReAsH/peptidea, TOl/scFvb, MG/scFvc, SNAP tag, SNAPf tag, CLIP tag, CLIPftag, Halo tag, TMP tag, SunTag, GFP1-10/GFP11 and sfCherryl-10/sfCherryl l, RNA tags, F30-Broccoli, Mango, DNB aptamer, JX1, DsRed, DsRed2, DsRed-Express, Dendra2, tdTomato, LSSmOrange, EBFP, ECFP, and EYFP. In some embodiments, the fluorescent tag is mNeonGreen. In some embodiments, the fluorescent tag is BFP. In some embodiments, the fluorescent tag is mtagBFP2. In some embodiments, the fluorescent tag is tagFRP. In some embodiments, the fluorescent tag is mCherry. In some embodiments, the fluorescent tag is tagRFP658. In some embodiments, the fluorescent tag is mTurquois. In some embodiments, the fluorescent tag is mBeRFP. In some embodiments, the fluorescent tag is mEGFP. In some embodiments, the fluorescent tag is mOrange2. In some embodiments, the fluorescent tag is mGrape2. In some embodiments, the fluorescent tag is detectable by flow cytometry.

[0235] In some embodiments, the selection tag of a polypeptide of a receptor complex is not expressed extracellularly on a cell that lacks a sequence encoding at least one polypeptide that makes up the receptor complex.

[0236] For example, the selection tag of a first polypeptide of a receptor complex made up of two polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex.

[0237] For example, the selection tag of a first polypeptide of a receptor complex made up of three polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of three polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the third polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of three polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex and that lacks a sequence encoding the third polypeptide of the receptor complex.

[0238] For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the third polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the fourth polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex and that lacks a sequence encoding the third polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex and that lacks a sequence encoding the fourth polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the third polypeptide of the receptor complex and that lacks a sequence encoding the fourth polypeptide of the receptor complex. For example, the selection tag of a first polypeptide of a receptor complex made up of four polypeptides may not be expressed extracellularly on a cell that lacks a sequence encoding the second polypeptide of the receptor complex, that lacks a sequence encoding the third polypeptide of the receptor complex, and that lacks a sequence encoding the fourth polypeptide of the receptor complex.

[0239] In some embodiments, the selection tag of the first polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide. In some embodiments, the selection tag of the first polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second, third, and/or fourth polypeptide. In some embodiments, the selection tag of the second polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the second polypeptide that lacks a sequence encoding the first polypeptide. In some embodiments, the selection tag of the second polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the second polypeptide that lacks a sequence encoding the first, third, and/or fourth polypeptide. In some embodiments, the selection tag of the third polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the third polypeptide that lacks a sequence encoding the first polypeptide. In some embodiments, the selection tag of the third polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the third polypeptide that lacks a sequence encoding the first, second, and/or fourth polypeptide. In some embodiments, the selection tag of the fourth polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the fourth polypeptide that lacks a sequence encoding the first polypeptide. In some embodiments, the selection tag of the fourth polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the fourth polypeptide that lacks a sequence encoding the first polypeptide, second polypeptide, and/or third polypeptide.

[0240] In some embodiments, at least one polypeptide of the receptor complex comprises a selection tag that is expressed extracellularly on a cell. In some embodiments, at least two, three, or four polypeptides of the receptor complex comprise a selection tag that is expressed extracellularly on a cell. In some embodiments, only the first polypeptide comprises a selection tag that is expressed extracellularly on a cell. In some embodiments, only the second polypeptide comprises a selection tag that is expressed extracellularly on a cell. In some embodiments, only the third polypeptide comprises a selection tag that is expressed extracellularly on a cell. In some embodiments, only the fourth polypeptide comprises a selection tag that is expressed extracellularly on a cell.

[0241] In some embodiments, the first polypeptide and the second polypeptide each comprise a different selection tag that is expressed extracellularly on the cell. In some embodiments, the first polypeptide and the third polypeptide each comprise a different selection tag that is expressed extracellularly on the cell. In some embodiments, the first polypeptide and the fourth polypeptide each comprise a different selection tag that is expressed extracellularly on the cell. In some embodiments, the second polypeptide and the third polypeptide each comprise a different selection tag that is expressed extracellularly on the cell. In some embodiments, the first polypeptide and the second polypeptide each comprise the same selection tag that is expressed extracellularly on the cell. In some embodiments, the first polypeptide and the third polypeptide each comprise the same selection tag that is expressed extracellularly on the cell. In some embodiments, the first polypeptide and the fourth polypeptide each comprise the same selection tag that is expressed extracellularly on the cell. In some embodiments, the second polypeptide and the third polypeptide each comprise the same selection tag that is expressed extracellularly on the cell.

[0242] In some embodiments, both the first polypeptide and second polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is different from the selection tag of the second polypeptide. In some embodiments, the first polypeptide, second polypeptide, and third polypeptide each comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is different from the selection tag of the second polypeptide and the selection tag of the third polypeptide. In some embodiments, the first polypeptide, second polypeptide, third polypeptide, and fourth polypeptide each comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is different from the selection tag of the second polypeptide, the selection tag of the third polypeptide, and the selection tag of the fourth polypeptide. In some embodiments, both the first polypeptide and third polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is different from the selection tag of the third polypeptide. In some embodiments, both the first polypeptide and fourth polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is different from the selection tag of the fourth polypeptide. In some embodiments, both the second polypeptide and third polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the second polypeptide is different from the selection tag of the third polypeptide. In some embodiments, both the second polypeptide and fourth polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the second polypeptide is different from the selection tag of the fourth polypeptide.

[0243] In some embodiments, both the first polypeptide and second polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is the same as the selection tag of the second polypeptide. In some embodiments, the first polypeptide, second polypeptide, and third polypeptide each comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is the same as the selection tag of the second polypeptide and the selection tag of the third polypeptide. In some embodiments, the first polypeptide, second polypeptide, third polypeptide, and fourth polypeptide each comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is the same as the selection tag of the second polypeptide, the selection tag of the third polypeptide, and the selection tag of the fourth polypeptide. In some embodiments, both the first polypeptide and third polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is the same as the selection tag of the third polypeptide. In some embodiments, both the first polypeptide and fourth polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the first polypeptide is the same as the selection tag of the fourth polypeptide. In some embodiments, both the second polypeptide and third polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the second polypeptide is the same as the selection tag of the third polypeptide. In some embodiments, both the second polypeptide and fourth polypeptide comprise a selection tag that is expressed extracellularly on a cell, wherein the selection tag of the second polypeptide is the same as the selection tag of the fourth polypeptide.

[0244] In some embodiments, a polypeptide of the receptor complex may comprise an extracellular terminus. In some embodiments, a polypeptide of the receptor complex may comprise an extracellular terminus that may be the N-terminus. In some embodiments, a polypeptide of the receptor complex may comprise an extracellular terminus that may be the C-terminus. In some embodiments, the selection tag may be fewer than 60 (or alternatively, fewer than 10, 15, 25, 30, 35, 40, 45, or 50) amino acid residues away from the extracellular terminus of a polypeptide of the receptor complex. In some embodiments, the selection tag may be zero amino acid residues away from the transmembrane terminus of a polypeptide of the receptor complex. In some embodiments, the selection tag may be serially concatenated directly to the extracellular terminus of a polypeptide of the receptor complex.

Transmembrane domain

[0245] Provided herein are constructs containing polypeptides that make up a receptor complex, wherein each of the polypeptides that make up a receptor complex comprises a transmembrane domain. In some embodiments the receptor complex is formed via interactions between the transmembrane domains of the polypeptides such that the receptor complex is formed at the cell surface. In some embodiments, expression of one or more or each of the polypeptides that make up a given receptor complex is dependent on their transmembrane domains’ interaction with each other such that the receptor complex is formed at the cell surface when all polypeptides that make up the receptor complex are expressed.

[0246] In some embodiments, each of the polypeptides of a given receptor complex comprises a transmembrane domain. In some embodiments, the transmembrane domains of each of the polypeptides of a given receptor complex interact with the transmembrane domain of another polypeptide of the receptor complex.

[0247] In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex comprises a transmembrane domain. In some embodiments, the transmembrane domain of a polypeptide comprises a sequence of any one of SEQ ID NOs: 18-26. In some embodiments, the transmembrane domain of a polypeptide of the receptor complex comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18- 26. In some embodiments, the transmembrane domain of a polypeptide of the receptor complex consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26.

[0248] In some embodiments, the extracellular domain of the receptor complex is operably connected to a transmembrane domain. In some embodiments, the extracellular domain may be connected directly to the transmembrane terminus of a polypeptide of the receptor complex. Optionally, in some embodiments, the extracellular domain is connected to the transmembrane domain by a short polypeptide linker. Optionally, in some embodiments, the short polypeptide linker may be fewer than 30 (or alternatively 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 19, 20, 25, 27) ammo acids long.

[0249] In some embodiments, the first, second, third, and/or the fourth transmembrane domain can be the transmembrane domain from DNAX Activating Protein of 12 kDA (DAP12), CD79a, CD79[3, Immunoglobulin M (IgM), Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), TREM2, IREM2, KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5A), CD300 Molecule Like Lamily Member B (CD300LB), Sialic Acid Binding Ig Like Lectin 14 (SIGLEC14), SIGLEC15, SIGLEC16, CD8a, CD8b, CD4, CD28, glycophorm A, EPOR, CD79, DAP10, IL21R or a modified IL21R, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, or PDGFRalpha. In some embodiments, the transmembrane domain can be any suitable transmembrane domain known in the art.

[0250] In some embodiments, the transmembrane domain is a TREM1 transmembrane domain. In some embodiments, the transmembrane domain is a TREM2 transmembrane domain. In some embodiments, the transmembrane domain is an IREM2 transmembrane domain. In some embodiments, the transmembrane domain is a KIR2DS2 transmembrane domain. In some embodiments, the transmembrane domain is a DAP12 transmembrane domain. In some embodiments, the transmembrane domain is an IgM CH2, CH3, CH4 transmembrane domain. In some embodiments, the transmembrane domain is an IgM CH2, CH4 transmembrane domain. In some embodiments, the transmembrane domain is an IgM CH3, CH4 transmembrane domain. In some embodiments, the transmembrane domain is an IgM CH2 transmembrane domain. In some embodiments, the transmembrane domain is a CD79a transmembrane domain. In some embodiments, the transmembrane domain is a CD79P transmembrane domain.

[0251] In some embodiments, the transmembrane domain comprises the transmembrane domain of TREM1. In some embodiments, the transmembrane domain of TREM1 comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the transmembrane domain of TREM1 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain of TREM1 consists or consists essentially of an amino acid sequence of SEQ ID NO: 18. In some embodiments, the transmembrane domain of TREM1 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18.

[0252] In some embodiments, the transmembrane domain comprises the transmembrane domain of TREM2. In some embodiments, the transmembrane domain of TREM2 comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the transmembrane domain of TREM2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. In some embodiments, the transmembrane domain of TREM2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 19. In some embodiments, the transmembrane domain of TREM2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

19.

[0253] In some embodiments, the transmembrane domain comprises the transmembrane domain of IREM2. In some embodiments, the transmembrane domain of IREM2 comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the transmembrane domain of IREM2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. In some embodiments, the transmembrane domain of IREM2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 20. In some embodiments, the transmembrane domain of IREM2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

20.

[0254] In some embodiments, the transmembrane domain comprises the transmembrane domain of KIR2DS2. In some embodiments, the transmembrane domain of KIR2DS2 comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, the transmembrane domain of KIR2DS2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21. In some embodiments, the transmembrane domain of KIR2DS2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 21. In some embodiments, the transmembrane domain of KIR2DS2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

21.

[0255] In some embodiments, the transmembrane domain comprises the transmembrane domain of DAP12. In some embodiments, the transmembrane domain of DAP12 comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, the transmembrane domain of DAP12 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22. In some embodiments, the transmembrane domain of DAP12 consists or consists essentially of an amino acid sequence of SEQ ID NO: 22. In some embodiments, the transmembrane domain of DAP12 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

22.

[0256] In some embodiments, the transmembrane domain comprises the transmembrane domain of IgM CH2, CH3, CH4. In some embodiments, the transmembrane domain of IgM CH2, CH3, CH4 comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the transmembrane domain of IgM CH2, CH3, CH4 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23. In some embodiments, the transmembrane domain of IgM CH2, CH3, CH4 consists or consists essentially of an amino acid sequence of SEQ ID NO: 23. In some embodiments, the transmembrane domain of IgM CH2, CH3, CH4 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23.

[0257] In some embodiments, the transmembrane domain comprises the transmembrane domain of IgM CH2. In some embodiments, the transmembrane domain of IgM CH2 comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24. In some embodiments, the transmembrane domain of IgM CH2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 24. In some embodiments, the transmembrane domain of IgM CH2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24.

[0258] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD79a. In some embodiments, the transmembrane domain of CD79a comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the transmembrane domain CD79a comprises an amino acid sequence of CD79a with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25. In some embodiments, the transmembrane domain of CD79a consists or consists essentially of an amino acid sequence of SEQ ID NO: 25. In some embodiments, the transmembrane domain consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25.

[0259] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD79p. In some embodiments, the transmembrane domain of CD79P comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26. In some embodiments, the transmembrane domain of CD79P n consists or essentially of an amino acid sequence of SEQ ID NO: 26. In some embodiments, the transmembrane domain of CD79P consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.

[0260] In some embodiments, each transmembrane domain of either the first, second, third, or fourth polypeptide in the receptor complex comprises a different amino acid sequence. In some embodiments, the receptor complex may comprise at least two polypeptides that each have transmembrane domains of the same amino acid sequence.

[0261] In some embodiments, the first polypeptide comprises the first transmembrane domain. In some embodiments, the second polypeptide comprises the second transmembrane domain. In some embodiments, the second polypeptide comprises the third transmembrane domain. In some embodiments, the third polypeptide comprises the third transmembrane domain. In some embodiments, the fourth polypeptide comprises the fourth transmembrane domain.

[0262] In some embodiments, the first polypeptide consists of the first transmembrane domain. In some embodiments, the second polypeptide consists of the second transmembrane domain. In some embodiments, the second polypeptide consists of the third transmembrane domain. In some embodiments, the third polypeptide consists of the third transmembrane domain. In some embodiments, the fourth polypeptide consists of the fourth transmembrane domain.

[0263] In some embodiments, the first transmembrane domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26. In some embodiments, the second transmembrane domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26. In some embodiments, the third transmembrane domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26.

[0264] In some embodiments, the first transmembrane domain consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26. In some embodiments, the second transmembrane domain consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26. In some embodiments, the third transmembrane domain consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 18-26.

Intracellular Domain

[0265] In some embodiments, none of the polypeptides that make up a given receptor complex comprises a functional intracellular signaling domain.

[0266] In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex does not comprise an intracellular domain.

[0267] In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex comprises an intracellular domain. In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex comprises a truncated intracellular domain. In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex comprises a full-length intracellular domain.

[0268] In some embodiments, the intracellular domain of a polypeptide of the receptor complex comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 27-37. In some embodiments, the intracellular domain of a polypeptide of the receptor complex consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 27- 37.

[0269] In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex does not comprise an intracellular domain comprising an intracellular signaling domain. In some embodiments, the first, second, third, and/or fourth polypeptide of the receptor complex does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor. In some embodiments, the intracellular domain of the first, second, third, and/or fourth polypeptide does not transduce extracellular signals to activate downstream signaling cascades. In some embodiments, the intracellular domain of a polypeptide of a receptor complex may be fewer than 75 (or alternatively 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70) ammo acids long. In some embodiments, the intracellular domain of a polypeptide of a receptor complex may be fewer than 30 (or alternatively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) amino acids long. In some embodiments, the intracellular domain of a polypeptide of a receptor complex may be longer than 75 (or alternatively, 100, 110, 120, 125, 130, 150, 185, 200, 225, 250, 275, 300, 320, 340) amino acids long. In some embodiments, the intracellular domain of a polypeptide of a receptor complex may be at least 1 (or alternatively 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 19, 20, 25) amino acid long. In some embodiments, the intracellular domain of a polypeptide of a receptor complex is from about 1 to about 100 amino acids in length. In some embodiments, each intracellular domain of the first, second, third, and/or fourth polypeptides in the receptor complex comprise a different amino acid sequence. In some embodiments, at least two polypeptides in the receptor complex comprise intracellular domains comprising the same amino acid sequence.

[0270] In some embodiments, the first, second, third, and/or the fourth intracellular domain can be the intracellular domain from DNAX Activating Protein of 12 kDa (DAP 12), CD79a, CD79J3, Immunoglobulin M (IgM), Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), TREM2, IREM2, KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5A), CD300 Molecule Like Lamily Member B (CD300LB), Sialic Acid Binding Ig Like Lectin 14 (SIGLEC14), SIGLEC15, SIGLEC16, CD8a, CD8b, CD4, CD28, glycophorm A, EPOR, CD79, DAP10, IL21R or a modified IL21R, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, or PDGFRalpha. In some embodiments, the intracellular domain can be any suitable intracellular domain known in the art.

[0271] In some embodiments, the intracellular domain is a TREM1 intracellular domain. In some embodiments, the intracellular domain is a TREM2 intracellular domain. In some embodiments, the intracellular domain is an IREM2 intracellular domain. In some embodiments, the intracellular domain is a KIR2DS2 intracellular domain.

[0272] In some embodiments, the intracellular domain is a DAP12 intracellular domain. In some embodiments, the intracellular domain is an IgM CH2, CH3, CH4 intracellular domain. In some embodiments, the intracellular domain is an IgM CH2 intracellular domain. In some embodiments, the intracellular domain is a CD79a intracellular domain. In some embodiments, the intracellular domain is a CD79J3 intracellular domain.

[0273] In some embodiments, the intracellular domain comprises the intracellular domain of TREM1. In some embodiments, the intracellular domain of TREM1 comprises an amino acid sequence of SEQ ID NO:

27. In some embodiments, the intracellular domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27. In some embodiments, the intracellular domain of TREM1 consists or consists essentially of an amino acid sequence of SEQ ID NO: 27. In some embodiments, the intracellular domain of TREM1 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27.

[0274] In some embodiments, the intracellular domain comprises the intracellular domain of TREM2. In some embodiments, the intracellular domain of TREM2 comprises an amino acid sequence of SEQ ID NO:

28. In some embodiments, the intracellular domain of TREM2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28. In some embodiments, the intracellular domain of TREM2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 28. In some embodiments, the intracellular domain of TREM2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28.

[0275] In some embodiments, the intracellular domain comprises the intracellular domain of IREM2. In some embodiments, the intracellular domain of IREM2 comprises an amino acid sequence of SEQ ID NO:

29. In some embodiments, the intracellular domain of IREM2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 29. In some embodiments, the intracellular domain of IREM2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 29. In some embodiments, the intracellular domain ofIREM2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 29.

[0276] In some embodiments, the intracellular domain comprises the intracellular domain of KIR2DS2. In some embodiments, the intracellular domain of KIR2DS2 comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the intracellular domain of KIR2DS2 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 30. In some embodiments, the intracellular domain of KIR2DS2 consists or consists essentially of an amino acid sequence of SEQ ID NO: 30. In some embodiments, the intracellular domain of KIR2DS2 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 30.

[0277] In some embodiments, the intracellular domain comprises the intracellular domain of DAP12. In some embodiments, the intracellular domain comprises of DAP12 an amino acid sequence of SEQ ID NO: 31. In some embodiments, the intracellular domain of DAP12 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31. In some embodiments, the intracellular domain of DAP12 consists or consists essentially of an amino acid sequence of SEQ ID NO: 31. In some embodiments, the intracellular domain of DAP12 consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31.

[0278] In some embodiments, the intracellular domain comprises a truncated intracellular domain of DAP12. In some embodiments, the truncated intracellular domain of DAP12 comprises an amino acid sequence of either SEQ ID NO: 32 or 33. In some embodiments, the intracellular domain comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to either SEQ ID NO: 32 or 33. In some embodiments, the intracellular domain consists of an amino acid sequence of either SEQ ID NO: 32 or 33. In some embodiments, the intracellular domain consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to either SEQ ID NO: 32 or 33.

[0279] In some embodiments, the intracellular domain comprises the intracellular domain of CD79a. In some embodiments, the intracellular domain of CD79a comprises an amino acid sequence of SEQ ID NO: 34 or 35. In some embodiments, the intracellular domain of CD79a comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34 or 35. In some embodiments, the intracellular domain of CD79a consists or consists essentially of an amino acid sequence of SEQ ID NO: 34 or 35. In some embodiments, the intracellular domain of CD79a consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34 or 35.

[0280] In some embodiments, the intracellular domain comprises the intracellular domain of CD79[3. In some embodiments, the intracellular domain of CD79J3 comprises an amino acid sequence of SEQ ID NO: 36 or 37. In some embodiments, the intracellular domain of CD79|3 comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36 or 37. In some embodiments, the intracellular domain of CD79J3 consists or consist essentially of an amino acid sequence of SEQ ID NO: 36 or 37. In some embodiments, the intracellular domain of CD79P consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36 or 37.

ER Retention and Cell Surface Expression

[0281] In some embodiments, a polypeptide of the receptor complex is retained in the endoplasmic reticulum (ER) of the cell if the receptor complex comprises at least one polypeptide whose expression is dependent on the expression of a second, third, and/or fourth polypeptide, and the second, third, and/or fourth polypeptide is not expressed.

[0282] In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the second polypeptide by the cell. In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the third polypeptide by the cell. In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the fourth polypeptide by the cell. In some embodiments, expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the second polypeptide and third polypeptide by the cell.

[0283] In some embodiments, expression of the second polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the first polypeptide by the cell. In some embodiments, expression of the second polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the third polypeptide by the cell. In some embodiments, expression of the second polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the fourth polypeptide by the cell. In some embodiments, expression of the second polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the first polypeptide and third polypeptide by the cell.

[0284] In some embodiments, expression of the third polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the first polypeptide by the cell. In some embodiments, expression of the third polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the second polypeptide by the cell. In some embodiments, expression of the third polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the fourth polypeptide by the cell. In some embodiments, expression of the third polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the first polypeptide and second polypeptide by the cell.

[0285] In some embodiments, the first polypeptide is retained in the ER of a cell that does not express the second, third, and/or fourth polypeptide. In some embodiments, the second polypeptide is retained in the ER of a cell that does not express the first, third, and/or fourth polypeptide. In some embodiments, the third polypeptide is retained in the ER of a cell that does not express the first, second, and/or fourth polypeptide. In some embodiments, the fourth polypeptide is retained in the ER of a cell that does not express the first, second, and/or third polypeptide.

[0286] In some embodiments, cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first and second polypeptides. In some embodiments, cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, and third polypeptides. In some embodiments, cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the fourth polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, third, and fourth polypeptides.

[0287] In some embodiments, cell surface expression of the selection tag of the second polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the second polypeptide that lacks a sequence encoding the first polypeptide compared to cell surface expression of the selection tag of the second polypeptide on a cell comprising the recombinant polynucleic acid encoding the first and second polypeptides. In some embodiments, cell surface expression of the selection tag of the second polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the second polypeptide that lacks a sequence encoding the third polypeptide compared to cell surface expression of the selection tag of the second polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, and third polypeptides. In some embodiments, cell surface expression of the selection tag of the second polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the second polypeptide that lacks a sequence encoding the fourth polypeptide compared to cell surface expression of the selection tag of the second polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, third, and fourth polypeptides.

[0288] In some embodiments, cell surface expression of the selection tag of the third polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the third polypeptide that lacks a sequence encoding the first polypeptide compared to cell surface expression of the selection tag of the third polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, and third polypeptides. In some embodiments, cell surface expression of the selection tag of the third polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the third polypeptide that lacks a sequence encoding the second polypeptide compared to cell surface expression of the selection tag of the third polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, and third polypeptides. In some embodiments, cell surface expression of the selection tag of the third polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the third polypeptide that lacks a sequence encoding the fourth polypeptide compared to cell surface expression of the selection tag of the third polypeptide on a cell comprising the recombinant polynucleic acid encoding the first, second, third, and fourth polypeptides.

[0289] In some embodiments, retention of the first polypeptide in the ER is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide. In some embodiments, retention of the first polypeptide in the ER is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the third polypeptide. In some embodiments, retention of the first polypeptide in the ER is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first, second, and third polypeptides. In some embodiments, retention of the first polypeptide in the ER is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the fourth polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first, second, third, fourth polypeptides.

[0290] In some embodiments, at least one polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. In some embodiments, all the polypeptides of a receptor complex are non-functional receptors or are not capable of signaling to a cell in which they are expressed. In some embodiments, the first polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. In some embodiments, the second polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. In some embodiments, the third polypeptide is a nonfunctional receptor or is not capable of signaling to a cell in which it is expressed. In some embodiments, the fourth polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed.

Embodiments

[0291] In some embodiments, a receptor complex comprises receptors comprising at least two different polypeptides. For example, a receptor complex can comprise a polypeptide with a TREM1 transmembrane domain and a polypeptide with a DAP12 transmembrane domain. In some embodiments, a receptor complex comprises receptors comprising at least three polypeptides wherein at least two polypeptides comprise different amino acid sequences. For example, a receptor complex can comprise a polypeptide with a TREM1 transmembrane domain and two polypeptides each with a DAP12 transmembrane domain, wherein one of the polypeptides with a DAP12 transmembrane domain comprises a tag. In some embodiments, a receptor complex comprises receptors comprising at least three polypeptides wherein each of the three polypeptides comprise different amino acid sequences. For example, a receptor complex can comprise a polypeptide with a CD79a transmembrane domain, a polypeptide with a CD79b transmembrane domain and a polypeptide with an IgM transmembrane domain. In some embodiments, a receptor complex comprises receptors comprising at least four polypeptides, wherein at least three polypeptides comprise different amino acid sequences. For example, a receptor complex can comprise a polypeptide with a CD79a transmembrane domain, a polypeptide with a CD79b transmembrane domain and two polypeptides each with an IgM transmembrane domain. In some embodiments, the first, second, third, and/or fourth polypeptides in a receptor complex interact with each other (e.g., via their transmembrane domains) to form a multimeric complex. In some embodiments, the first polypeptide and the second polypeptide form a complex. In some embodiments, the first, second, and third polypeptides form a complex. In some embodiments, the first, second, third, and fourth polypeptides form a complex. In some embodiments, the first polypeptide and the second polypeptide form a dimer. For example, a receptor complex can comprise a polypeptide with a TREM1 transmembrane domain and a polypeptide with a DAP12 transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide form a trimer. For example, a receptor complex can comprise a polypeptide with a TREM1 transmembrane domain and two polypeptides each with a DAP12 transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide form a tetramer. For example, a receptor complex can comprise a polypeptide with a CD79a transmembrane domain, a polypeptide with a CD79b transmembrane domain and a polypeptide each with an IgM transmembrane domain. In some embodiments, the first polypeptide and the second polypeptide form a pentamer. In some embodiments, the first, second, and third polypeptides form a trimer. For example, a receptor complex can comprise a polypeptide with a CD79a transmembrane domain, a polypeptide with a CD79b transmembrane domain and a polypeptide with an IgM transmembrane domain. In some embodiments, the first, second, and third polypeptides form a tetramer. For example, a receptor complex can comprise a polypeptide with a CD79a transmembrane domain, a polypeptide with a CD79b transmembrane domain and two polypeptides each with an IgM transmembrane domain.

[0292] In some embodiments, the first polypeptide and the second polypeptide are each different molecules. In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide are each different molecules. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are each different molecules. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the second polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42- 69. In some embodiments, the second polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the third polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the third polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the fourth polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69. In some embodiments, the fourth polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-69.

[0293] In some embodiments, the first receptor and the second receptor are different receptors. In some embodiments, the first receptor and the third receptor are different receptors. In some embodiments, the second receptor and the third receptor are different receptors. In some embodiments, the second receptor and the fourth receptor are the same receptors. In some embodiments, the first receptor and the fourth receptor are different receptors.

[0294] In some embodiments, a receptor complex that comprises a polypeptide comprising a transmembrane domain from DAP12 will also comprise a polypeptide comprising a transmembrane domain from either TREM1, TREM2, IREM1, or KIR2DS2. In some embodiments, any cell comprising a polypeptide comprising TREM1 is more likely to have TREM1 retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes DAP12. In some embodiments, any cell comprising a polypeptide comprising TREM2 is more likely to have TREM2 retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes DAP12. In some embodiments, any cell comprising a polypeptide comprising IREM1 is more likely to have IREM1 retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes DAP12. In some embodiments, any cell comprising a polypeptide comprising KIR2DS2 is more likely to have KIR2DS2 retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes DAP12. [0295] In some embodiments, any cell comprising a polypeptide comprising a TREM1 transmembrane domain is more likely to have the TREM1 transmembrane domain-containing polypeptide retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes the transmembrane domain of DAP12. In some embodiments, any cell comprising a polypeptide comprising a TREM2 transmembrane domain is more likely to have the TREM2 transmembrane domain-containing polypeptide retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes the transmembrane domain of DAP12. In some embodiments, any cell comprising a polypeptide comprising a IREM1 transmembrane domain is more likely to have the IREM1 transmembrane-containing polypeptide retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes the transmembrane domain of DAP12. In some embodiments, any cell comprising a polypeptide comprising a KIR2DS2 transmembrane domain is more likely to have KIR2DS2 retained in the ER when the same cell lacks at least one other recombinant polynucleic acid that also encodes the transmembrane domain of DAP12.

[0296] In some embodiments, the first receptor is TREM1 and the second receptor is DAP12. In some embodiments, the first receptor is TREM1, the second receptor is DAP12, and the third receptor is DAP12. In some embodiments, the first receptor is DAP12 and the second receptor is TREM1. In some embodiments, the first receptor is DAP12, the second receptor is TREM1, and the third receptor is DAP12. In some embodiments, the first receptor is DAP12, the second receptor is DAP12, and the third receptor is TREM1. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-50. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 42-50. In some embodiments, the second polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the second polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the third polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the third polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 51-55.

[0297] In some embodiments, the first polypeptide comprises EGFRt concatenated to TREM1. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 42. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 42.

[0298] In some embodiments, the first polypeptide comprises CD34t concatenated to TREM1. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 43-46. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 43- 46.

[0299] In some embodiments, the first polypeptide comprises EGFRt concatenated to the extracellular domain of DAP12. In some embodiments, the first polypeptide comprises CD34t concatenated to the extracellular domain of DAP12. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of DAP12. In some embodiments, the second polypeptide comprises CD34t concatenated to the extracellular domain of DAP12. In some embodiments, the third polypeptide comprises EGFRt concatenated to the extracellular domain of DAP12. In some embodiments, the third polypeptide comprises CD34t concatenated to the extracellular domain of DAP12.

[0300] In some embodiments, a receptor complex that comprises a polypeptide comprising a transmembrane domain from CD79a will also comprise a polypeptide comprising a transmembrane domain from CD79 and a polypeptide comprising a transmembrane domain from IgM. In some embodiments, the first receptor is IgM and the second receptor is CD79a. In some embodiments, the first receptor is EGFRt concatenated to IgM and the second receptor is CD79a. In some embodiments, the first receptor is IgM, the second receptor is CD79a, and the third receptor is CD79 . In some embodiments, the first receptor is EGFRt concatenated to IgM, the second receptor is CD79a, and the third receptor is CD79|3. In some embodiments, the first receptor is IgM and the second receptor is CD79|3. In some embodiments, the first receptor is IgM, the second receptor is CD79 , and the third receptor is CD79a. In some embodiments, the first receptor is EGFRt concatenated to IgM, the second receptor is CD79 , and the third receptor is CD79a. In some embodiments, the first receptor is CD79a and the second receptor is CD79[3. In some embodiments, the first receptor is EGFRt concatenated to CD79a and the second receptor is CD79J3. In some embodiments, the first receptor is CD79a, the second receptor is CD79|3, and the third receptor is IgM. In some embodiments, the first receptor is EGFRt concatenated to CD79a, the second receptor is CD79P, and the third receptor is IgM. In some embodiments, the first receptor is CD79a, the second receptor is EGFRt concatenated to CD79|3, and the third receptor is IgM. In some embodiments, the first receptor is CD79a, the second receptor is CD79|3, and the third receptor is EGFRt concatenated to IgM. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, or any combination thereof, lacks an antigen binding domain. In some embodiments, the IgM lacks one or more fragment antigen binding (Fab) domains. In some embodiments, the IgM polypeptide is a truncated IgM polypeptide sequence. In some embodiments, the IgM polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 56-62 and 69. In some embodiments, a receptor complex that comprises a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 56-62 and 69 also comprises a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 63, 64, or 67 and a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 65, 66, or 68.

[0301] In some embodiments, any cell comprising a polypeptide comprising IgM is more likely to have IgM retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes CD79a and at least one other recombinant polynucleic acid that also encodes CD790. In some embodiments, any cell comprising a polypeptide comprising CD79a is more likely to have CD79a retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes IgM and at least one other recombinant polynucleic acid that also encodes CD790. In some embodiments, any cell comprising a polypeptide comprising CD790 is more likely to have CD790 retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes IgM and at least one other recombinant polynucleic acid that also encodes CD79a.

[0302] In some embodiments, any cell comprising a polypeptide comprising an IgM transmembrane domain is more likely to have the IgM-containing polypeptide retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes a transmembrane domain of CD79a and at least one other recombinant polynucleic acid that also encodes a transmembrane domain of CD790. In some embodiments, any cell comprising a polypeptide comprising a transmembrane domain of CD79a is more likely to have the CD79a transmembrane domain-containing polypeptide retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes an IgM transmembrane domain and at least one other recombinant polynucleic acid that also encodes a CD790 transmembrane domain. In some embodiments, any cell comprising a polypeptide comprising a transmembrane domain of CD790 is more likely to have the CD790 transmembrane domain-containing polypeptide retained in the ER when the same cell lacks at least one recombinant polynucleic acid that also encodes an IgM transmembrane domain and at least one other recombinant polynucleic acid that also encodes a CD79a transmembrane domain.

[0303] In some embodiments, the first, second, or third polypeptide comprises EGFRt concatenated to the extracellular domain of IgM. In some embodiments, the first, second, or third polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69. In some embodiments, the first, second, or, third polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.

[0304] In some embodiments, the first polypeptide comprises EGFRt concatenated to the extracellular domain of CD79a. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

67. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD79|3. In some embodiments, the second polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the second polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

68. In some embodiments, the third polypeptide comprises EGFRt concatenated to the extracellular domain of IgM. In some embodiments, the third polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69. In some embodiments, the third polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.

[0305] In some embodiments, the first polypeptide comprises EGFRt concatenated to the extracellular domain of CD79p. In some embodiments, the first polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the first polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 68. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD79a. In some embodiments, the second polypeptide comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67. In some embodiments, the second polypeptide consists of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 67.

[0306] In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain of CD79a, and the third polypeptide comprises at least the transmembrane domain of CD79|3. In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain of CD79 , and the third polypeptide comprises at least the transmembrane domain of CD79a. In some embodiments, when the first polypeptide comprises CD79a, the second polypeptide comprises at least the transmembrane domain of CD79 , and the third polypeptide comprises at least the transmembrane domain of IgM. In some embodiments, when the first polypeptide comprises CD79|3, the second polypeptide comprises at least the transmembrane domain of CD79a, and the third polypeptide comprises at least the transmembrane domain of IgM.

[0307] In some embodiments, a receptor complex that comprises a polypeptide comprising a transmembrane domain from CD79a will also comprise a polypeptide comprising a transmembrane domain CD79J3 and a polypeptide comprising a transmembrane domain from either IgM CH2, CH3, CH4 or IgM CH2. In some embodiments, a receptor complex that comprises a polypeptide comprising a transmembrane domain from CD79 will also comprise a polypeptide comprising a transmembrane domain CD79a and a polypeptide comprising a transmembrane domain from either IgM CH2, CH3, CH4 or IgM CH2. In some embodiments, a receptor complex that comprises a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25 will also comprise a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26 and a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to either SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, a receptor complex that comprises a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26 will also comprise a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25 and a polypeptide comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to either SEQ ID NO: 23 or SEQ ID NO: 24.

[0308] In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain of CD79a, and the third polypeptide comprises at least the transmembrane domain of CD79 . In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain of CD79 , and the third polypeptide comprises at least the transmembrane domain of CD79a. In some embodiments, when the first polypeptide comprises CD79a, the second polypeptide comprises at least the transmembrane domain of CD79J3, and the third polypeptide comprises at least the transmembrane domain of IgM. In some embodiments, when the first polypeptide comprises CD79J3, the second polypeptide comprises at least the transmembrane domain of CD79a, and the third polypeptide comprises at least the transmembrane domain of IgM.

[0309] In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain and extracellular domain of CD79a, and the third polypeptide comprises at least the transmembrane domain and extracellular domain of CD79 . In some embodiments, when the first polypeptide comprises IgM, the second polypeptide comprises at least the transmembrane domain and extracellular domain of CD79|3, and the third polypeptide comprises at least the transmembrane domain and extracellular domain of CD79a. In some embodiments, when the first polypeptide comprises CD79a, the second polypeptide comprises at least the transmembrane domain and extracellular domain of CD79P, and the third polypeptide comprises at least the transmembrane domain and extracellular domain of IgM. In some embodiments, when the first polypeptide comprises CD790, the second polypeptide comprises at least the transmembrane domain and extracellular domain of CD79a, and the third polypeptide comprises at least the transmembrane domain and extracellular domain of IgM.

[0310] In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD79a. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD790. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD79a. In some embodiments, the second polypeptide comprises EGFRt concatenated to the extracellular domain of CD790. In some embodiments, the second polypeptide comprises CD34t concatenated to the extracellular domain of CD79a. In some embodiments, the second polypeptide comprises CD34t concatenated to the extracellular domain of CD79 . In some embodiments, the second polypeptide comprises CD34t concatenated to the extracellular domain of CD79a. In some embodiments, the second polypeptide comprises CD34t concatenated to the extracellular domain of CD790.

[0311] In some embodiments, the first transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0312] In some embodiments, the second transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0313] In some embodiments, the third transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26.

[0314] For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-21, and the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 18, and the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 19, and the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 20, and the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 21, and the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22.

[0315] For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-21, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 18, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 19, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 20, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 21, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 22.

[0316] For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 23 and 24, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 25, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 26. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 23, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 25, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 26. For example, the first transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 24, the second transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 25, and the third transmembrane domain can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 26.

[0317] In some embodiments, the first polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69. [0318] In some embodiments, the second polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69.

[0319] In some embodiments, the third polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69.

[0320] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42 and 44-50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0321] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0322] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0323] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0324] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0325] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0326] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0327] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55.

[0328] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50, and the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55. [0329] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42 and 44-50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0330] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0331] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 42; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0332] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 44; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0333] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 45; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: [0334] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 46; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0335] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 47; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0336] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 48; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0337] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 49; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0338] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 51 ; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 52; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 53; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 54; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 50; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 55; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 51-55.

[0339] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 56-62 and 69; the second polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 63, 64 and 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0340] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 56; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 56; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 56; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0341] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 57; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 57; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 57; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0342] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 58; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 58; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 58; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0343] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 59; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 59; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 59; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0344] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 60; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 60; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 60; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0345] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 61; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 61; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 61; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0346] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 62; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 62; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 62; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

[0347] For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 69; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 63; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 69; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 64; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68. For example, the first polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 69; the second polypeptide can comprise a sequence with at least about 80% sequence identity to SEQ ID NO: 67; and the third polypeptide can comprise a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 65, 66 and 68.

Recombinant Polynucleic Acids

[0348] In some aspects, the present disclosure describes polynucleic acid sequences encoding any of the transmembrane receptor complex polypeptides disclosed herein.

[0349] In some embodiments, the recombinant polynucleic acids of a receptor complex comprise a first recombinant polynucleic acid comprising a sequence encoding the first polypeptide and a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide. In some embodiments, the recombinant polynucleic acids of a receptor complex further comprise a third recombinant polynucleic acid comprising a sequence encoding the third polypeptide. In some embodiments, the recombinant polynucleic acids of a receptor complex further comprise a fourth recombinant polynucleic acid comprising a sequence encoding the fourth polypeptide.

[0350] In some embodiments, the first recombinant polynucleic acid encodes a first transmembrane domain of a receptor complex. In some embodiments, the second recombinant polynucleic acid encodes a second transmembrane domain of a receptor complex. In some embodiments, the third recombinant polynucleic acid encodes a third transmembrane domain of a receptor complex. In some embodiments, the fourth recombinant polynucleic acid encodes a fourth transmembrane domain of a receptor complex.

[0351] In some embodiments, the first recombinant polynucleic acid encodes a first polypeptide comprising a selection tag and a transmembrane domain. In some embodiments, the first recombinant polynucleic acid encodes a first polypeptide comprising a selection tag, an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the second recombinant polynucleic acid encodes a second polypeptide comprising a selection tag and a transmembrane domain. In some embodiments, the second recombinant polynucleic acid encodes a second polypeptide comprising a selection tag, an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the third recombinant polynucleic acid encodes a third polypeptide comprising a selection tag and a transmembrane domain. In some embodiments, the third recombinant polynucleic acid encodes a third polypeptide comprising a selection tag, an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the fourth recombinant polynucleic acid encodes a fourth polypeptide comprising a selection tag and a transmembrane domain. In some embodiments, the fourth recombinant polynucleic acid encodes a fourth polypeptide comprising a selection tag, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0352] In some embodiments, the first, second, third, and/or fourth polynucleic acids each comprise a sequence encoding a gene of interest. In some embodiments, the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest. In some embodiments, the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. In some embodiments, the third recombinant polynucleic acid further comprises a sequence encoding a third gene of interest. In some embodiments, the fourth recombinant polynucleic acid further comprises a sequence encoding a fourth gene of interest. In some embodiments, the first gene of interest comprises a chimeric antigen receptor (CAR). In some embodiments, the second gene of interest comprises a CAR. In some embodiments, the third gene of interest comprises a CAR. In some embodiments, the fourth gene of interest comprises a CAR. In some embodiments, the first, second, third, and fourth CARs each target a different antigen. [0353] In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the first, second, third, and/or fourth polypeptides of the receptor complex receptor may further comprise a CAR. In some embodiments, the first recombinant polynucleic acid comprises a sequence encoding a first CAR. In some embodiments, the second recombinant polynucleic acid comprises a sequence encoding a second CAR. In some embodiments, the third recombinant polynucleic acid comprises a sequence encoding a third CAR. In some embodiments, the fourth recombinant polynucleic acid comprises a sequence encoding a fourth CAR.

[0354] In some embodiments, the sequence encoding the first gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the fourth gene of interest encodes for a fourth protein of interest, wherein the fourth polypeptide and the fourth protein of interest are expressed as a single polypeptide molecule.

[0355] In some embodiments, the first polypeptide and the first CAR are expressed as a single polypeptide molecule. In some embodiments, the first polypeptide is linked to the sequence encoding the first CAR by a sequence encoding a linker. In some embodiments, the second polypeptide and the second CAR are expressed as a single polypeptide molecule. In some embodiments, the second polypeptide is linked to the sequence encoding the second CAR by a sequence encoding a linker. In some embodiments, the third polypeptide and the third CAR are expressed as a single polypeptide molecule. In some embodiments, the third polypeptide is linked to the sequence encoding the third CAR by a sequence encoding a linker. In some embodiments, the fourth polypeptide and the fourth CAR are expressed as a single polypeptide molecule. In some embodiments, the fourth polypeptide is linked to the sequence encoding the fourth CAR by a sequence encoding a linker.

Recombinant Polynucleic Acids - Linkers

[0356] In some embodiments, at least one sequence encoding a polypeptide of the receptor complex is linked to the sequence encoding a gene of interest by a sequence encoding a linker. In some embodiments, the sequence encoding the first polypeptide is linked to the sequence encoding the first protein of interest by a sequence encoding a linker. In some embodiments, the sequence encoding the second polypeptide is linked to the sequence encoding the second protein of interest by a sequence encoding a linker. In some embodiments, the sequence encoding the third polypeptide is linked to the sequence encoding the third protein of interest by a sequence encoding a linker. In some embodiments, the sequence encoding the fourth polypeptide is linked to the sequence encoding the fourth protein of interest by a sequence encoding a linker.

[0357] In some embodiments, the linker comprises a protease cleavage site or a 2A self-cleaving peptide. In some embodiments, the linker may comprise any self-cleaving peptide known in the art. In some embodiments, the linker may comprise more than one self-cleaving peptide concatenated in series. In some embodiments, the linker comprises at least one self-cleaving peptide. In some embodiments, the linker comprises two different self-cleaving peptides. In some embodiments, the linker comprises two of the same self-cleaving peptides.

[0358] Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional spacer 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.

[0359] Various protease cleavage sites include, but are not limited to, protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and/or the aspartic acid protease family, and/or the glutamic acid protease family. In some embodiments, serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtilisin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some embodiments, the protease cleavage site is TEV protease cleavage site.

[0360] In some embodiments, the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the first polypeptide and further comprises a sequence encoding a first gene of interest, wherein the first polypeptide and the first gene of interest are expressed as a single polypeptide molecule, and wherein the sequence encoding the first polypeptide is linked to the sequence encoding the first gene of interest by a 2A self-cleaving peptide. In some embodiments, the recombinant polynucleic acid comprises a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide and further comprises a sequence encoding a second gene of interest, wherein the second polypeptide and the second gene of interest are expressed as a single polypeptide molecule, and wherein the sequence encoding the second polypeptide is linked to the sequence encoding the second gene of interest by a 2A self-cleaving peptide. In some embodiments, the recombinant polynucleic acid comprises a third recombinant polynucleic acid comprising a sequence encoding the third polypeptide and further comprises a sequence encoding a third gene of interest, wherein the third polypeptide and the third gene of interest are expressed as a single polypeptide molecule, and wherein the sequence encoding the third polypeptide is linked to the sequence encoding the third gene of interest by a 2A self-cleaving peptide. In some embodiments, the recombinant polynucleic acid comprises a fourth recombinant polynucleic acid comprising a sequence encoding the fourth polypeptide and further comprises a sequence encoding a fourth gene of interest, wherein the fourth polypeptide and the fourth gene of interest are expressed as a single polypeptide molecule, and wherein the sequence encoding the fourth polypeptide is linked to the sequence encoding the fourth gene of interest by a 2A self-cleaving peptide.

[0361] In some embodiments, the sequence encoding a first gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as separate polypeptide molecules. In some embodiments, the sequence encoding a second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules. In some embodiments, the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as separate polypeptide molecules. In some embodiments, the sequence encoding a fourth gene of interest encodes for a fourth protein of interest, wherein the fourth polypeptide and the fourth protein of interest are expressed as separate polypeptide molecules.

[0362] In some embodiments, the sequence encoding the first polypeptide and the sequence encoding the first gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the sequence encoding the third polypeptide and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the sequence encoding the fourth polypeptide and the sequence encoding the fourth gene of interest are separated or linked by an IRES or have different promoters.

[0363] In some embodiments, the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the first polypeptide and further comprises a sequence encoding a first gene of interest, wherein the first polypeptide and the first gene of interest are expressed as separate polypeptide molecules, and wherein the sequence encoding the first polypeptide and the sequence encoding the first gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the recombinant polynucleic acid comprises a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide and further comprises a sequence encoding a second gene of interest, wherein the second polypeptide and the second gene of interest are expressed as separate polypeptide molecules, and wherein the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the recombinant polynucleic acid comprises a third recombinant polynucleic acid comprising a sequence encoding the third polypeptide and further comprises a sequence encoding a third gene of interest, wherein the third polypeptide and the third gene of interest are expressed as separate polypeptide molecules, and wherein the sequence encoding the third polypeptide and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters. In some embodiments, the recombinant polynucleic acid comprises a fourth recombinant polynucleic acid comprising a sequence encoding the fourth polypeptide and further comprises a sequence encoding a fourth gene of interest, wherein the fourth polypeptide and the fourth gene of interest are expressed as separate polypeptide molecules, and wherein the sequence encoding the fourth polypeptide and the sequence encoding the fourth gene of interest are separated or linked by an IRES or have different promoters.

Vectors and Expression Cassettes

[0364] In some embodiments, the recombinant nucleic acid is operably linked to a heterologous nucleic acid sequence, such as, for example, a structural gene that encodes a protein of interest or a regulatory sequence (e.g., a promoter sequence). In some embodiments, the recombinant nucleic acid is further defined as an expression cassette or a vector. In some embodiments, the vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.

[0365] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule as disclosed herein. An expression cassette is a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell. As such, the term expression cassette may be used interchangeably with the term “expression construct.”

[0366] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid sequences encoding any of the receptor complex polypeptides and the CARs disclosed herein. The nucleic acid sequences described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al, Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al, “Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989).

Chimeric Antigen Receptors (CARs)

[0367] In some embodiments, the first protein of interest, the second protein of interest, the third protein of interest, and/or the fourth protein of interest each comprise a chimeric antigen receptor (CAR). In some embodiments, the first protein of interest, the second protein of interest, the third protein of interest, and/or the fourth protein of interest each comprise a different CAR. [0368] In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 andHCDR3 ofDYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain comprises an scFv with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NO: 75 or 76. In some embodiments, the anti-CD19 binding domain is an scFv comprising a light chain variable domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1 , LCDR2 and LCDR3 sequences in Tables 12-14; and a heavy chain variable domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 and HCDR3 sequences in Tables 9-11. In some embodiments, the scFv comprises a heavy chain vanable region (VH) with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the VH sequences in Table 8. In some embodiments, the scFv comprises a light chain variable region (VL) with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the VL sequences in Table 8. In some embodiments, the scFv comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the sequences in Table 7.

[0369] In some embodiments, the antigen binding domain is an anti-CD20 binding domain. In some embodiments, the anti-CD20 binding domain is an scFv comprising a light chain variable domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1 , LCDR2 and LCDR3 sequences in Tables 20-22; and a heavy chain variable domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 andHCDR3 sequences in Tables 17-19. In some embodiments, the scFv comprises a heavy chain vanable region (VH) with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the VH sequences in Table 16. In some embodiments, the scFv comprises a light chain variable region (VL) with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the VL sequences in Table 16. In some embodiments, the scFv comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from the group consisting of the VL sequences in Table 15. [0370] In some embodiments, the antigen binding domain is an anti-CD22 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a variable heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. In some embodiments, the antigen binding domain comprises an scFv with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 77.

[0371] In some embodiments, the antigen binding domain is an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. Alternatively, the antigen binding domain can be a receptor or a receptor fragment that binds specifically to the target antigen. The antigen binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners). Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, but are not limited to, 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. Bamhashemi et al., Iran J Basic Med Sci (2018) 21(5):455-64 (CD19 nanobody); F. Rahbarizadeh et al Adv Drug Deliv Rev (2019) 141:41-46 (sdAb);S.M. Kipriyanov et al., Int J Cancer (1998) 77(5):763-72 (diabody); F. Le Gall et al., FEBS Lett (1999) 453(1-2): 164-68 (tnabody); M.A. Ghetie et al., Blood (1994) 83(5): 1329- 36 (F(ab')2); and M.A. Ghetie et al., Clin Cancer Res (1999) 5(12):3920-27 (F(ab')2 and Fab'). Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody. Antibody derivatives can also be designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0372] In some embodiments, the antigen binding domain binds to an antigen that can be glioma- associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, LI CAM, EGFR, mesothehn, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23H, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, R0R1, CD2, or GD2. In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain from CD2. In some embodiments, the intracellular domain of the CAR comprises one or more intracellular signaling domains selected from the group consisting of CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79 , CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70.

[0373] In some domains the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises a hinge domain and a transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. In some embodiments, the transmembrane domain of the CAR consists or consists essentially of a transmembrane domain from CD8 or CD28. In some embodiments, the hinge domain of the CAR comprises a hinge domain from CD8 or CD28. In some embodiments, the CAR comprises a hinge domain and a transmembrane domain. In some embodiments the CAR comprises a CD8 hinge domain and a CD8 transmembrane domain. In some embodiments, the CAR comprises a CD28 hinge domain and a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD8 hinge domain and a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD28 hinge domain and a CD8 transmembrane domain. In some embodiments, the extracellular domain of the CAR consists of a hinge domain from CD8 or CD28. In some embodiments, the hinge domain of a CAR provided herein has a CD28 hinge with the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In some embodiments, the hinge domain of a CAR or provided herein is from CD8. In some embodiments, the hinge domain of a CAR provided herein has a CD8 hinge with the sequence

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY.

[0374] In certain embodiments, a hinge or spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and/or CH3 of IgG 1, lgG4, or IgD. Illustrative spacer domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD28, which may be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain includes a CD8a or CD28 hinge region. In some embodiments, the hinge is a PD-1 hinge or CD152 hinge.

[0375] In some embodiments, the CAR further includes an extracellular spacer domain, which may include a hinge domain. The hinge domain is generally a flexible polypeptide connector region disposed between the targeting moiety and the transmembrane domain. Exemplary hinge domain sequences include those from IgG subclasses (such as IgGl and IgG4), IgD, CD28, and CD8 domains. In some embodiments, the hinge domain provides structural flexibility to flanking polypeptide regions. The hinge domain may consist of natural or synthetic polypeptides. It will be appreciated by those skilled in the art that hinge domains may improve the function of the CAR by promoting optimal positioning of the antigen binding domain in relationship to the portion of the antigen recognized by it. In some embodiments, a hinge domain may not be required for optimal CAR activity. In some embodiments, a hinge domain comprising a short sequence of amino acids promotes CAR activity by facilitating antigen-binding by, for example, relieving steric constraints that could otherwise alter antibody binding kinetics. In some embodiments, the hinge domain is linked downstream of the antigen-binding domain of a CAR and upstream of the transmembrane domain of a CAR.

[0376] Non-limiting examples of suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgGl. In some embodiments, the hinge domain can include regions derived from a human CD8a (also known as CD8a) molecule, a CD28 molecule, and any other receptors that provide a similar function in providing flexibility to flanking regions. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain. In some embodiments, the hinge domain has about at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4 hinge domain.

[0377] In some embodiments, the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and the extracellular hinge domain. In some embodiments, the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain. In principle, there are no particular limitations to the length and/or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another to achieve a desired activity of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another can be varied and/or optimized as a “tuning” tool or effect to enhance or reduce the efficacy of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the hinge domain to one another can be varied and/or optimized to create a partially functional version of the CAR. In some embodiments, the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2- CH3 domain.

[0378] Alternatively, the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGSn”) sequence, or a variation thereof such as (SGG)n, (GGGS)n, (SGGG)n, (GSGGG)n, and the like, where n can range from about 1 to about 15. The synthetic polypeptide spacer domain can also include a naturally occurring sequence, such as a hinge domain derived from CD8a, IgG, and the like.

[0379] 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 transmembrane domain known in the art, including but not limited to, CD3^ transmembrane domain, 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. Examples of the transmembrane domain include the transmembrane regions of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0380] In some embodiments, the CAR comprises a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8a, CD28, CD154, and PD-1; and one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: 4-1BB, CD28, CD 134, and CD 137; and an intracellular signaling domain from a polypeptide selected from the group consisting of: FcyRI, FcRy, FcR, CD3y, CD35, CD3s, CD3zeta, CD35, CD22, CD79a, CD79, and CD665. Such a CAR may further include a spacer domain between the antigen-binding portion and the transmembrane domain, e.g., a CD8a hinge. In some embodiments, the CAR comprises a transmembrane domain from CD28. In some embodiments, the CAR comprises a transmembrane domain having the sequence FWVLWVGGVLACYSLLVTVAFIIFWV. In some embodiments, the CAR comprises a transmembrane domain from CD8. In some embodiments, the CAR comprises a transmembrane domain having the sequence IWAPLAGTCGVLLLSLVITLYC.

[0381] The transmembrane domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the TM domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T cell receptor, CD3y, CD35, CD3s, CD35, CD3zeta, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and/or PD-1.

[0382] The transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3zeta chain), CD28, CD2, CD4, 0X40, 4-1BB (CD137), ICOS (CD278), ILRB (CD 122), IL-2RG (CD 132), CTLA-4, PD-1, or CD40, or a sequence derived from such a transmembrane domain. The cytoplasmic signaling domain in general comprises a domain that transduces the event of ligand binding into an intracellular signal that activates the T cell. The CD3z intracellular domain/activating domain is frequently used, although others such as MyD88 can be used. In an embodiment, the transmembrane domain is the transmembrane domain from CD3eta, CD2, CD8, or CD28. In an embodiment, the transmembrane domain is derived from the transmembrane domain from CD2 or CD28. In some embodiments, the transmembrane domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD3zeta, CD28, CD2, CD4, 0X40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.

[0383] According to some embodiments, a CAR includes a transmembrane domain derived from CD8a or CD28 and a short polypeptide linker, e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine linker may be employed as such a linker, for example.

[0384] The transmembrane domain of the CAR is operably connected to the intracellular domain. The intracellular domain serves to transduce the received external signal to kick-start the downstream signaling cascade. The intracellular domain comprises an intracellular signaling domain. In some embodiments, the intracellular domain comprises one or more intracellular signaling domains selected from the group consisting of CD3^, 4-1BB (CD137) CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79p, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP12, LAT, KD2C, SLP76, TRIM, and ZAP70.

[0385] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4- IBB (CD 137). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain having the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0386] 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 having the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain having the sequence

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0387] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).

[0388] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2 and an intracellular signaling domain from CD3zeta. In some embodiments, the CAR comprises an intracellular signaling domain from CD3zeta. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPAT. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH.

[0389] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3 epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI. In some embodiments, the CAR comprises an intracellular domain comprising a truncated CD3 epsilon intracellular domain.

[0390] Signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of the T cell and a secondary or costimulatory signal may also be required. Thus, T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen- independent manner to provide a secondary or costimulatory signal. As such, the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.

[0391] Primary signaling domains can regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a CAR include those derived from FcyRI, FcRy, FcR, CD3y, CD35, CD3E, CD3zeta, CD35, CD22, CD79a, CD79P, and CD665. In certain embodiments, a CAR includes a CD3zeta primary signaling domain and one or more costimulatory signaling domains. In certain embodiments, a CAR includes a 4-1BB costimulatory signaling domain. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain. In certain embodiments, a CAR lacks a CD2 intracellular signaling domain.

[0392] In some embodiments, the CAR includes one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR. Exemplary costimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP12, LAT, KD2C, SLP76, TRIM, and/or ZAP70. In some embodiments, the costimulatory signaling domain has at least about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a costimulatory signaling domain from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP12, LAT, KD2C, SLP76, TRIM, and/or ZAP70 domain. In some embodiments, a CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD2, 4-1BB/CD137, CD28, and CD134, and a CD3zeta primary signaling domain. A costimulatory molecule can be selected from any one of the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. In certain embodiments, the CAR comprises two or more intracellular signaling domains. For example, the CAR may comprise a first signaling domain and a second signaling domain or fragments thereof independently selected from a CD3zeta intracellular signaling domain, a CD28 intracellular signaling domain, a 4- IBB 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 fusion intracellular signaling domain. In some embodiments, the CAR comprises a first intracellular signaling domain or fragment thereof comprising a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof comprising a CD28 intracellular signaling domain. In some embodiments, the CAR comprises a first intracellular signaling domain or fragment thereof comprising a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof comprising a 4-1BB intracellular signaling domain. In some embodiments, the CAR comprises a first intracellular signaling domain or fragment thereof comprising a CD3zeta intracellular signaling domain, a second intracellular signaling domain or fragment thereof comprising a 4-1BB intracellular signaling domain, and a third intracellular signaling domain or fragment thereof comprising a CD3 epsilon intracellular signaling domain. In some embodiments, the CAR comprises a first intracellular signaling domain or fragment thereof comprising a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof comprising a CD2 intracellular signaling domain.

[0393] CARs of the disclosure may comprise a CD3^, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD790, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP12, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an CD3i;, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD790, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, DAP12, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. CARs of the disclosure may comprise a CD2 co-stimulatory domain, and one or more additional co-stimulatory domains to increase cytokine production or sensitivity, reduce or prevent anergy, and/or to increase proliferation and cytotoxic activity. These additional co-stimulatory domains can be derived from co-stimulatory proteins such as B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 4 IBB (CD 137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integnn a4, CD82, mtegrin a4bl, CD90, mtegrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 4 IBB (CD 137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F- 10, NTB-A, CD48, SLAM(CD150), CD58, ikaros, CD53, mtegrin a4, CD82, mtegrin a4bl, CD90, mtegrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR domains.

[0394] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a CD28 costimulatory signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a CD28 costimulatory signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CDl 9 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments the CAR comprises an anti-CD19 binding domain, a CD8a hinge domain, a CD8a transmembrane domain, and a cytoplasmic domain comprising a costimulatory domain from CD28 and an intracellular signaling domain from CD3zeta.

[0395] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a CD28 intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a CD28 intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments the third CAR comprises an anti-CD22 binding domain, a CD8a hinge domain, a CD8a transmembrane domain, and a cytoplasmic domain comprising a costimulatory domain from 4-1BB and an intracellular signaling domain from CD3zeta.

[0396] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD20 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a CD28 costimulatory signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD20 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a CD28 costimulatory signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD20 binding domain, a CD28 hinge and a CD28 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD20 binding domain, a CD8 hinge and a CD8 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain and a CD3zeta intracellular signaling domain. In some embodiments the CAR comprises an anti-CD20 binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta.

[0397] In some embodiments the first CAR comprises an anti-CD19 scFv, a hinge domain from CD8a, a transmembrane domain from CD8a, and a cytoplasmic domain comprising a costimulatory domain from CD28 and an intracellular signaling domain from CD3zeta. In some embodiments the second CAR comprises an anti-CD20 scFv, a hinge domain from CD28, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta. In some embodiments the third CAR comprises an anti-CD22 scFv, a hinge domain from CD8a, a transmembrane domain from CD8a, a cytoplasmic domain comprising a costimulatory domain from 4-1BB and an intracellular signaling domain from CD3zeta.

[0398] In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. In some embodiments, the CAR comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 78-80. In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. In some embodiments, the CAR consists of a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 78-80. In some embodiments, the CAR comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of sequences in Table 24. In some embodiments, the CAR comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of sequences in Table 25. In some embodiments, the CAR comprises a sequence with at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of sequences in Table 26.

[0399] In some embodiments, the CAR further comprises a protein localization tag operably linked to the intracellular domain of the CAR or to the extracellular domain of the CAR. The protein localization tag comprises a tag selected from the group consisting of an ER localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, and a nuclear localization tag. In some embodiments, the protein localization tag is an ER localization tag. In some embodiments, the ER localization tag comprises an amino acid sequence of any one of SEQ ID NOs: 81-88. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 81-88.

[0400] In some embodiments, the protein localization tag is a Golgi localization tag. In some embodiments, the Golgi localization tag comprises the amino acid sequence YQRL. In some embodiments, the Golgi localization tag consists or consists essentially of the amino acid sequence YQRL. 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. In some embodiments, the lysosome localization tag consists or consists essentially of the amino acid sequence KFERQS.

[0401] In some embodiments, the protein localization tag is an ER localization tag. In some embodiments, the ER localization tag comprises the amino acid sequence LYKYKSRRSFIDEKKMP. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to LYKYKSRRSFIDEKKMP. In some embodiments, the ER localization tag consists or consists essentially of the amino acid sequence LYKYKSRRSFIDEKKMP. In some embodiments, the ER localization tag comprises the amino acid sequence KKMP. In some embodiments, the ER localization tag consists or consists essentially of an amino acid sequence of KKMP.

[0402] In some embodiments, a protease cleavage site is disposed between the protein localization tag and the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the intracellular domain of the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the extracellular domain of the CAR. Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional amino acids can be present at the N- terminus and/or C-terminus of the cleavage site. A protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized/cleaved by the protease.

[0403] Exemplary protease cleavage sites include, but are not limited to, protease cleavage sites for proteases from the serine protease family, for metalloproteases, for proteases from the cysteine protease family, the aspartic acid protease family, and/or the glutamic acid protease family. In some embodiments, serine protease cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtilisin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some embodiments, the protease cleavage site is a TEV protease cleavage site. In some embodiments, the TEV protease cleavage site comprises the amino acid sequence ENLYFQS. In some embodiments, the TEV protease cleavage site comprises the amino acid sequence GGGGSGGGENLYFQS.

[0404] The sequences encoding the receptor complex polypeptides disclosed herein and the sequences encoding the CARs disclosed herein can be single polynucleotide sequences. In some embodiments, the single polynucleotide sequences comprise a self-cleaving site (e.g., 2A peptides) separating the sequence encoding the receptor complex polypeptide and the sequence encoding the CAR. In some embodiments, the sequence encoding the receptor complex polypeptide and the sequence encoding the CAR are separate sequences. The sequence encoding the receptor complex polypeptide or the sequence encoding the CAR can be inserted to a vector for expression in a cell. In some embodiments, the sequence encoding the receptor complex polypeptide and the sequence encoding the CAR are inserted into the same vector. In some embodiments, the sequence encoding the receptor complex polypeptide and the sequence encoding the CAR are inserted into different vectors. In some embodiments, the vector comprising the sequence encoding the receptor complex polypeptide is different from the vector comprising the sequence encoding the CAR. In some embodiments, the vector comprising the sequence encoding the receptor complex polypeptide and the vector comprising the sequence encoding the CAR are the same vector.

Delivery Vectors

[0405] In some embodiments, the receptor complex polypeptides and the CARs of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors, lentiviral or other retroviral vectors). Expression vectors are designed to express coding sequences they are delivering to the host cell(s) of interest. Frequently, expression vectors used in recombinant DNA techniques are in the form of plasmids. However, other types of expression vectors are also used, including, as anon-limiting example, viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) are also included.

[0406] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1 89) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0407] Vectors suitable for use include the pMSXND expression vector for use in mammalian cells. In some embodiments, nucleic acid inserts encoding the receptor complex polypeptides and/or the CARs in such vectors, are operably linked to a promoter. In some embodiments, the promoter is selected based on the host cell type in which the nucleic acid insert of interest will be expressed. Viral vectors that may be used in any of the various embodiments disclosed and described herein include, but are not limited to, for example, retroviral, lentiviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0408] In some embodiments, the expression vector is a viral vector. The term “viral vector” is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s). Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus).

[0409] Viral vectors that can be used to express any of the various polynucleic acids disclosed and described in this specification include, but are not limited to, for example, retroviral vectors (including lentiviral vectors), adenoviral vectors, and adeno-associated virus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0410] In some embodiments, the nucleic acid molecules are delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for stable or transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Although stable integration can be accomplished using integrating vectors, such as lentiviral vectors and retroviral vectors, stable integration can also be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA-directed CRISPR/Cas9, DNA- guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for stable or transient expression. [0411] Lentiviral systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host cell genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy -target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile (e.g. , by targeting a site for integration that has little or no oncogenic potential); and (vii) a relatively easy system for vector manipulation and production.

[0412] In some embodiments, the sequence encoding the CAR and the sequence encoding the first, second, third, and/or fourth polypeptides is delivered in a single vector. In some embodiments, the single vector is a lentiviral vector. In some embodiments, the sequence encoding the CAR and the sequence encoding the first, second, third, and/or fourth polypeptides is delivered in two or more vectors. In some embodiments, both vectors are lentiviral vectors. In some embodiments, each of the first, second, third, and/or fourth polypeptides further comprises a CAR, and each of the first, second, third, and/or fourth polypeptides further comprising a CAR are delivered to a cell in a separate vector. In some embodiments, each of the separate vectors is a lentiviral vector.

Cells and Methods of Transforming Cells

[0413] Also provided herein are cells comprising the receptor complex polypeptides disclosed herein, cells comprising the recombinant polynucleic acids disclosed herein, cells comprising the compositions comprising the receptor complex polypeptides disclosed herein, and cells comprising the compositions comprising the recombinant polynucleic acids disclosed herein. In some embodiments, the cells express the receptor complex polypeptides disclosed herein. In some embodiments, the receptor complex polypeptide(s) is(are) expressed on the surface of the cell. By “expressed on the surface of the cell” or “cell surface expression” is meant the receptor complex polypeptide - when no longer associated with the protein localization tag (e.g. , ER localization tag, Golgi localization tag, or the like) has been trafficked to the cell membrane such that - 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, so that the intracellular signaling domain transduces a signal upon binding of the extracellular binding domain to the target ligand/antigen, thereby activating the cell.

[0414] In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, or a suppressor T cell. In some embodiments, the T cell is a CD8+ cell. In some embodiments, the T cell is a CD4+ cell. In some embodiments, the T cell is a CD8+CD4+ cell. In some embodiments, the T cell is a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, or a naive T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a B cell. In some embodiments, the B cell is a naive mature B cell, a plasmablast, a plasma cell, or a memory B cell. In some embodiments, the B cell is a CD 19+ cell.

[0415] In some embodiments, a cell expresses a first polypeptide and a second polypeptide of a receptor complex. In some embodiments, a cell expresses a first polypeptide, a second polypeptide and a third polypeptide of a receptor complex. In some embodiments, a cell expresses a first, second, third, and fourth polypeptides of a receptor complex.

[0416] Further disclosed herein includes a composition comprising a cell comprising a recombinant polynucleic acid comprising at least two sequences that each encode a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides may each comprise an extracellular domain that comprises a selection tag, and a transmembrane domain, wherein the first receptor and the second receptor are different receptors. In some embodiments, the composition comprising a cell comprising a recombinant polynucleic acid comprises at least three sequences each encoding a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides may each comprise an extracellular domain that comprises a selection tag, and a transmembrane domain, wherein the first receptor and the second receptor are different receptors. In some embodiments, the composition comprises a cell comprising a recombinant polynucleic acid comprising at least three sequences each encoding a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides may each comprise an extracellular domain that comprises a selection tag, and a transmembrane domain, wherein the first receptor, the second receptor, and the third receptor are different receptors.

[0417] In some embodiments, the composition comprises a cell comprising a recombinant polynucleic acid comprising at least two sequences each encoding a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides each comprise an extracellular domain that comprises a selection tag, a transmembrane domain, and an intracellular signaling domain, wherein the first receptor and the second receptor are different receptors. In some embodiments, the composition comprises a cell comprising a recombinant polynucleic acid comprising at least three sequences that each encode a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides each comprise an extracellular domain that comprises a selection tag, a transmembrane domain, and an intracellular domain, wherein the first receptor and the second receptor are different receptors. In some embodiments, the composition comprises a cell comprising a recombinant polynucleic acid comprising at least three sequences that each encode a receptor complex polypeptide, wherein the cell expresses the receptor complex polypeptides, wherein the receptor complex polypeptides may each comprise an extracellular domain that comprises a selection tag, a transmembrane domain, and an intracellular domain, wherein the first receptor, the second receptor, and the third receptor are different receptors.

[0418] In some embodiments, host cells can be genetically engineered (e.g. , transduced, transformed, or transfected) with, for example, a vector construct of the present disclosure that can 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 can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. In some embodiments, the host cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell. In some embodiments, the genetically engineered host cells are autologous or allogeneic.

[0419] Host cells can be transduced with any of the polynucleic acids described herein, such as those encoding one or more receptor complex polypeptides and/or a CAR, with or without the protein localization tag. In some embodiments, a host cell can be transduced with a nucleic acid encoding a receptor complex polypeptide. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a receptor complex polypeptide and a CAR. In some embodiments, a host cell can be transduced with a nucleic acid encoding a receptor complex polypeptide and an additional nucleic acid encoding a CAR. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a receptor complex polypeptide with a protein localization tag and a CAR. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a receptor complex polypeptide and a CAR with a protein localization tag. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a receptor complex polypeptide with a protein localization tag and a CAR with another protein localization tag. In some embodiments, the host 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.

[0420] In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, mammalian cell is a mouse, rat, rabbit, human, canine, feline, bovine, pig or primate cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer (NK) cell, natural killer T (NKT) cell, a B cell, a plasma cell, a tumorinfiltrating lymphocyte (TIL)), a monocyte, a macrophage, or a dendritic cell, or a precursor cell, i. e. , a cell that is capable of differentiating into an immune cell. In some embodiments, the immune system cell is selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells. In some embodiments, the immune system cell is a T lymphocyte. In some embodiments, the immune system cell is a B cell. In some embodiments, the T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, or a suppressor T cell. In some embodiments, the T cell is a CD8+ cell. In some embodiments, the T cell is a CD4+ cell. In some embodiments, the T cell is a CD8+CD4+ cell. In some embodiments, the T cell is a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, or a naive T cell. In some embodiments, the B cell is a naive mature B cell, a plasmablast, a plasma cell, or a memory B cell. In some embodiments, the B cell is a CD19+ cell.

[0421] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. In some embodiments, the nucleic acid molecule is introduced into a host cell by transduction, electroporation, or biolistics. Accordingly, cell cultures including at 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. [0422] Transformed cells of the present disclosure may be autologous/autogeneic (“self’) or non- autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells derived from the same individual to which they are subsequently administered. “Allogeneic” as used herein refers to cells of the same species that differ genetically from the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison. [0423] T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g. , FICOLL™ separation.

[0424] In some embodiments, an isolated or purified population of T cells is used. In some embodiments, CD8+ T cells (“cytotoxic T cells” or “TCTL”) and CD4+ T cells (“helper T cells” or TH”) lymphocytes are purified from PBMCs. In some embodiments, the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification. Suitable approaches for such sorting are known and include, but are not limited to, for example, magnetic- activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L- CD95+. An exemplaiy approach for such sorting is described in Wang et al. (2016) Blood 127(24):2980- 90.

[0425] A specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, is further isolated by positive or negative selection techniques. In some embodiments, the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.

[0426] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primate cell. In some embodiments, the primate cell is a human cell. In some embodiments, the human cell is a blood cell. In some embodiments, the blood cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells is a population of blood cells. In some embodiments the population of blood cells is a population of lymphocytes. In some embodiments, the population of lymphocytes is a population of T cells. In some embodiments, the population of T cells is a population of cytotoxic T cells, effector T cells, helper T cells, natural killer T cells, or suppressor T cells. In some embodiments, the population of T cells is a population of CD8+ cells. In some embodiments, the population of T cells is a population of CD4+ cells. In some embodiments, the population of T cells is a population of CD8+CD4+ cells. In some embodiments, the population of T cells is a population of regulatory T cells, memory T cells, stem cell memory T cells, central memory T cells, effector memory T cells, stem cell memory T cells, central memory T cells, effector memory T cells, or a naive 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 I xlO⁵ cells. In some embodiments, the population of cells comprises at least about I xlO⁶ cells. In some embodiments, the population of cells comprises at least about I xlO⁷ cells. In some embodiments, the population of cells comprises at least about I xlO⁸ cells. In some embodiments, the population of cells comprises at least about IxlO⁹ cells. In some embodiments, the population of cells comprises from about I xlO⁵ cells to about I x lO⁹ cells. In some embodiments, the population of cells comprises from about I xlO⁵ cells to about IxlO⁸ cells. In some embodiments, the population of cells comprises from about IxlO⁵ cells to about I xlO⁷ cells. In some embodiments, the population of cells comprises from about I x lO⁵ cells to about I xlO⁶ cells. In some embodiments, at least 50% or more of the cells in the population of cells express the first protein of interest and the second protein of interest. In some embodiments, at least 50% or more of the cells in the population of cells express the first protein of interest, the second protein of interest, and the third protein of interest. In some embodiments, at least 50% or more of the cells in the population of cells express at least two CARs. In some embodiments, at least 50% or more of the cells in the population of cells express at least three CARs.

[0427] In some embodiments, using the methods described herein a higher percentage of the cells can be obtained that express both a first protein of interest (e.g. , a first CAR) encoded by a first vector, and a second protein of interest (e.g, a second CAR) encoded by a second vector compared to conventional methods known in the art. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g, a first CAR) and a sequence encoding a transmembrane domain of TREM1 polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a transmembrane domain and an extracellular domain of DAP12 polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a transmembrane domain of TREM1 polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a transmembrane domain and an extracellular domain of DAP12 polypeptide can produce a population of cells in which a higher percentage of the cells in the population express both the first protein of interest e.g. , a first CAR) and the second protein of interest e.g. , a second CAR) compared to the percentage of the cells that express both the first protein of interest e.g., a first CAR) and the second protein of interest e.g., a second CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g., a first CAR) but without the sequence encoding the TREM1 transmembrane domain polypeptide, and a second vector encoding the second protein of interest (e.g., a second CAR) but without the sequence encoding the DAP12 polypeptide. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a TREM1 transmembrane domain polypeptide, and a second vector having a sequence encoding a second protein of interest e.g., a second CAR) and a sequence encoding a transmembrane and extracellular domains of DAP12 can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest e.g., a first CAR) and the second protein of interest e.g., a second CAR) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold higher than the percentage of the cells that express both the first protein of interest e.g., a first CAR) and the second protein of interest e.g., a second CAR) when the cell population is transduced with a first vector encoding the first protein of interest e.g. , a first CAR) but without the sequence encoding the transmembrane domain of TREM1 polypeptide, and a second vector encoding the second protein of interest (e.g., a second CAR) but without the sequence encoding the transmembrane and extracellular domain of DAP12 polypeptide.

[0428] In some embodiments, using the methods described herein a higher percentage of the cells can be obtained that express each of a first protein of interest e.g., a first CAR) encoded by a first vector, and a second protein of interest (e.g., a second CAR) and a third protein of interest (e.g., a third CAR) encoded by the second vector compared to conventional methods known in the art. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a transmembrane domain of TREM1 polypeptide, and a second vector having a sequence encoding a second protein of interest e.g. , a second CAR), a sequence encoding a third protein of interest (e.g., a third CAR) and a sequence encoding a transmembrane and extracellular domain of DAP12 polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest e.g., a first CAR), the second protein of interest e.g. , a second CAR) and the third protein of interest e.g. , a third CAR) is atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a transmembrane domain of TREM1 polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR), a sequence encoding a third protein of interest (e.g. , a third CAR) and a sequence encoding a transmembrane and an extracellular domain of DAP12 can produce a population of cells in which the percentage of the cells in the population that express each of the first protein of interest (e.g, a first CAR), the second protein of interest (e.g, a second CAR) and the third protein of interest (e.g., a third CAR) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold higher than the percentage of the cells that express each of the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g. , a first CAR) but without the sequence encoding the transmembrane domain of TREM1 , and a second vector encoding the second protein of interest (e.g. , a second CAR) and the third protein of interest (e.g., a third CAR) but without the sequence encoding the transmembrane and extracellular domain of DAP 12.

[0429] In some embodiments, using the methods described herein a higher percentage of the cells can be obtained that express each of a first protein of interest (e.g., a first CAR) encoded by a first vector, and a second protein of interest (e.g., a second CAR) encoded by a second vector, and a third protein of interest (e.g., a third CAR) encoded by the third vector compared to conventional methods known in the art. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding an IgM polypeptide, a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a CD79a polypeptide, and a third vector encoding a sequence encoding a third protein of interest (e.g., a third CAR) and a sequence encoding a CD79|3 polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding an IgM polypeptide, a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a CD79a polypeptide, and a third vector encoding a sequence encoding a third protein of interest (e.g. , a third CAR) and a sequence encoding a CD79 polypeptide can produce a population of cells in which the percentage of the cells in the population that express each of the first protein of interest (e.g., a first CAR), the second protein of interest e.g. , a second CAR) and the third protein of interest (e.g. , a third CAR) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold higher than the percentage of the cells that express each of the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g., a first CAR) but without the sequence encoding the IgM polypeptide, and a second vector encoding the second protein of interest (e.g. , a second CAR) but without the sequence encoding the CD79a polypeptide, and a third vector encoding the third protein of interest (e.g., a third CAR) but without the sequence encoding the CD79P polypeptide.

Pharmaceutical compositions

[0430] In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the receptor complex polypeptides disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the cells disclosed herein, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of the cells disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a pharmaceutical composition of the present disclosure includes from 1 * 10⁵ to 5 * 10¹⁰ of the cells of the present disclosure.

[0431] 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. 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.

[0432] Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0433] An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g. , on the buffer and the desired tonicity of the formulation.

[0434] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g. , in an amount of 100 mM to 350 mM.

[0435] 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, but are not limited to, poly oxy ethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- poly oxy propylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan- fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.

[0436] In some embodiments, the pharmaceutical composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).

[0437] In some embodiments, the pharmaceutical composition comprises an engineered cell comprising any of the first, second, third, and/or fourth polypeptides disclosed and described in detail herein. In some embodiments, the engineered cell is an engineered T cell. In some embodiments, the engineered T cell is an engineered cytotoxic T cell, an engineered effector T cell, an engineered helper T cell, an engineered natural killer T cell, or an engineered suppressor T cell. In some embodiments, the engineered T cell is an engineered CD8+ cell. In some embodiments, the engineered T cell is an engineered CD4+ cell. In some embodiments, the engineered T cell is an engineered CD8+CD4+ cell. In some embodiments, the engineered T cell is an engineered regulatory T cell, an engineered memory T cell, an engineered stem cell memory T cell, an engineered central memory T cell, an engineered effector memory T cell, an engineered stem cell memory T cell, an engineered central memory T cell, an engineered effector memory T cell, or an engineered naive T cell. In some embodiments, the engineered cell is an engineered B cell. In some embodiments, the engineered B cell is an engineered naive mature B cell, an engineered plasmablast, an engineered plasma cell, or an engineered memory B cell. In some embodiments, the engineered B cell is an engineered CD 19+ cell.. In some embodiments, the pharmaceutical composition comprises a population of cells. In some embodiments, the pharmaceutical composition comprises a population of engineered cells. In some embodiments, a pharmaceutical composition comprises a population of engineered cells comprising at least l *10^A5 cells. In some embodiments, a pharmaceutical composition comprises a population of engineered T cells comprising at least l * 10^A5 cells. In some embodiments, a pharmaceutical composition comprises a population of engineered B cells comprising at least l *10^A5 cells.

Methods of Treatment

[0438] Further disclosed here include methods for treating a disease, disorder, or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. The pharmaceutical composition can be administered alone or in combination with other agents (e.g. , an antibody or an antigen binding fragment thereof, or a molecule). In some embodiments, the other agent comprises a vaccine, an oncolytic virus, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and/or a bispecific antibody. In some embodiments, the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells). Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anti-cancer agent” are used interchangeably herein. Various classes of anticancer agents can be used. Non-limiting examples include, but are not limited to, 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.

[0439] In some embodiments, a method of treating a disease, disorder, or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein and administering a therapeutically effective amount of T cells comprising the recombinant polynucleic acids described herein.

[0440] In some embodiments, a therapeutically effective amount of the pharmaceutical composition is administered as a method of treating cancer in a subject in need. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, 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, but is not limited to, a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). [0441] 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.

[0442] 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 infdtrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer; melanoma; multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated/small cell/atypical lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and mycosis fungoides/Sezary Syndrome; non-melanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intra-abdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hurthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and/or uterine sarcoma.

[0443] Methods for administering immune cells for therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described in US 2003/0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli et al., Nat Biotechnol (2013) 31 (10):928-33 ; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(1): 84-89.

Cell Selection

[0444] Further disclosed herein include methods for selecting cells expressing receptor complexes and genes of interest.

[0445] In some embodiments, a method of multiplexed cell selection comprises (a) introducing the recombinant polynucleic acid into a population of cells; (b) culturing the cell, the engineered cell, the engineered T cell, or the engineered B cell, and (c) selecting the cells based on cell surface expression of the selection tag, thereby obtaining a population of selected cells. In some embodiments, selecting comprises purifying or isolating cells. In some embodiments, multiplexed cell selection comprises selecting cells with the same selection tag. In some embodiments, multiplexed cell selection comprises selecting cells with two or more different selection tags. In some embodiments, multiplexed cell selection comprises selecting a mixed population of cells with two or more different selection tags. In some embodiments, multiplexed cell selection comprises selecting a mixed population of cells with the same selection tags.

[0446] In some embodiments, the selection tag is a fluorescent tag and selecting comprises flow cytometry. In some embodiments, the selection tag is a purification tag and selecting comprises contacting the cells with an agent comprising a moiety that binds to the selection tag. In some embodiments, the agent further comprises, for example, a bead or solid surface that is attached to the moiety that binds to the selection tag. In some embodiments, the agent comprises further comprises a magnetic bead that is attached to the moiety that binds to the selection tag. In some embodiments, the moiety that binds to the selection tag comprises an antibody. [0447] In some embodiments, selecting comprises contacting a sample containing a heterogeneous population of cells, comprising cells comprising recombinant nucleic acids encoding receptor complex polypeptides, with magnetic beads comprising a binding agent or moiety that binds to the selection tag expressed on one or more receptor complex polypeptides; allowing the mixture of beads and cells to stand at a temperature appropriate for preservation of cell function; removing other substances in the mixture, and leaving only the magnetic beads and captured material, which may comprise, for example, one or more receptor complex polypeptides.

[0448] In still other embodiments, the binding agent can comprise, for example, antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins, and fragments of the same. In more particular embodiments, the antibody is a F(ab')2 fragment and is even more particularly an F(ab')2 fragment that specifically binds to the Fc portion of an antibody. Binding agents can be disposed on a derivatized solid support through methods well known in the art. Solid supports include, but are not limited to, beads, magnetic beads, microarray chips, nitrocellulose membranes, nylon membranes, multiwell plates, and PVDF membranes. In some embodiments, the solid support is a plate in which an electrode is disposed beneath the plate, which creates a magnetic field that attracts a binding agent bound to a magnetic material. In some embodiments, the solid support comprises cellulose, Sepharose, polyacrylamide, glass, polystyrene, magnetic metals such as iron, or any inert support that does not affect the binding agents’ ability to bind a protein.

[0449] In some embodiments, a binding agent may be attached to a resin. In certain embodiments, the binding agent can comprise, for example, antibodies, ligands, receptors, fusion proteins, subunits of proteins, recombinant proteins, and fragments of the same. In some embodiments, the protein is eluted from the reagent using a vacuum. In other embodiments, the protein is eluted by centrifugation. In still other embodiments, the protein is eluted by gravity flow through the resin. In many embodiments, the screening of the incubated cell lines utilizes an automated workstation.

[0450] In some embodiments, multiplexed cell selection comprises introducing the recombinant nucleic acid into the cell prior to culturing the cell, the engineered cell, the engineered T cell, or the engineered B cell.

[0451] In some embodiments, at least 50% of the cells in the population of selected express the first polypeptide and the second polypeptide. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first polypeptide and the second polypeptide. In some embodiments, at least 50% of the cells in the population of selected cells express the first protein of interest and the second protein of interest. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first protein of interest and the second protein of interest. [0452] In some embodiments, at least 50% of the cells in the population of selected express the first polypeptide, the second polypeptide, and the third polypeptide. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first polypeptide, the second polypeptide, and the third polypeptide. In some embodiments, at least 50% of the cells in the population of selected cells express the first protein of interest, the second protein of interest, and the third protein of interest. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first protein of interest, the second protein of interest, and the third protein of interest.

[0453] In some embodiments, at least 50% of the cells in the population of selected express the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide. In some embodiments, at least 50% of the cells in the population of selected cells express the first protein of interest, the second protein of interest, the third protein of interest, and the fourth protein of interest. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of selected cells express the first protein of interest, the second protein of interest, the third protein of interest, and the fourth protein of interest.

[0454] In some embodiments, the population of selected cells is a population of chimeric antigen receptor (CAR)-expressing immune effector cells.

[0455] Immune effector cells can be obtained from a number of sources, including, but are not limited to, 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. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g. , by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells. [0456] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

[0457] In some embodiments, at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least two different CARs. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least two different CARs.

[0458] In some embodiments, at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least three different CARs. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least three different CARs.

[0459] In some embodiments, at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least four different CARs. In some embodiments, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least four different CARs.

EXAMPLES

[0460] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited herein.

[0461] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

Example 1 - Exemplary receptor polypeptide constructs and domains

[0462] Exemplary receptor polypeptides and domains and are listed in Table 1.

Table 1: Exemplary Receptor Polypeptide Constructs and Domains

Table 2: Exemplary Purification Tag Constructs

Example 3 - Antigen Binding Domains for CARs

[0464] Exemplary antigen binding domains are listed in Table 3.

Table 3: Exemplary Antigen Binding Domains for CARs

Example 4 -CARs

[0465] Exemplary CAR constructs are listed in Table 4. Table 4: Exemplary CAR Constructs

Example 5 - Exemplary constructs comprising a CD34 truncated tag with a TREM1

transmembrane domain or an IgM domain

[0466] Exemplary constructs comprising a CD34t tag with a TREM1 transmembrane domain or an IgM domain are listed in Table 5. Table 5: Exemplary constructs comprising a CD34t tag with a TREM1 transmembrane domain or an IgM domain

Example 6 - Exemplary constructs expressing anti-CD19, anti-CD20, or anti-CD22 CARs with various transmembrane domains

[0467] Exemplary constructs expressing anti-CD19, anti-CD20, or anti-CD22 CAR with transmembrane domains from TREM1, DAP12, IgM, CD79a, or CD79 are listed in Table 6.

Table 6: Exemplary constructs expressing anti-CD19, anti-CD20, or anti-CD22 CAR with TREM1 or DAP12

Example 7 - Co-expressing EGFRt/DAP12 increases EGFRt on cell surface

[0468] A vector expressing the TREM1 transmembrane domain and the truncated EGFRt tag (EGFRt/TREMl) and a vector expressing the DAP12 extracellular domain and transmembrane domain (DAP12EC+TM) were designed. Cells were transduced with the EGFRt/TREMl vector only or both the EGFRt/TREMl vector and the DAP12EC+TM vector. Transduced cells were cultured and expanded for 6 days. Cells were collected, and the percentage of cells expressing the EGFRt tag on the surface were assessed by flow cytometry. As shown in FIG. IB, only 35.4% of the cell population transduced with the EGFRt/TREMl vector alone expressed the EGFRt tag on the surface. On the contrary, 94.5% of the cell population expressed the EGFRt tag on the surface when transduced with both the EGFRt/TREMl vector and the DAP12EC+TM vector (FIG. 1C). This result suggests that transduction of a population of T cells with a first vector having a sequence encoding the EGFRt tag and a sequence encoding a TREM1 transmembrane polypeptide, and a second vector having a sequence encoding a DAP 12 transmembrane domain and an extracellular domain polypeptide produced a population of T cells in which a higher percentage of the T cells in the population expressed the EGFRt tag on cell surface compared to the percentage of the T cells that expressed EGFRt when the T cell population was transduced with just the first vector encoding the EGFRt but without the second vector encoding DAP 12 TM+EC domain.

[0469] Example 8 - Purification of cells co-expressing EGFRt/DAP12

[0470] A vector expressing EGFRt-TREMl and NeonGreen (Vector 1) and a vector expressing DAP 12 and blue fluorescent protein (BFP) (Vector 2) were designed (FIG.2A). Cells were transduced with Vector 1 and Vector 2. Transduced cells were cultured and expanded for 6 days. Cells were collected and purified by MACS beads. The percentage of cells expressing BFP, NeonGreen and the EGFRt tag on the surface was assessed by flow cytometry. As shown in FIG. 2B, only 59.3% of the cells expressed both BFP and NeonGreen prior to purification (meaning they expressed both constructs on the cell surface), and 33.3% of the cells expressed both BFP and NeonGreen/EGFRt (meaning they also expressed both constructs) in the flow-through portion that were not captured by MACS beads. On the contrary, 96.8% of the cells captured by the MACS beads expressed both BFP and NeonGreen (FIG. 2B, right panel). As shown in FIG. 2C, 60% of the cells expressing both vectors were recovered in the eluted samples after MACS purification using a biotinylated anti-EGFR antibody followed by anti-biotin MACS beads.

[0471] Example 9 - Co-expressing IgM/CD79«/CD79B increases IgM/CD79a/CD79B complex on cell surface

[0472] Vectors expressing IgM (Vector IgM), CD79a (Vector CD79a), or CD790 (Vector CD79 ) were designed. Cells were transduced with Vector IgM only, with Vector CD79a and Vector CD790, or with all three vectors. Transduced cells were cultured, expanded for 6 days, and collected. Cell surface expression of IgM, CD79a, and CD790 were measured by flow cytometry. As shown in FIG. 3B (upper panel), while only 2.3% of the cells transduced with Vector IgM only expressed IgM on cell surface, 65.9% of the cells transduced with all three vectors expressed IgM on cell surface. FIG. 3B (bottom panel) shows that while only 0.9% of the cells transduced with Vector CD79a and Vector CD790 expressed both CD79a and CD790 on cell surface, 59.8% of the cells transduced with all three vectors expressed both CD79a and CD790. This result suggests that co-expressing IgM, CD79a, and CD790 in the cells increases localization of the IgM/CD79a/CD790 complex to cell surface.

[0473] Example 10 - Purification of cells co-expressing IgM/CD79o/CD79B

[0474] Vectors expressing an EGFRt tag and IgM (Vector EGFRt/IgM), an EGFRt tag and CD79a (V ector EGFRt/CD79a), or an EGFRt tag and CD790 (Vector EGFRt/CD790) were designed. Cells were transduced with Vector EGFRt/IgM, Vector EGFRt/CD79a, or Vector EGFRt/CD790. Transduced cells were cultured and expanded for 6 days. Cells were collected and purified by MACS beads. Cell-surface expression of the EGFRt tag was measured by flow cytometry. As shown in FIG. 4A, after purification, 61.3% of cells transduced with Vector EGFRt/CD79a expressed the EGFRt tag, 85.9% of cells transduced with Vector EGFRt/CD79p expressed the EGFRt tag, and 76.9% of cells transduced with Vector EGFRt/IgM expressed the EGFRt tag.

[0475] FIG. 4B shows the enrichment of EGFRt positive cells that was achieved for each EGFRt configuration during the MACS purification step. The result suggests that EGFRt fused to CD79 gives the best purity while EGFRt fused to CD79a gives the lowest purity. FIG. 4C shows the yield of EGFRt positive cells and suggests that EGFRt fused to CD79a gives the highest yield while EGFRt fused to membrane IgM gives the lowest yield.

[0476] Example 11 - Selection of trispecific CAR-T cells using TREM1/DAP12 two-vector system

[0477] Vectors expressing TREM1 fused to a EGFRt or CD34t tag, a CD19 CAR, and a CD20CAR (Vector 2878: SEQ ID NO: 94; Vector 2879: SEQ ID NO: 95; Vector 2880: SEQ ID NO: 96; Vector 3050: SEQ ID NO: 99; Vector 3051: SEQ ID NO: 100; Vector 3057: SEQ ID NO: 102; Vector 3137: SEQ ID NO: 103; Vector 3138: SEQ ID NO: 104; Vector 3139: SEQ ID NO: 105; Vector 3140: SEQ ID NO: 106) and vectors expressing DAP12 and a CD22 CAR (Vector 2883: SEQ ID NO: 97; Vector 2884: SEQ ID NO: 98) were designed (FIG. 6A). See Table 6 for construct details. Cells were transduced with Vector 2883 and one of Vector 2878, Vector 2879, and Vector 2880. Transduced cells were cultured, expanded for 6 days, collected, and purified by MACS beads. Cell-surface expression of CD22 CAR and the EGFRt or CD34t tag was measured by flow cytometry. After MACS beads purification, more than 75% of cells expressed both the CD22 CAR and the tag on the cell surface (FIG. 6B) and more than 80% of cells expressed both the CD22 CAR and the CD 19 CAR on the cell surface (FIG. 6C).

[0478] Cells were transduced with Vector 2883 and one of Vector 3050, Vector 3051, Vector 3056 and Vector 3057. Transduced cells were cultured, expanded for 6 days, collected, and purified by MACS beads using the CD34t tag. Cell-surface expression of CD22 CAR and the CD34t tag was measured by flow cytometry. As shown in FIG. 7A (bottom panel), more than 80% of the cells in the population post MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface. On the contrary, less than 65% of the cells in the population prior to MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 7A, top panel), and less than 35% of the cells in the flow-through (FT) not captured by the MACS beads expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 7A, middle panel).

[0479] Cells were transduced with Vector 2884 and one of Vector 3050, Vector 3051, Vector 3056 and Vector 3057. Transduced cells were cultured, expanded for 6 days, collected, and purified by MACS beads using the CD34t tag. Cell-surface expression of CD22 CAR and the CD34t tag was measured by flow cytometry. As shown in FIG. 7B (bottom panel), more than 80% of the cells in the population post MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface. On the contrary, less than 65% of the cells in the population prior to MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 7B, top panel), and less than 35% of the cells in the flow-through (FT) not captured by the MACS beads expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 7B, middle panel).

[0480] Cells were transduced with Vector 2884 and one of Vector 3137, Vector 3138, Vector 3139, Vector 3140, Vector 2879 and Vector 2880 containing structural variations to the CD34t tag. Transduced cells were cultured, expanded for 6 days, collected, and purified by MACS beads using the CD34t tag. Cellsurface expression of CD22 CAR and the CD34t tag was measured by flow cytometry. As shown in FIGS. 8A & 8B (bottom panel), more than 70% of the cells in the population post MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface. On the contrary, less than 55% of the cells in the population prior to MACS purification expressed both the CD34t tag and the CD22 CAR on cell surface (FIGS. 8A & 8B, top panel), and less than 25% of the cells in the flow-through (FT) not captured by the MACS beads expressed both the CD34t tag and the CD22 CAR on cell surface (FIGS. 8A & 8B, middle panel).

[0481] A vector expressing TREM1 with a CD34t tag and a CD20 CAR (Vector 3748: SEQ ID NO: 110) and a vector expressing DAP12, a CD19 CAR, and a CD22 CAR (Vector 3750: SEQ ID NO: 111) were designed. Cells were transduced with both vectors. Transduced cells were cultured, expanded for 10 days, collected, and purified by MACS beads using the CD34t tag. Cell-surface expression of CD22 CAR and the CD34t tag was measured by flow cytometry. As shown in FIG. 9A, only 32.2% of cells in the population prior to purification expressed both the CD34t tag and the CD22 CAR. On the contrary, 86.5% of cells in the population after purification expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 9B), and 62.2% of cells in the population after purification expressed both the CD19 CAR and the CD22 CAR on cell surface (FIG. 9C).

[0482] Overall, the results demonstrate that using the methods described herein a purer T cell population expressing multiple CARs can be obtained compared to conventional methods. For example, using the methods described herein a higher percentage of the T cells that express both a first CAR and a second CAR encoded by a first vector (CD19 CAR and CD22 CAR), and a third CAR encoded by a second vector (CD22 CAR) can be obtained compared to conventional methods.

[0483] Example 12 - Selection of trispecific CAR-T using IgM/CD79a/CD79B three-vector system [0484] A vector expressing tCD79a and a CD22 CAR (Vector 3588: SEQ ID NO: 107), a vector expressing tCD79[3 and a CD19 CAR (Vector 3589: SEQ ID NO: 108), and a vector expressing mlgM with a CD34t tag and a CD20 CAR (Vector 3590: SEQ ID NO: 109) were designed (FIG. 10A). Cells were transduced with all three vectors. Transduced cells were cultured, expanded for 10 days, collected, and purified by MACS beads using the CD34t tag. Cell-surface expression of the CD22 CAR, the CD34t tag, the CD 19 CAR, or the CD20 CAR was measured by flow cytometry. As shown in FIG. 10B, only 16.3% of cells in the population prior to purification expressed both the CD34t tag and the CD22 CAR. After purification, 95.7% of cells expressed both the CD34t tag and the CD22 CAR on cell surface (FIG. 10C), 90.3% of cells in the population expressed both the CD19 CAR and the CD22 CAR on cell surface (FIG. 10D), and 96.2% of cells in the population expressed both the CD20 CAR and the CD22 CAR on cell surface (FIG. 10E). The results suggest that tri-specific CAR T cells can be produced using the three- vector mIgM/CD79a/CD79p system, and using the methods of the MACS beads purification yields a higher percentage of cells expressing all three CARs.

[0485] Example 13 - Exemplary anti-CD19 scFv constructs

[0486] Exemplary full length anti-CD19 scFv constructs are listed in Table 7.

Table 7: Exemplary full length anti-CD19 scFv sequences

[0487] Exemplary anti-CD19 scFv heavy chain variable region (VH) and light chain variable region

(VL) sequences are listed in Table 8.

Table 8: Exemplary full length anti-CD19 scFv sequences

[0488] Exemplary anti-CD19 scFv heavy chain Chothia CDR sequences are listed in Table 9.

Table 9: Exemplary Anti-CD19 Heavy Chain Chothia CDR sequences

[0489] Exemplary anti-CD19 scFv heavy chain Kabat CDR sequences are listed in Table 10.

Table 10: Exemplary Anti-CD19 Heavy Chain Kabat CDR sequences

[0490] Exemplary anti-CD19 scFv heavy chain IMGT CDR sequences are listed in Table 11.

Table 11: Exemplary Anti-CD19 Heavy Chain IMGT CDR sequences

[0491] Exemplary anti-CD19 scFv light chain Chothia CDR sequences are listed in Table 12.

[0492] Exemplary anti-CD19 scFv light chain Kabat CDR sequences are listed in Table 13.

Table 13: Anti-CD19 Light Chain Kabat CDRs

[0493] Exemplary anti-CD19 scFv light chain IMGT CDR sequences are listed in Table 14.

[0494] Example 14 - Exemplary anti-CD20 scFv constructs

[0495] Exemplary full length anti-CD20 scFv constructs are listed in Table 15.

Table 15: Exemplary full length anti-CD20 scFv Constructs

[0496] Exemplary anti-CD20 heavy chain variable region (VH) and light chain variable region (VL) are listed in Table 16.

Table 16: Exemplary anti-CD20 heavy chain variable region (VH) and light chain variable region (VL) sequences

[0497] Exemplary anti-CD20 heavy chain Chothia CDRs are listed in Table 17.

[0498] Exemplary Anti-CD20 heavy chain Kabat CDRs are listed in Table 18.

Table 18: Exemplary Anti-CD20 heavy chain Kabat CDRs

[0499] Exemplary Anti-CD20 heavy chain IMGT CDRs are listed in Table 19.

Table 19: Exemplary Anti-CD20 heavy chain IMGT CDRs

[0500] Exemplary Anti-CD20 light chain Chothia CDRs are listed in Table 20.

[0501] Exemplary Anti-CD20 light chain Kabat CDRs are listed in Table 21.

[0502] Exemplary Anti-CD20 light chain IMGT CDRs are listed in Table 22.

Table 22: Exemplary Anti-CD20 light chain IMGT CDRs

Example 15 - Exemplary anti-CD22 scFv constructs

[0503] Exemplary anti-CD22 scFv construct, the heavy chain sequence, the light chain sequence, the variable regions, and the CDRs are listed in Table 23.

Table 23: Exemplary anti-CD22 scFv Constructs

Example 16 - Exemplary CAR components sequences

[0504] Exemplary sequences in the CAR components are listed in Table 24.

Table 24: Exemplary CAR Components

Example 17 - Exemplary anti-CD19., anti-CDZO, and anti-CD22 CAR constructs [0505] Exemplary anti-CD19, anti-CD20, and anti-CD22 CAR constructs are listed in Table 25.

Table 25: Exemplary anti-CD19, anti-CD20, and anti-CD22 CAR constructs

Example 18 - Exemplary bispecific and trispecific CAR constructs

[0506] Exemplary bispecific and trispecific CAR constructs are listed in Table 26.

Table 26: Exemplary bispecific and trispecific CAR constructs

Example 19 - Exemplary ER Localization Tag constructs

[0507] Exemplary ER localization tag constructs are listed in Table 27.

Table 27: Exemplary ER Tag Constructs

[0508] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a receptor complex and a second sequence encoding a second polypeptide comprising a second portion of the receptor complex, wherein the first polypeptide comprises:

(a) a first extracellular domain comprising a selection tag, and

(b) a first transmembrane domain from a first receptor, wherein the second polypeptide comprises:

(c) a second transmembrane domain from a second receptor; wherein the first receptor and the second receptor are different receptors; and wherein

(i) the selection tag is not a cytokine binding domain from a cytokine receptor, and/or

(ii) wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor; and wherein cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide.

2. The composition of claim 1, wherein the selection tag of the first polypeptide is not expressed extracellularly on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide.

3. The composition of claim 1 or 2, wherein the selection tag comprises a purification tag or a fluorescent tag.

4. The composition of claim 3, wherein the selection tag comprises a purification tag selected from the group consisting of: ahistidine-glycine tag, poly-arginine tag, poly-aspartate tag, poly-cysteine tag, poly -phenylalanine tag, c-myc tag, Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, a tag consisting of the amino acid sequence WSHPQFEK, a tag consisting of the amino acid sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK, albuminbinding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, CD34 tag, CD 133 tag, CD304 tag, chloramphenicol acetyl transferase (CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, epidermal growth factor receptor (EGF) tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP) tag, glutathione-S- transferase (GST) tag, Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag, Protein C tag, S 1 -tag, S-tag, biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, a VSV-G epitope tag derived from the Vescular Stomatis viral glycoprotein, or a V5 tag derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus of simian virus 5 (SV5). The composition of claim 4, wherein the selection tag comprises multiple copies of a purification tag sequence. The composition of claim 3, wherein the selection tag comprises a fluorescent tag detectable by flow cytometry. The composition of claim 3 or 6, wherein the selection tag comprises a fluorescent tag selected from the group consisting of mNeonGreen, mtagBFP2, tagRFP, mCherry, tagRFP658, mTurquois, mBeRFP, mEGFP, m0range2, mGrape2. The composition of any one of claims 1-5, wherein the selection tag comprises a truncated receptor. The composition of claim 8, wherein the truncated receptor comprises a truncated receptor selected from the group consisting of truncated epidermal growth factor receptor (EGFRt), truncated CD34 (CD34t), and truncated human epidermal growth factor receptor 2 (HER2t). The composition of any one of claims 1-9, wherein the first transmembrane domain is a Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) transmembrane domain. The composition of any one of claims 1-9, wherein the first transmembrane domain is a transmembrane domain from a protein selected from the group consisting of Killer Cell Immunoglobulin Like Receptor Two Ig Domains And Short Cytoplasmic Tail 2 (KIR2DS2), TREM2, IREM2, KIR3DS1, KIR2DS5, KIR2DS1, KIR2DS4, KLRD1, KLRC2, Natural Cytotoxicity Triggering Receptor 2 (NCR2), Signal Regulatory Protein Beta 1 (SIRPB1), Paired Immunoglobin Like Type 2 Receptor Beta (PILRB), C-Type Lectin Domain Containing 5A (CLEC5A), CD300 Molecule Like Family Member B (CD300LB), Sialic Acid Binding Ig Like Lectin 14 (SIGLEC14), SIGLEC15, and SIGLEC16. The composition of any one of claims 1-11, wherein the second transmembrane domain is a DNAX Activating Protein of 12 kDA (DAP12) transmembrane domain. The composition of any one of claims 1-12, wherein the first polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the second polypeptide. The composition of any one of claims 1-13, wherein expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the second polypeptide by the cell. The composition of any one of claims 1-14, wherein retention of the first polypeptide in the endoplasmic reticulum (ER) is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the second polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide and the second polypeptide. The composition of any one of claims 1-15, wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain. The composition of any one of claims 1-16, wherein the second polypeptide does not comprise a full-length intracellular domain. The composition of any one of claims 1-17, wherein the second polypeptide does not comprise an intracellular domain. The composition of any one of claims 1-18, wherein the second polypeptide consists of the second transmembrane domain. The composition of any one of claims 1-19, wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor. The composition of any one of claims 1-19, wherein the selection tag is not a cytokine binding domain from a cytokine receptor. The composition of any one of claims 1-19, wherein the selection tag is not a cytokine binding domain from a cytokine receptor, and wherein the second polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain from a cytokine receptor. The composition of any one of claims 1-17 and 20-22, wherein the second polypeptide comprises an intracellular domain that is at most about 70 amino acids in length. The composition of any one of claims 1-23, wherein the second polypeptide comprises a second selection tag, wherein the second selection tag is the same as the selection tag of the first polypeptide. The composition of any one of claims 1-23, wherein the second polypeptide does not comprise a selection tag. The composition of any one of claims 1-23 and 25, wherein the second polypeptide does not comprise an extracellular domain. The composition of any one of claims 1-24, wherein the second polypeptide comprises an extracellular domain that is at most about 350 amino acids in length. The composition of any one of claims 1-27, wherein the first receptor is TREM1, the second receptor is DAP12. The composition of any one of claims 1-28, wherein the recombinant polynucleic acid further comprises a third sequence encoding a third polypeptide. The composition of claim 29, wherein the first polypeptide, the second polypeptide, and the third polypeptide are each different molecules. The composition of claim 29 or 30, wherein the third polypeptide comprises a third transmembrane domain from a third protein. The composition of claim 31, wherein the second receptor and the third receptor are the same. The composition of claim 31, wherein the first receptor is TREM1, the second receptor is DAP12, and the third receptor is DAP12. The composition of claim 31, wherein the third receptor and the second receptor are different receptors. The composition of claim 34, wherein the third receptor and the first receptor are different receptors. The composition of claim 34 or 35, wherein the first receptor is IgM and the second receptor is CD79a. The composition of claim 36, wherein the third receptor is CD79 . The composition of claim 34 or 35, wherein the first receptor is IgM and the second receptor is CD79P. The composition of claim 38, wherein the third receptor is CD79a. The composition of claim 34 or 35, wherein the first receptor is CD79a and the second receptor is

CD79P. The composition of claim 40, wherein the third receptor is IgM. The composition of any one of claims 1-41, wherein the first polypeptide, the second polypeptide, the third polypeptide, or any combination thereof, lacks an antigen binding domain. The composition of any one of claims 36-41, wherein the IgM lacks one or more fragment antigen binding (Fab) domains. The composition of any one of claims 29-43, wherein the third polypeptide does not comprise an intracellular domain comprising an intracellular signaling domain. The composition of any one of claims 29-44, wherein the third polypeptide does not comprise a full-length intracellular domain. The composition of any one of claims 29-45, wherein the third polypeptide does not comprise an intracellular domain. The composition of any one of claims 29-46, wherein the third polypeptide consists of the third transmembrane domain. The composition of any one of claims 29-45, wherein the third polypeptide comprises an intracellular domain that is at most about 70 amino acids in length. The composition of any one of claims 29-48, wherein the third polypeptide comprises a third selection tag, wherein the third selection tag is the same as the selection tag of the first polypeptide and/or the second polypeptide. The composition of any one of claims 29-48, wherein the third polypeptide does not comprise a selection tag. The composition of any one of claims 29-48 and 50, wherein the third polypeptide does not comprise an extracellular domain. The composition of any one of claims 29-48, wherein the third polypeptide comprises an extracellular domain that is at most about 350 amino acids in length. The composition of any one of claims 29-52, wherein cell surface expression of the selection tag of the first polypeptide is lower on a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to cell surface expression of the selection tag of the first polypeptide on a cell comprising the recombinant polynucleic acid encoding the first polypeptide, the second polypeptide and the third polypeptide. The composition of any one of claims 29-53, wherein the first polypeptide is retained in the endoplasmic reticulum (ER) of a cell that does not express the third polypeptide. The composition of any one of claims 29-54, wherein expression of the first polypeptide on a cell surface of a cell comprising the recombinant polynucleic acid is dependent on expression of the third polypeptide by the cell. The composition of any one of claims 29-55, wherein retention of the first polypeptide in the endoplasmic reticulum (ER) is higher in a cell comprising a recombinant polynucleic acid encoding the first polypeptide that lacks a sequence encoding the third polypeptide compared to retention of the first polypeptide in a cell comprising the recombinant polynucleic acid encoding the first polypeptide, the second polypeptide and the third polypeptide. The composition of any one of claims 1-56, wherein the first polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. The composition of any one of claims 1-57, wherein the second polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. The composition of any one of claims 29-58, wherein the third polypeptide is a non-functional receptor or is not capable of signaling to a cell in which it is expressed. The composition of any one of claims 1-59, wherein the first polypeptide and the second polypeptide form a complex. The composition of any one of claims 29-60, wherein the first polypeptide, the second polypeptide and the third polypeptide form a complex. The composition of claim 60 or 61, wherein the complex is a non-functional receptor complex or is not capable of signaling to a cell in which it is expressed. The composition of any one of claims 1-62, wherein the first polypeptide and the second polypeptide form a dimer. The composition of any one of claims 1-62, wherein the first polypeptide and the second polypeptide form a trimer. The composition of any one of claims 29-62, wherein the first polypeptide, the second polypeptide, and the third polypeptide form a trimer. The composition of any one of claims 1-65, wherein the first transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26. The composition of any one of claims 1-66, wherein the second transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26. The composition of any one of claims 29-67, wherein the third transmembrane domain comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 18-26. The composition of any one of claims 1-65, wherein the first polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69. The composition of any one of claims 1-66, wherein the second polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69. The composition of any one of claims 29-67, wherein the third polypeptide comprises a sequence with at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-69. The composition of any one of claims 1-71, wherein the recombinant polynucleic acid comprises a first recombinant polynucleic acid comprising a sequence encoding the first polypeptide and a second recombinant polynucleic acid comprising a sequence encoding the second polypeptide. The composition of claim 72, wherein the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest. The composition of claim 73, wherein the sequence encoding the first gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as a single polypeptide molecule. The composition of claim 74, wherein the sequence encoding the first polypeptide is linked to the sequence encoding the first protein of interest by a sequence encoding a linker. The composition of claim 75, wherein the linker comprises a protease cleavage site or a 2A selfcleaving peptide. The composition of claim 73, wherein the sequence encoding the first gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as separate polypeptide molecules. The composition of claim 77, wherein the sequence encoding the first polypeptide and the sequence encoding the first gene of interest are separated or linked by an IRES or have different promoters. The composition of any one of claims 72-78, wherein the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. The composition of claim 79, wherein the sequence encoding a second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as a single polypeptide molecule. The composition of claim 80, wherein the sequence encoding the second polypeptide is linked to the sequence encoding the second protein of interest by a sequence encoding a linker. The composition of claim 81, wherein the linker comprises a protease cleavage site or a 2A selfcleaving peptide. The composition of claim 79, wherein the sequence encoding the second gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as separate polypeptide molecules. The composition of claim 83, wherein the sequence encoding the second polypeptide and the sequence encoding the second gene of interest are separated or linked by an IRES or have different promoters. The composition of any one of claims 72-84, wherein the recombinant polynucleic acid comprises a third recombinant polynucleic acid comprising a sequence encoding the third polypeptide. The composition of claim 85, wherein the third recombinant polynucleic acid further comprises a sequence encoding a third gene of interest. The composition of claim 86, wherein the sequence encoding the third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as a single polypeptide molecule. The composition of claim 87, wherein the sequence encoding the third polypeptide is linked to the sequence encoding the third protein of interest by a sequence encoding a linker. The composition of claim 88, wherein the linker comprises a protease cleavage site or a 2A selfcleaving peptide. The composition of claim 86, wherein the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as separate polypeptide molecules. The composition of claim 90, wherein the sequence encoding the third polypeptide and the sequence encoding the third gene of interest are separated or linked by an IRES or have different promoters. The composition of any one of claims 73-91, wherein the first protein of interest, the second protein of interest, and/or the third protein of interest is a chimeric antigen receptor (CAR). The composition of any one of claims 1-91, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR). The composition of one of claim 92 or 93, wherein the CAR comprises

(a) an extracellular domain comprising an antigen-binding domain or an antigen-binding domain and a hinge domain;

(b) a transmembrane domain; and

(c) an intracellular domain comprising an intracellular signaling domain. The composition of claim 94, wherein the antigen binding domain is an anti-CD19 binding domain. The composition of claim 95, 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. The composition of claim 96, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 75 or 76. The composition of claim 95, wherein the anti-CD19 binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1, LCDR2 and LCDR3 sequences in Tables 12- 14; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 and HCDR3 sequences in Tables 9-11 The composition of claim 98, wherein the scFv comprises a heavy chain variable region (VH) with at least about 80% sequence identity to a sequence selected from the group consisting of the VH sequences in Table 8. The composition of claim 98, wherein the scFv comprises a light chain variable region (VL) with at least about 80% sequence identity to a sequence selected from the group consisting of the VL sequences in Table 8. The composition of any one of claims 98-100, wherein the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 7. The composition of claim 94, wherein the antigen binding domain is an anti-CD20 binding domain. The composition of claim 102, wherein the anti-CD20 binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 selected from the group consisting of the LCDR1, LCDR2 and LCDR3 sequences in Tables 20- 22; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 selected from the group consisting of the HCDR1, HCDR2 and HCDR3 sequences in Tables 17-19 The composition of claim 103, wherein the scFv comprises a heavy chain variable region (VH) with at least about 80% sequence identity to a sequence selected from the group consisting of the VH sequences in Table 16. The composition of claim 103, wherein the scFv comprises a light chain variable region (VL) with at least about 80% sequence identity to a sequence selected from the group consisting of the VL sequences in Table 16. The composition of any one of claims 103-105, wherein the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 15. The composition of claim 94, wherein the antigen binding domain is an anti-CD22 binding domain. The composition of claim 107, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 andHCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. The composition of claim 107, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 77. The composition of claim 94, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY- ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)- I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, LI CAM, EGFR, mesothehn, MART- 1, gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catemn, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, ROR1, CD2 and GD2. The composition of any one of claims 94-110, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD2. The composition of any one of claims 94-111, wherein the intracellular domain of the CAR comprises one or more intracellular signaling domains selected from the group consisting of: CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79P, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. The composition of any one of claims 94-112, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. The composition of any one of claims 94-113, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28. The composition of any one of claims 94-114, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 and the transmembrane domain of the CAR comprises a transmembrane domain from CD 8. The composition of any one of claims 94-114, wherein the extracellular domain of the CAR comprises a hinge domain from CD28 and the transmembrane domain of the CAR comprises a transmembrane domain from CD28. The composition of any one of claims 94-116, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 24. The composition of any one of claims 94-116, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 78-80. The composition of any one of claims 94-116, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 25. The composition of any one of claims 94-116, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 26. The composition of any one of claims 94-116, wherein the CAR comprises an anti-CD19 scFv, a hinge domain from CD8a, a transmembrane domain from CD8a, and a cytoplasmic domain comprising a costimulatory domain from CD28 and an intracellular signaling domain from CD3zeta. The composition of any one of claims 94-116, wherein the CAR comprises an anti-CD20 scFv, a hinge domain from CD28, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta. The composition of any one of claims 94-116, wherein the CAR comprises an anti-CD22 scFv, a hinge domain from CD8a, a transmembrane domain from CD8a, and a cytoplasmic domain comprising a costimulatory domain from 4-1BB and an intracellular signaling domain from CD3zeta. A composition comprising a cell comprising the composition of any one of claims 1-123, wherein the cell expresses the first polypeptide and the second polypeptide. The composition of claim 124, wherein the cell expresses the third polypeptide. The composition of claim 124 or 125, wherein the cell is an engineered cell. The composition of any one of claims 124-126, wherein the cell is autologous. The composition of any one of claims 124-126, wherein the cell is allogeneic. The composition of any one of claims 124-128, wherein the cell is a lymphocyte. The composition of any one of claims 124-128, wherein the cell is a T cell. The composition of claim 130, wherein the T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, a naive T cell, or a suppressor T cell. The composition of any one of claims 124-128, wherein the cell is a B cell. The composition of claim 132, wherein the B cell is a naive mature B cell, a plasmablast, a plasma cell, or a memory B cell. The composition of any one of claims 124-133, wherein the cell is a population of cells. The composition of claim 134, wherein the population of cells comprises at least l*10^A5 cells. The composition of any one of claims 134-135, wherein at least 50% of the cells in the population of cells express the first protein of interest and the second protein of interest. The composition of any one of claims 134-135, wherein at least 50% of the cells in the population of cells express the first protein of interest, the second protein of interest, and the third protein of interest. The composition of any one of claims 134-135, wherein at least 50% of the cells in the population of cells express at least two CARs. The composition of any one of claims 134-135, wherein at least 50% of the cells in the population of cells express at least three CARs. A pharmaceutical composition comprising the composition of any one of claims 1-139. A method of treating cancer in a subj ect in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 140. The method of claim 141, wherein the cancer is lymphoma or leukemia. The method of claim 141, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). A method of multiplex cell selection, the method comprising

(a) culturing the cell of any one of claims 124-139;

(b) selecting cells from (a) based on cell surface expression of the selection tag, thereby obtaining a population of selected cells. The method of claim 144, wherein selecting comprises purifying or isolating cells. The method of claim 144, wherein the selection tag is a fluorescent tag and selecting comprises flow cytometry. The method of claim 144, wherein the selection tag is a purification tag and selecting comprises contacting the cells with an agent comprising a moiety that binds to the selection tag. The method of claim 147, wherein the agent further comprises a bead or solid surface that is attached to the moiety that binds to the selection tag. The method of claim 147, wherein the agent comprises further comprises a magnetic bead that is attached to the moiety that binds to the selection tag. The method of claim 147-149, wherein the moiety that binds to the selection tag comprises an antibody. The method of claim 150, wherein the method comprises introducing the recombinant nucleic acid into the cell prior to culturing the cell, the engineered cell, the engineered T cell, or the engineered B cell. The method of claim 151, wherein the engineered T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, a naive T cell, or a suppressor T cell. The method of claim 151, wherein the engineered B cell is a naive mature B cell, a plasmablast, a plasma cell, or a memory B cell. The method of claim 144, wherein at least 50% of the cells in the population of selected cells express the first polypeptide and the second polypeptide. The method of claim 144 or 154, wherein at least 50% of the cells in the population of selected cells express the first protein of interest and the second protein of interest. The method of claim 144, wherein at least 50% of the cells in the population of selected cells express the first polypeptide, the second polypeptide and the third polypeptide. The method of claim 144 or 156, wherein at least 50% of the cells in the population of selected express the first protein of interest, the second protein of interest, and the third protein of interest. A method of multiplex cell selection, comprising

(a) introducing the recombinant polynucleic acid of any one of claims 1-139 into a population of cells

(b) culturing the population of cells from (a); and

(c) selecting cells from the population of cells from (b) based on cell surface expression of the selection tag, thereby obtaining a population of selected cells. The method of claim 158, wherein the population of selected cells is a population of chimeric antigen receptor (CAR)-expressing immune effector cells. The method of claim 159, wherein at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least two different CARs. The method of claim 159, wherein at least 50% of the cells in the population of chimeric antigen receptor (CAR)-expressing immune effector cells express at least three different CARs.