Compositions and methods for allogeneic immune cells

Abstract

The present disclosure relates to the field of adoptive immune cells, and particularly relates to recombinant nucleic acids encoding molecules to reduce TCR-CD3 complex expression, MHC class I and class II expression, and CD58 expression on immune cells, the use of such nucleic acid molecules and compositions for production of allogeneic immune cells, and pharmaceutical compositions containing the allogeneic immune cells. Also provided are methods for treating cancer.

Description

Mintz Ref. No.: 063384-521001WO COMPOSITIONS AND METHODS FOR ALLOGENEIC IMMUNE CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application SerialNos.63 / 641,310, filed on May 1, 2024, and 63 / 744,140, filed on January 10, 2025. The contents of the above-referenced applications are herein expressly incorporated by reference in their entirety, including all drawings. FIELD

[0002] The present disclosure relates to the field of immunology and cell therapy, andparticularly relates to recombinant nucleic acid molecules, compositions, and systems thereof, allogeneic immune cells including the recombinant nucleic acid molecules, pharmaceutical compositions containing the allogeneic immune cells, and methods for making and using allogeneic immune cells. BACKGROUND

[0003] Adoptive immune cell therapies have shown considerable efficacy in the treatment ofcancers and autoimmune related diseases. Immune cells such as T cells can be harvested and modified to express synthetic chimeric antigen receptors (CARs) that can recognize antigens on target cells (i.e., malignant or cancer cells) and activate the T cells to induce T cell cytotoxicity toward the malignant cells expressing the target antigen. Most existing immune cell therapies approved for use in humans are autologous, comprising immune cells isolated from the patient being treated, modified to express CARs of desired antigen specificity, and re- infused into the same patient. However, autologous immune cell therapies are expensive due to the requirement for bespoke manufacturing of the drug product (i.e., the autologous CAR T cell infusion) for each individual patient. Additionally, not all patients are healthy enough to donate immune cells for autologous treatments and the quality of the immune cells obtained can vary widely. The development of off-the-shelf allogeneic cells for use with these immunotherapies is an attractive alternative approach, however, major challenges still remain for the use of allogeneic immunotherapies, including graft rejection, low graft persistence, and conditions such as graft vs. host disease (GvHD).

[0004] Host rejection of allogeneic cells occurs when host cells recognize the allogeneic cellsand direct an immune response against the allogeneic cells, while GvHD occurs when a grafted 1Mintz Ref. No.: 063384-521001WO allogeneic cell recognizes the host cells as foreign and directs an immune response against the host. Host rejection of the allogeneic graft can lead to loss of efficacy and / or complete loss of the cell transplant and the need for additional treatment. In GvHD, donor T cells can attack the patients’ tissues, for example skin, gastrointestinal tract, liver, lungs, kidneys, eyes, and hematopoietic system and cause severe organ damage, cancer, and death if the GvHD cannot be controlled. Thus, an urgent need exists to increase the effectiveness of the off-the-shelf allogeneic cells immunotherapies by reducing undesired immune response against the host and preventing graft rejection (e.g., GvHD).

[0005] The disclosure provides solutions to the problems existing with previous attempts toprovide allogeneic immune cells and potentially offers improved methods for the treatment of many diseases, most notably cancer and autoimmune disease. SUMMARY

[0006] The present disclosure relates generally to the development of immunotherapies, suchas recombinant nucleic acids and compositions thereof, methods of making engineered immune cells by using the recombinant nucleic acids, methods of using the engineered immune cells, and pharmaceutical compositions including the engineered immune cells for use in the management of various health conditions such as GvHD. The recombinant nucleic acids can be used in combination with other recombinant nucleic acid constructs to further modify immune cells to treat conditions such as cancer or autoimmune disease.

[0007] In particular, as described in greater detail below, some embodiments of the disclosureprovide recombinant nucleic acids including, but not limited to (1) a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; (2) a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and (3) a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell. Also disclosed are immune cells that have been engineered to include one or more of the recombinant nucleic acids disclosed herein, methods for making engineered immune cells, and pharmaceutical compositions including the engineered immune cells of the disclosure. Further provided in particular aspects of the disclosure are compositions and methods for the treatment of various health conditions, including cancers or autoimmune disease in a subject in need thereof. 2Mintz Ref. No.: 063384-521001WO

[0008] The foregoing summary is illustrative only and is not intended to be in any way limiting.In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects, and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

[0009] In one aspect of the disclosure, provided herein is a recombinant nucleic acidcomprising a first nucleotide sequence encoding a first molecule capable of reducing TCR- CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of an immune cell; wherein the recombinant nucleic acid is multicistronic.

[0010] In a second aspect of the disclosure, provided herein is a recombinant nucleic acidcomprising a first nucleotide sequence encoding a first molecule capable of reducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell; wherein the recombinant nucleic acid is bicistronic.

[0011] In some embodiments, the recombinant nucleic acid comprises at least one or more ofan exogenous promoter, a post-transcriptional response element (PRE), and a polyadenylation signal sequence.

[0012] In some embodiments, the recombinant nucleic acid further comprises a ribosomeskipping sequence disposed between each cistron.

[0013] In some embodiments, the recombinant configuration is tricistronic and a ribosomeskipping sequence is disposed between the first cistron and the second cistron and between the second cistron and the third cistron.

[0014] In some embodiments, the ribosome skipping sequence disposed between the firstcistron and the second cistron is different from the ribosome skipping sequence disposed between the second cistron and the third cistron.

[0015] In some embodiments, the ribosome skipping sequence disposed between the firstcistron and the second cistron is the same as the ribosome skipping sequence disposed between the second cistron and the third cistron.

[0016] In some embodiments, the recombinant configuration is bicistronic and a ribosomeskipping sequence is disposed between the first cistron and the second cistron. 3Mintz Ref. No.: 063384-521001WO

[0017] In some embodiments, the first nucleotide sequence encoding a first molecule capableof reducing TCR-CD3 complex expression on the surface of an immune cell encodes a first molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein subunit of the TCR-CD3 complex capable of reducing assembly of a functional TCR-CD3 complex; the second nucleotide sequence encoding a second molecule capable of reducing MHC / HLA expression on the surface of an immune cell encodes a second molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative protein capable of regulating transcription of MHC class I and / or MHC class II; and the third nucleic acid encoding a third molecule capable of reducing CD58 expression on the surface of an immune cell encodes a third molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein capable of reducing the binding of CD58 to its cognate ligand CD2.

[0018] In some embodiments, the first nucleotide sequence encodes a first moleculecomprising an anti-TCR-CD3 PEBL comprising an scFv capable of binding a TCR-CD3 complex component. In some embodiments, the TCR-CD3 complex component comprises a TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta. In some embodiments, the scFv is capable of binding CD3zeta. In some embodiments, the scFv is capable of binding CD3epsilon. In some embodiments, the scFv comprises a sequence selected from the sequences in Table 3 or Table 4.

[0019] In some embodiments, the protein localization sequence or tag of the PEBL is selectedfrom the group consisting of: an endoplasmic reticulum (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 sequence or tag comprises an ER localization tag. In some embodiments, the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). 4Mintz Ref. No.: 063384-521001WO

[0020] In some embodiments, the first molecule comprises a dominant negative mutant TCR-CD3 complex component capable of binding an unmutated or wild-type TCR-CD3 complex component.

[0021] The recombinant nucleic acid of claim 18, wherein the dominant negative mutant TCR-CD3 complex component is a dominant negative mutant TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta. In some embodiments, the dominant negative mutant TCR-CD3 complex component is a dominant negative mutant CD3epsilon. In some embodiments, the dominant negative mutant TCR-CD3 complex component is a dominant negative mutant CD3zeta. In some embodiments, the dominant negative mutant TCR-CD3 complex component comprises a sequence selected from the sequences in Table 5.

[0022] In some embodiments, the first molecule comprises an shRNA targeting a TCR-CD3complex component. In some embodiments, the shRNA targets TCRalpha, TCRbeta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta. In some embodiments, the shRNA targets CD3zeta. In some embodiments, the shRNA targets CD3epsilon.

[0023] In some embodiments, the second nucleotide sequence encodes a second moleculecapable of inhibiting MHC class I or MHC class II expression.

[0024] In some embodiments, the second molecule comprises a mutated RFX5 polypeptide.In some embodiments, the mutated RFX5 polypeptide comprises a truncated RFX5 molecule. In some embodiments, the RFX5 polypeptide comprises a sequence selected from the sequences listed in Table 7.

[0025] In some embodiments, the third nucleotide sequence encodes a third moleculecomprising an anti-CD58 PEBL comprising an scFv capable of binding CD58 (anti-CD58 scFv). In some embodiments, the anti-CD58 scFv comprises a sequence selected from the sequences listed Table 8, Table 9, or Table 10. In some embodiments, the protein localization sequence or tag of the PEBL comprises an ER localization tag. In some embodiments, the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). 5Mintz Ref. No.: 063384-521001WO

[0026] In some embodiments, the third molecule comprises an CD58 shRNA.

[0027] In some embodiments, the CD58 shRNA comprises a sequence selected from thesequences listed in Table 13.

[0028] In some embodiments, the third molecule comprises an UL148 polypeptide. In someembodiments, the UL148 polypeptide comprises the amino acid sequence of SEQ ID NO: 244.

[0029] In some embodiments, according to the second aspect of the disclosure, the firstmolecule comprises a nucleotide sequence encoding a tandem PEBL comprising an scFv capable of binding a TCR-CD3 complex component and an scFv capable of binding CD58. In some embodiments, the two scFvs are operably linked together. In some embodiments, the protein localization sequence or tag of the PEBL comprises an ER localization tag. In some embodiments, the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

[0030] In some embodiments, according to the second aspect of the disclosure, the firstmolecule comprises a nucleotide sequence encoding a dominant negative mutant TCR-CD3 complex component and a PEBL comprising an scFv capable of binding CD58. In some embodiments, the dominant negative mutant TCR-CD3 complex component and the scFv are operably linked together.

[0031] In some embodiments, according to the second aspect of the disclosure, therecombinant nucleic acid comprises from 5’ to 3’ end: (a) the second nucleotide sequence and (b) the first nucleotide sequence. In some embodiments, the first nucleotide sequence comprises a nucleotide sequence encoding a dominant negative mutant TCR-CD3 complex component, a PEBL comprising an scFv capable of binding CD58, and a CD3z shRNA. In some embodiments, the dominant negative mutant TCR-CD3 complex component and the scFv are operably linked together. In some embodiments, the second nucleotide sequence comprises a mutated RFX5 polypeptide. In some embodiments, the mutated RFX5 polypeptide comprises a truncated RFX5 molecule. In some embodiments, the RFX5 polypeptide comprises a sequence selected from the sequences listed in Table 7. In some embodiments, the protein localization sequence or tag of the PEBL comprises an ER localization tag. In some 6Mintz Ref. No.: 063384-521001WO embodiments, wherein the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

[0032] Also provided herein is a recombinant nucleic acid encoding, from 5’ end to 3’ end, amutated RFX5, a T2A ribosome skipping sequence, a fusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon, and a CD3z shRNA. In some embodiments, the mutated RFX5 polypeptide comprises a truncated RFX5 molecule. In some embodiments, the RFX5 polypeptide comprises a sequence selected from the sequences listed in Table 7. In some embodiments, the protein localization sequence or tag of the PEBL comprises an ER localization tag. In some embodiments, the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). In some embodiments, the recombinant nucleic acid comprises the sequence of SEQ ID NO: 806.

[0033] In some embodiments, the scFvs are fully humanized.

[0034] In some embodiments, the recombinant nucleic acid comprises from 5’ to 3’ end: thefirst nucleotide sequence; the second nucleotide sequence; and the third nucleotide sequence; or wherein the nucleic acid comprises from 5’ to 3’ end:the first nucleotide sequence; the third nucleotide sequence; and the second nucleotide sequence; or wherein the nucleic acid comprises from 5’ to 3’ end: the second nucleotide sequence; the first nucleotide sequence; and the third nucleotide sequence; or wherein the nucleic acid comprises from 5’ to 3’ end: the second nucleotide sequence; the third nucleotide sequence; and the first nucleotide sequence; or wherein the nucleic acid comprises from 5’ to 3’ end: the third nucleotide sequence; the first nucleotide sequence; and the second nucleotide sequence; or wherein the nucleic acid comprises from 5’ to 3’ end: the third nucleotide sequence; the second nucleotide encoding; 7Mintz Ref. No.: 063384-521001WO and the first nucleotide sequence. In some embodiments, the nucleic acid comprises one or more ribosome skipping sequences located between the first, second, and / or third nucleotide sequence.

[0035] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end aPEBL comprising an scFv capable of binding a TCR-CD3 complex component, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and PEBL comprising an scFv capable of binding CD58.

[0036] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end amutated RFX5, a 2A ribosome skipping sequence, and a fusion protein comprising a PEBL comprising an scFv capable of binding a TCR-CD3 complex component, and a PEBL comprising an scFv capable of binding CD58.

[0037] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end afusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon.

[0038] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end amutated RFX5 and a fusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon. In some embodiments, the recombinant nucleic acid further comprises the 3’ end, a 2A ribosome skipping sequence and a PEBL comprising an scFv capable of binding a TCR-CD3 complex component.

[0039] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end adominant negative mutant CD3epsilon, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a PEBL comprising an scFv capable of binding a CD58.

[0040] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end adominant negative mutant CD3zeta operably linked with a ubiquitin ligase, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a PEBL comprising an scFv capable of binding a CD58.

[0041] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end adominant negative mutant CD3epsilon, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a UL148 polypeptide.

[0042] Also provided herein is a recombinant nucleic acid encoding from 5’ end to 3’ end adominant negative mutant CD3zeta operably linked with a ubiquitin ligase, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a UL148 polypeptide. 8Mintz Ref. No.: 063384-521001WO

[0043] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding US11.

[0044] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding SerpinB9.

[0045] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding a cell selection molecule. In some embodiments, the cell selection molecule comprises a CD34 epitope. In some embodiments, the cell selection molecule comprises a CD34 epitope selected from Table 18.

[0046] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a promoter.

[0047] In some embodiments, the promoter is an MND promoter, an MNDU3 promoter, anEF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or an NFkB responsive promoter. In some embodiments, the recombinant nucleic acid further comprises a second promoter. In some embodiments, each of the promoters is operably linked to a nucleotide sequence in the same direction. In some embodiments, each of the promoters is operably linked to a nucleotide sequence in opposite directions. In some embodiments, one promoter is operably linked to the nucleotide sequences encoding an shRNA and the other promoter is operably linked to the nucleotide sequences encoding a PEBL or a dominant negative protein.

[0048] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises an shRNA. In some embodiments, the shRNA is a CD3zeta shRNA. In some embodiments, the shRNA is located in an intron of a promoter. In some embodiments, the promoter is an EF-1alpha, a MND, MNDU3, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or a hUbC promoter.

[0049] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a Woodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE) sequence, a polyA sequence, and / or an LTR sequence.

[0050] the recombinant nucleic acid of the present disclosure further comprises a fourthnucleotide sequence encoding a fourth molecule capable of blocking the CD8-MHC class I interaction.

[0051] In some embodiments, the fourth molecule capable of blocking the CD8-MHC class Iinteraction comprises an MHC class I binder. In some embodiments, the MHC class I binder 9Mintz Ref. No.: 063384-521001WO comprises an CD8alpha extracellular domain with a cysteine to serine (C to S) mutation at amino acid position 54 and a serine to asparagine (S to N) mutation at amino acid position 74 in SEQ ID NO: 407. In some embodiments, the MHC class I binder comprises SEQ ID NO: 411. In some embodiments, the MHC class I binder additionally comprises a B2M polypeptide or a fragment thereof.

[0052] In some embodiments, the recombinant nucleic acid of the present disclosure furthercomprises a fifth nucleotide sequence encoding one or more shRNAs capable of reducing expression of CD3zeta, FasR, B2M, CIITA, CD3epsilon, TRAC, RFX5 and / or CD58. In some embodiments, the shRNA is capable of reducing TCR-CD3 complex expression on the surface of the immune cell.

[0053] In some embodiments, the first molecule is a CD3zeta ubiquitin ligase fusion proteinand the fifth molecule is a CD3zeta shRNA located in a promoter region.

[0054] In some embodiments, the first, second, and / or third molecule comprises a ubiquitinligase fusion protein.

[0055] Also provided herein is a recombinant nucleic acid encoding, from 5’ end to 3’ end, amutated RFX5, a 2A ribosome skipping sequence, a 54mer MHC class I binder fused to B2M, a 2A ribosome skipping sequence, a fusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon , a CD3z shRNA, and an RFX5 shRNA. In some embodiments, the mutated RFX5 comprises a truncated RFX5 molecule. In some embodiments, the RFX5 comprises a sequence selected from the sequences listed in Table 7. In some embodiments, the protein localization sequence or tag of the PEBL comprises an ER localization tag. In some embodiments, the ER localization tag is selected from the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). In some embodiments, the nucleic acid sequence comprises the sequence of SEQ ID NO: 811.

[0056] Also provided herein is a vector comprising the recombinant nucleic acid of the presentdisclosure. 10Mintz Ref. No.: 063384-521001WO

[0057] Also provided herein is a lentiviral expression vector comprising the recombinantnucleic acid of the present disclosure.

[0058] Also provided herein is a virus particle comprising the recombinant nucleic acid of thepresent disclosure. In some embodiments, the virus is a lentivirus.

[0059] Also provided herein is an engineered immune cell comprising the recombinant nucleicacid of the present disclosure, the vector of the present disclosure, or the virus of the present disclosure. In some embodiments, the cell is a T cell. In some embodiments, the T cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the T cell is a CD8- positive T cell, a CD4-positive T cell, a regulatory T cell (TREG), a cytotoxic T cell (TCTL), a central memory T cell (TCM), an effector memory T cell (TEM), a tissue-resident memory T cell (TRM), a stem cell-like memory T cell (TSCM) or a tumor infiltrating lymphocyte (TIL). In some embodiments, the engineered immune cell is a CAR T cell.

[0060] Also provided herein is a population of engineered immune cells comprising therecombinant nucleic acid of the present disclosure, or the vector of the present disclosure.

[0061] Also provided herein is a composition comprising the recombinant nucleic acid of thepresent disclosure, or the vector of the present disclosure, or the virus of the present disclosure, and additionally comprising a nucleic acid encoding a CAR construct.

[0062] Also provided herein is an engineered immune cell comprising the composition of thepresent disclosure.

[0063] Also provided herein is a pharmaceutical composition comprising the virus particle ofthe present disclosure or engineered immune cell of the present disclosure and a pharmaceutically acceptable excipient.

[0064] Also provided herein is a method of producing an engineered immune cell, the methodcomprising, contacting an immune cell with the recombinant nucleic acid of the present disclosure, the vector of the present disclosure, the virus of the present disclosure, or the composition of the present disclosure in vitro.

[0065] Also provided herein is a method of reducing or abolishing TCR-CD3 complexexpression on the surface of an immune cell, the method comprising contacting the immune cell with the recombinant nucleic acid of the present disclosure, the vector of the present disclosure, the virus of the present disclosure, or the composition of the present disclosure in vitro.

[0066] Also provided herein is a method of reducing MHC expression on the surface of animmune cell, the method comprising contacting the immune cell with the recombinant nucleic 11Mintz Ref. No.: 063384-521001WO acid of the present disclosure, the vector of the present disclosure, the of the present disclosure, or the composition of the present disclosure in vitro.

[0067] Also provided herein is a method of reducing CD58 expression on the surface of animmune cell, the method comprising contacting the immune cell with the recombinant nucleic acid of the present disclosure, the vector of the present disclosure, the virus of the present disclosure, or the composition of the present disclosure in vitro.

[0068] Also provided herein is a method of reducing TCR-CD3 complex expression, MHCexpression, and CD58 expression on the surface of an immune cell, the method comprising contacting the immune cell with the recombinant nucleic of the present disclosure, the vector of the present disclosure, the virus of the present disclosure, or the composition of the present disclosure in vitro.

[0069] Also provided herein is a method of preventing or reducing graft versus host disease ina subject in need of engineered immune cell, the method comprising administering the engineered immune cell or population of immune cells of the present disclosure to the subject.

[0070] Also provided herein is a method of treating a subject in need thereof with anengineered immune cell, the method comprising administering the engineered immune cell or population of immune cells of the present disclosure to the subject.

[0071] Also provided herein is a method of increasing allogeneic immune cell survival in asubject, the method comprising administering the engineered immune cell or population of immune cells of the present disclosure to the subject.

[0072] Also provided herein is a method of reducing immune rejection or aiding in preventionof immune rejection in a subject in need of an allogeneic graft, the method comprising administering the engineered immune cell or population of immune cells of the present disclosure to the subject.

[0073] In some embodiments of the method of the disclosure, the immune cell is a cell from abiological sample. In some embodiments, the biological sample is from a human subject.

[0074] In some embodiments of the method of the disclosure, the immune cell is an allogeneiccell.

[0075] In some embodiments of the method of the disclosure, the engineered immune cell is aT cell. In some embodiments, the T cell is a CD4+ cell, CD8+ cell, TREGcell, TSCMcell, or TCMcell.

[0076] In some embodiments of the method of the disclosure, the immune cell is a CAR T cell.12Mintz Ref. No.: 063384-521001WO

[0077] In yet another aspect, provided herein is an allogeneic system comprising, a firstrecombinant nucleic acid of the present disclosure; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

[0078] In yet another aspect, provided herein is an allogeneic system comprising, a firstrecombinant nucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

[0079] In yet another aspect, provided herein is an allogeneic system comprising, recombinantnucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

[0080] In yet another aspect, provided herein is an allogeneic system comprising, a first viralparticle comprising a first recombinant nucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second viral particle comprising a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

[0081] In yet another aspect, provided herein is an allogeneic system comprising, a first viralparticle comprising a first recombinant nucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell; and a second viral particle comprising a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors. 13Mintz Ref. No.: 063384-521001WO

[0082] In some embodiments, the first and second viral particles are lentiviral particles.

[0083] In some embodiments, the first recombinant nucleic acid is multicistronic.

[0084] In some embodiments, the first recombinant nucleic acid comprises at least one or moreof an exogenous promoter, a post-transcriptional response element (PRE), and a polyadenylation signal sequence.

[0085] In some embodiments, the first recombinant nucleic acid further comprises a ribosomeskipping sequence disposed between each cistron.

[0086] In some embodiments, the first recombinant nucleic acid configuration is tricistronicand a ribosome skipping sequence is disposed between the first cistron and the second cistron and between the second cistron and the third cistron.

[0087] In some embodiments, the first recombinant nucleic acid configuration is bicistronicand a ribosome skipping sequence is disposed between the first cistron and the second cistron.

[0088] In some embodiments, the first nucleotide sequence encoding a first molecule capableof reducing TCR-CD3 complex expression on the surface of an immune cell encodes a first molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein subunit of the TCR-CD3 complex capable of reducing assembly of a functional TCR-CD3 complex; the second nucleotide sequence encoding a second molecule capable of reducing MHC / HLA expression on the surface of an immune cell encodes a second molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative protein capable of regulating transcription of the MHC / HLA; and the third nucleic acid encoding a third molecule capable of reducing CD58 expression on the surface of an immune cell encodes a third molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein capable of reducing the binding of CD58 to its cognate ligand CD2.

[0089] In some embodiments, the first molecule comprises an scFv capable of binding a TCR-CD3 complex component.

[0090] In some embodiments, the TCR-CD3 complex component comprises a TCRalpha,TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta. In some embodiments, the scFv is capable of binding CD3zeta. In some embodiments, the scFv is capable of binding CD3epsilon. In some embodiments, the scFv comprises a sequence selected from the sequences in Table 3 or Table 4. In some embodiments, the scFv additionally comprises a protein localization sequence or tag. In some embodiments, the protein localization sequence or tag is selected from the group consisting of: an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a 14Mintz Ref. No.: 063384-521001WO plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, and a nuclear localization tag.

[0091] In some embodiments, the first molecule comprises a dominant negative mutant TCR-CD3 complex component capable of binding an unmutated or wild-type TCR-CD3 complex component.

[0092] In some embodiments, the dominant negative mutant TCR-CD3 complex componentis a dominant negative mutant TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta. In some embodiments, the dominant negative mutant TCR-CD3 complex component is a dominant negative mutant CD3epsilon. In some embodiments, the dominant negative mutant TCR-CD3 complex component is a dominant negative mutant CD3zeta. In some embodiments, the dominant negative mutant TCR-CD3 complex component comprises a sequence selected from the sequences in Table 5.

[0093] In some embodiments, the first molecule comprises an shRNA targeting a TCR-CD3complex component. In some embodiments, the shRNA targets TCRalpha, TCRbeta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta. In some embodiments, the shRNA targets CD3zeta. In some embodiments, the shRNA targets CD3epsilon.

[0094] In some embodiments, the first recombinant nucleic acid additionally comprises afourth nucleotide sequence encoding a fourth molecule capable of reducing TCR-CD3 complex expression on the surface of the immune cell. In some embodiments, the first molecule is a CD3zeta shRNA located in a promoter region and the fourth molecule is a CD3zeta ubiquitin ligase fusion protein.

[0095] In some embodiments, the first, second, and / or third molecule comprises a ubiquitinligase fusion protein.

[0096] In some embodiments, the second molecule is capable of inhibiting MHC class I orMHC class II expression.

[0097] In some embodiments, the second molecule is a mutated RFX5 polypeptide.

[0098] In some embodiments, the mutated RFX5 polypeptide is a truncated RFX5 molecule.

[0099] In some embodiments, the mutated RFX5 polypeptide comprises a sequence selectedfrom the sequences listed in Table 7.

[0100] In some embodiments, the third molecule comprises an anti-CD58 scFv capable ofbinding a CD58. In some embodiments, the anti-CD58 scFv comprises a sequence selected from the sequences listed Table 8, Table 9, or Table 10. 15Mintz Ref. No.: 063384-521001WO

[0101] In some embodiments, the third molecule comprises an CD58 shRNA. In someembodiments, the CD58 shRNA comprises a sequence selected from the sequences listed in Table 13.

[0102] In some embodiments, the third molecule comprises a UL148 polypeptide.

[0103] In some embodiments, the third molecule comprises the amino acid sequence of SEQID NO: 244.

[0104] In some embodiments, the first molecule and the third molecule are each an scFv. Insome embodiments, the two scFvs are operably linked together. In some embodiments, the first scFv is capable of binding a TCR-CD3 complex component and the second scFv is capable of binding CD58.

[0105] In some embodiments, the operably linked scFvs further comprise an ER localizationsequence or tag. In some embodiments, the ER localization sequence is at the C-terminus.

[0106] In some embodiments, the first molecule is a dominant negative mutant TCR-CD3complex component and the third molecule is an scFv capable of binding CD58. In some embodiments, the dominant negative mutant TCR-CD3 complex component and the scFv are operably linked together.

[0107] In some embodiments, the scFvs are fully humanized.

[0108] In some embodiments, the first recombinant nucleic acid comprises the recombinantnucleic acid of any one of claims 1 to 75.

[0109] In some embodiments, the first recombinant nucleic acid further comprises a promoter.In some embodiments, the promoter is a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or an NFkB responsive promoter.

[0110] In some embodiments, the first recombinant nucleic acid further comprises a secondpromoter. In some embodiments, each of the promoters is operably linked to a nucleotide sequence in the same direction. In some embodiments, each of the promoters is operably linked to a nucleotide sequence in opposite directions. In some embodiments, one promoter is operably linked to express shRNA and another promoter is operably linked to express the polypeptide molecules.

[0111] In some embodiments, the first recombinant nucleic acid further comprises an shRNA.16Mintz Ref. No.: 063384-521001WO

[0112] In some embodiments, the first recombinant nucleic acid further comprises a CD3zetashRNA. In some embodiments, the shRNA is located in an intronic region of a promoter. In some embodiments, the promoter is an EF-1alpha or a hUbC promoter.

[0113] In some embodiments, the first recombinant nucleic acid further comprises a WPREsequence, a polyA sequence, and / or an LTR sequence.

[0114] In some embodiments, the first recombinant nucleic acid further comprises a fourthmolecule comprising an MHC class I binder. In some embodiments, the MHC class I binder comprises an CD8alpha extracellular domain with amino acid mutations S74N and C54S at amino acid positions 74 and 54 in SEQ ID NO: 407. In some embodiments, the MHC class I binder comprises SEQ ID NO: 411. In some embodiments, the MHC class I binder additionally comprises a B2M polypeptide.

[0115] In some embodiments, the one or more CARs encoded by the second recombinantnucleic acid comprises a CD22-specific CAR. In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a spacer, a hinge domain, a transmembrane domain, a peptide linker, and an intracellular domain. In some embodiments, the CD22-specific binding domain comprises an antibody that binds CD22 or an antigen-binding fragment thereof. In some embodiments, the CD22-specific binding domain comprises an antibody that binds human CD22. In some embodiments, the CD22-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD22. In some embodiments, the antigen-binding fragment of an antibody that binds human CD22 is a single chain variable fragment (scFv) that binds CD22.

[0116] In some embodiments, the scFv that binds CD22 has the sequence ofQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 559). In some embodiments, the scFv that binds CD22 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 559.

[0117] In some embodiments, the CD22-specific CAR comprises a CD8α hinge domaincomprising the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 560) 17Mintz Ref. No.: 063384-521001WO and a CD8α transmembrane domain comprising the sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 694) and optionally a peptide linker having the sequence of LYC.

[0118] In some embodiments, the CD22-specific CAR comprises a CD8α hinge domaincomprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 560 and a CD8α transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 694.

[0119] In some embodiments, the CD8α transmembrane domain further comprises a spacerhaving the sequence of LYC.

[0120] In some embodiments, the CD22-specific CAR comprises an intracellular domaincomprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain. In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3ζ intracellular signaling domain. In some embodiments, the CD3ζ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 562). In some embodiments, the CD3ζ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562. In some embodiments, the costimulatory signaling domain comprises a 4-1BB / CD137 signaling domain. In some embodiments, the 4-1BB / CD137 signaling domain comprises the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 561). In some embodiments, the 4-1BB / CD137 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 561.

[0121] In some embodiments, the CD22-specific CAR comprises the sequence ofQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW 18Mintz Ref. No.: 063384-521001WO APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 646).

[0122] In some embodiments, the CD22-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 646.

[0123] In some embodiments, the CD22-specific CAR comprises the sequence ofMLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQ AEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 342).

[0124] In some embodiments, the CD22-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 342.

[0125] In some embodiments, the one or more CARs encoded by the second recombinantnucleic acid further comprises a CD19-specific CAR and a CD20-specific CAR. In some embodiments, the CD19-specific CAR comprises a CD19-specific binding domain, a transmembrane domain, and an intracellular domain.

[0126] In some embodiments, the CD19-specific CAR comprises a CD19-specific bindingdomain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain.

[0127] In some embodiments, the CD19-specific binding domain comprises an antibody thatbinds CD19 or an antigen-binding fragment thereof. In some embodiments, the CD19-specific binding domain comprises an antibody that binds human CD19. In some embodiments, the CD19-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD19. In some embodiments, the antigen-binding fragment of an antibody that binds human CD19 is a single chain variable fragment (scFv) that binds CD19. In some embodiments, the scFv capable of binding CD19 has the sequence of 19Mintz Ref. No.: 063384-521001WO EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 564). In some embodiments, the scFv capable of binding CD19 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 564.

[0128] In some embodiments, the CD19-specific CAR comprises a CD28 hinge domaincomprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 565) and a CD28 transmembrane domain comprising the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 566).

[0129] In some embodiments, the CD19-specific CAR comprises a CD28 hinge domaincomprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 565 and a CD28 transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 566.

[0130] In some embodiments, the CD19-specific CAR comprises an intracellular domainfurther comprising a primary T cell activating domain comprising an immunoreceptor tyrosine- based activation motif (ITAM) and a costimulatory signaling domain. In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3ζ intracellular signaling domain. In some embodiments, the CD3ζ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 562). In some embodiments, the CD3ζ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562. In some embodiments, the costimulatory signaling domain comprises a CD28 signaling domain. In some embodiments, the CD28 signaling domain comprises the sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 567). In some embodiments, the CD28 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 567. 20Mintz Ref. No.: 063384-521001WO

[0131] In some embodiments, the CD19-specific CAR comprises the sequence ofEVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 647).

[0132] In some embodiments, the CD19-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 647.

[0133] In some embodiments, the CD19-specific CAR comprises the sequence ofMALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIA WVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDT AMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSV LTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD RFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPP YLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 563).

[0134] In some embodiments, the CD19-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 563.

[0135] In some embodiments, the CD20-specific CAR comprises a CD20-specific bindingdomain, a transmembrane domain, and an intracellular domain.

[0136] In some embodiments, the CD20-specific binding domain comprises an antibody thatbinds CD20 or an antigen-binding fragment thereof. In some embodiments, the CD20-specific binding domain comprises an antibody that binds human CD20. In some embodiments, the CD20-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD20. In some embodiments, the antigen-binding fragment of an antibody that 21Mintz Ref. No.: 063384-521001WO binds human CD20 is a single chain variable fragment (scFv) that binds CD20. In some embodiments, the scFv capable of binding CD20 has the sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 569). In some embodiments, the scFv capable of binding CD20 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 569.

[0137] In some embodiments, the CD20-specific CAR comprises a CD28 hinge domaincomprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 565) and a CD28 transmembrane domain comprising the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 566).

[0138] In some embodiments, the CD20-specific CAR comprises a CD28 hinge domaincomprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 565 and a CD28 transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 566.

[0139] In some embodiments, the CD20-specific CAR comprises an intracellular domainfurther comprising a primary T cell activating domain comprising an immunoreceptor tyrosine- based activation motif (ITAM) and a costimulatory signaling domain. In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3ζ intracellular signaling domain. In some embodiments, the CD3ζ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 562). In some embodiments, the CD3ζ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562. In some embodiments, the costimulatory signaling domain comprises a CD2 signaling domain. In some embodiments, the CD2 signaling domain comprises the sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAP SHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN 22Mintz Ref. No.: 063384-521001WO (SEQ ID NO: 570). In some embodiments, the CD2 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 570.

[0140] In some embodiments, the CD20-specific CAR comprises the sequence ofMLLLVTSLLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKN YLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYC QQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSC TASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKR KKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNR VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO: 568).

[0141] In some embodiments, the CD20-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 568.

[0142] In some embodiments, the CD20-specific CAR comprises the sequence ofDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEELETRAHRVAT EERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPS GTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 648).

[0143] In some embodiments, the CD20-specific CAR comprises a sequence having 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 648. 23Mintz Ref. No.: 063384-521001WO

[0144] In some embodiments, the second recombinant nucleic acid comprises a polynucleotidesequence encoding the CD19-specific CAR, a polynucleotide sequence encoding the CD20- specific CAR, and a polynucleotide sequence encoding the CD22-specific CAR, wherein each CAR-encoding polynucleotide sequence is separated from the other CAR-encoding polynucleotide sequences by a sequence encoding a viral ribosome skipping peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide.

[0145] In some embodiments, the second recombinant nucleic acid comprises from 5’ to 3’, asequence encoding the CD22-specific CAR; a sequence encoding a viral P2A ribosome skipping peptide; a sequence encoding the CD19-specific CAR; a sequence encoding a viral T2A ribosome skipping peptide; and a sequence encoding the CD20-specific CAR.

[0146] In some embodiments, the one or more CARs encoded by the second recombinantnucleic acid is specific for a tumor antigen.

[0147] In some embodiments, the tumor antigen is selected from the group consisting ofglioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b- HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY- CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, GPC3, TGFBR2, and GD2, or any combinations thereof.

[0148] In some embodiments, the tumor antigen is a solid tumor antigen.

[0149] In some embodiments, the tumor antigen is BCMA, TGFBR2, and / or GPC3.24Mintz Ref. No.: 063384-521001WO

[0150] In some embodiments, the second recombinant nucleic acid encoding the one or moreCARs further comprises a promoter.

[0151] In some embodiments, the promoter is a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, a gamma retro promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, or an NFkB responsive promoter.

[0152] In some embodiments, the one or more CARs encoded by the second recombinantnucleic acid is a bicistronic CAR.

[0153] In some embodiments, the first recombinant nucleic acid further comprises a fourthnucleotide sequence encoding a fourth molecule capable of blocking the CD8-MHC class I interaction.

[0154] Also provided herein is an allogeneic system comprising, a first recombinant nucleicacid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a first nucleotide sequence encoding an anti-CD19 CAR; a second nucleotide sequence encoding an anti-CD20 CAR; and a third nucleotide sequence encoding an anti-CD22 CAR.

[0155] Also provided herein is an allogeneic system comprising, a first recombinant nucleicacid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding an anti-CD22 CAR.

[0156] Also provided herein is an an immune cell comprising the allogeneic system of thepresent disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] The features of the present disclosure are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative 25Mintz Ref. No.: 063384-521001WO embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0158] FIG. 1A is a graphical representation of an exemplary single nucleic acid system for arecombinant nucleic acid including a Chimeric Antigen Receptor expression cassette (CAR), a TCR-CD3 complex protein expression blocker (PEBL) expression cassette, an RFX5 expression cassette, and a CD58 PEBL expression cassette. The four expression cassettes are all expressed from a single promoter (Option 1).

[0159] FIG. 1B is a graphical representation of a dual nucleic acid system with a recombinantnucleic acid including a TCR-CD3 complex protein expression blocker (PEBL) expression cassette, an RFX5 dominant negative (DN) expression cassette, a CD58 PEBL expression cassette and an additional nucleic acid encoding the Chimeric Antigen Receptor (CAR) (Option 2). The expression cassettes are separated by 2A ribosomal skipping sequences. FIGs. 1C-F show graphs of protein expression (FIG. 1C CAR expression, FIG. 1D TCR-CD3 complex expression, FIG. 1E MHC I and MHC II expression, and FIG. 1F CD58 expression), as measured by flow cytometry, on T cells transduced with nucleic acids comprising single expression cassettes for the knockdown of TCR-CD3 complex, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58KO), two different CAR cassettes (CAR1 and CAR2), Option 1, Option 2, CAR1 + Option 2, and CAR2 + Option 2.

[0160] FIG. 2A is a graphical representation of an exemplary nucleic acid without (top panel)or with (lower panel) shRNA (CD3zeta shRNA) embedded in the intron region of the EF- 1alpha promoter. The EF-1alpha promoter drives the expression of a control protein (BFP) and a TCR dominant negative mutant protein (CD3epsilon). The expression cassettes are separated by a 2A ribosomal skipping sequence. FIG. 2B shows a graph comparing the rate (%) of transgene (BFP) expression in combination with T cell receptor alpha chain (TRAC) knockout (TRAC KO), no TCR-CD3 complex knockout (no shRNA) and with shRNA (nucleic acids from FIG.1A) as measured by flow cytometry on T cells transduced with the nucleic acids. FIG. 2C shows a graph comparing the rate of TCR-CD3 complex expression in the cells of FIG.2B.

[0161] FIG. 3 is a graphical representation of an exemplary recombinant nucleic acid withshRNA targeting CD3zeta embedded in the intron region of the EF-1alpha promoter, a TCR- CD3 complex transgene, an RFX5 dominant negative mutant protein, and a CD58 PEBL protein. The positions of the TCR-CD3 complex transgene and the RFX5 dominant negative 26Mintz Ref. No.: 063384-521001WO mutant protein (RFX5DN) can be swapped. The expression cassettes are separated by 2A ribosomal skipping sequences.

[0162] FIG. 4A is a graphical representation of an exemplary recombinant nucleic acid withan MND promoter (upper panel, Tri allo MND), or an EF-1alpha promoter (lower panel, Tri allo EF-1alpha) encoding a TCR PEBL, an RFX5 DN, and a CD58 PEBL. FIG.4B shows a graph comparing the rate (%) of TCR-CD3 complex expression for the single expression cassettes for the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58 PEBL), original allo and the two nucleic acids shown in FIG. 4A. FIG. 4C shows a graph comparing the rate (%) of MHC (HLA-DR) expression and CD58 expression for the single expression cassettes for the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58 PEBL), original allo and the two vectors shown in FIG. 4A as measured by flow cytometry on T cells transduced with the nucleic acids. The expression cassettes are separated by 2A ribosome skipping sequences.

[0163] FIG. 5A is a graphical representation of an exemplary recombinant nucleic acid for theknockdown of TCR, MHC, and CD58 encoding an EF-1alpha promoter, an RFX5DN, and a tandem PEBL consisting of a TCR PEBL (scFv) / CD58 PEBL (scFv) fusion protein with an additional endoplasmic reticulum (ER) tag at the C-terminus. FIG. 5B shows a graph comparing the rate (%) of TCR-CD3 complex expression for the single expression cassettes for the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58PEBL), and the Tri allo EF-1alpha shown in FIG.4A, and the Tandem nucleic acid of FIG.5A as measured by flow cytometry on T cells transduced with the nucleic acids. FIG. 5C shows a graph comparing the rate (%) of MHC (HLA-DR) expression and CD58 expression for the single expression cassettes for the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58PEBL), Tri allo EF-1alpha shown in FIG.4A, and the Tandem nucleic acid of FIG.5A.

[0164] FIG. 6A is a graphical representation of an exemplary recombinant nucleic acid forthe knockdown of TCR, MHC, and CD58 encoding an MND promoter, an RFX5DN, and a tandem PEBL consisting of a TCR PEBL (scFv) / CD58 PEBL (scFv) fusion protein with an additional endoplasmic reticulum (ER) tag at the C-terminus. FIG. 6B shows a graph comparing the rate (%) of TCR-CD3 complex expression for the single expression cassettes for the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58 PEBL), and the Tandem nucleic acid of FIG.6A (MND-RFX5-biPEBL) as measured by flow cytometry on T cells transduced with the nucleic acids. FIG. 6C shows a graph comparing the rate (%) of MHC (HLA-DR) expression and CD58 expression for the single expression cassettes for 27Mintz Ref. No.: 063384-521001WO the knockdown of TCR, MHC, and CD58 (TCR PEBL, RFX5DN, and CD58PEBL), and the Tandem nucleic acid of FIG.6A.

[0165] FIG. 7A is a graphical representation of an exemplary design for a recombinant dualpromoter nucleic acid. An inducible promoter and an MND promoter can be separated by an insulator sequence and express two transgene cassettes in opposite directions. FIG. 7B is a graphical representation of an exemplary design for a recombinant dual promoter nucleic acid expressing a shRNA and a transgene. Transgene expression (for example BFP / 2A / CD3zUb) is driven by the MND promoter, and shRNA expression is driven by a second promoter in the opposite direction. The two promoters can be separated by an HS4 insulator sequence. FIG. 7C shows a graph comparing the rate (%) of TCR-CD3 complex expression for exemplary dual promoter expression cassettes combining a control transgene (BFP) and CD3zeta Ubiquitin ligase domain fusion (Ub) (targeting the TCR-CD3 complex expression) in combination with a CD3zeta shRNA driven by an EF-1alpha promoter, PGK promoter, SFFV promoter, or U6 promoter as measured by flow cytometry on T cells transduced with the nucleic acids.

[0166] FIG. 8 is a graphical representation of an exemplary design for a universal vectorconstruct with three Allo transgenes (Allo1, Allo2, and Allo3) driven by the MND promoter. An additional cassette of multiplexed shRNA with flanking EF-1alpha splice donor / acceptor sequences can be inserted in non-coding sequences before the start site, after the stop codon, or after the WPRE site.

[0167] FIG. 9 are graphical representations of exemplary designs for universal vectorconstructs with the expression of the encoded molecules driven by the human Ubiquitin C promoter (hUbC) in reverse direction.

[0168] FIG. 10 are graphical representations of exemplary designs for recombinant dualpromoter nucleic acids with tandem fusion proteins. The figure shows a nucleic acid construct with an shRNA (CD3zeta shRNA) driven by a core EF-1alpha promoter, a PGK promoter, an NFkB responsive promoter, or a U6 promoter (with mir22), and a bicistronic construct in the opposite direction driven by the MND promoter with a tandem TCR-PEBL CD58 PEBL fusion protein (upper panel) or a CD58 PEBL CD3epsilon DN fusion protein (lower panel).

[0169] FIG. 11A are graphical representations of exemplary designs for universal vectorconstructs driven by the MND promoter. Additionally, the design can include a CD3zeta targeting shRNA flanked by EF-1alpha splice donor and acceptor sequences that can be inserted before the start site, after the STOP codon, or after the WPRE sequence. FIG. 11B shows a graphical representation of an exemplary design for universal vector constructs driven 28Mintz Ref. No.: 063384-521001WO by the MND promoter with humanized tandem PEBL targeting the TCR-CD3 complex and CD58 (TCR scFv, CD58 scFv and one ER tag).

[0170] FIG. 12 shows flow cytometry blots depicting CD58 expression on T cells aftertransduction with CD58 shRNA, CD58PEBL, or UL148.

[0171] FIG. 13 are graphical representations of exemplary designs for universal vectorconstructs that include a viral protein (UL148) targeting CD58 expression.

[0172] FIG. 14A are graphical representations of exemplary designs for universal vectorconstructs with tandem fusion proteins (CD58 scFv CD3epsilon DN fusion) with an ER tag at the C-terminus. The lower panel shows an exemplary dual knockdown approach for the TCR- CD3. The nucleic acid includes a CD3epsilon DN and an additional TCR PEBL or a CD3zeta ubiquitin fusion for dual TCR-CD3 complex knockdown. FIG.14B shows a graph comparing the frequency (%) of TCR-CD3 complex expression and CD58 expression for the CD3epsilon DN alone or the CD58scFv-CD3epsilon DN) on T cells as measured by flow cytometry.

[0173] FIG. 15A are graphical representations of exemplary designs for universal vectorconstructs that include CD58 scFv operably linked to CD3epsilon DN with varying lengths of the CD3epsilon ectodomain and an additional reporter transgene (tBFP). FIG. 15B shows knockdown of expression of CD3 complex components, TCRalpha / beta complex, and CD58 with these fusion proteins.

[0174] FIG. 16A are graphical representations of exemplary designs for universal vectorconstructs with tandem fusion proteins (tandem TCR-CD58 scFv-CD3epsilon DN fusion) with full length CD3epsilon DN or a truncated CD3epsilon ecto domain and an additional reporter transgene (BFP). FIG. 16B shows representative flow cytometry plots of CD3 complex components, TCR-CD3 complex, and CD58 expression on transduced T cells (BFP+). Note that these constructs replace RFX5 DN with BFP in FIG 16A.

[0175] FIG. 17A is a graphical representation of an exemplary design for universal vectorallogeneic nucleic acids with and without intron embedded shRNA, including an MND promoter, RFX5 DN, 2A ribosome skipping sequence, an operably linked TCR scFv and CD58scFv with an ER tag. The intron embedded shRNA can be positioned before the start codon or after the stop codon. FIG. 17B shows knockdown of expression of TCR-CD3 complex components, HLA-A / B / C (MHC class I), HLA-DR (MHC class II) and CD58 with these universal vector constructs.

[0176] FIG. 18A is a graphical representation of exemplary designs for universal vectorconstructs with intron embedded single or multiplexed shRNA, including an MND promoter, 29Mintz Ref. No.: 063384-521001WO RFX5 DN, 2A ribosome skipping sequence, an operably linked TCR scFv and CD58scFv with an ER tag. Multiplexed shRNAs included B2M shRNA, CD3zeta shRNA, CIITA shRNA, and FasR shRNA. FIG.18B shows knockdown of expression of TCR-CD3 complex components, HLA-A / B / C (MHC class I), HLA-DR (MHC class II) and CD58, and FasR with these universal vector constructs.

[0177] FIG.19 (upper panel) shows a graphical representation of the inhibition between CD8on host CD8+ T cells and a CD8 blocked MHC class I on a graft cell. FIG.19 (lower panel) shows graft survival in hours in a co-culture assay with host T cells for graft T cells with (1) B2M KO, (2) RFX5 KO and CD58 KO, (3) RFX5 KO and CD58 KO and mutCD8alpha- G4S6linker- B2M (CD8 block 1), and (4) RFX5 KO and CD58 KO and RFdiff-G4S6linker- B2M (CD8 block 2). All graft T cells are TRAC KO.

[0178] FIG. 20 (upper panel) shows a graphical representation of the knockdown of MHCclass I expression by the viral protein US11. FIG.20 (lower panel) shows graft survival a co- culture assay with host T cells for graft T cells with (1) B2M KO, (2) RFX5 KO and CD58 KO, (3) RFX5 KO and CD58 KO and US11. All graft T cells are TRAC KO.

[0179] FIG. 21(upper panel) shows a graphical representation of the inhibition of GranzymeB by the granzyme B inhibitor SerpinB9. FIG.21 (lower panel) shows graft survival in days in a co-culture assay with host PBMC cells for graft T cells with (1) B2M KO, (2) RFX5 KO and CD58 KO, (3) RFX5 KO and CD58 KO and SerpinB9. All graft T cells are TRAC KO.

[0180] FIG. 22A is a graphical representation of an exemplary design for a universal vectorconstruct with 2 transgenes (RFX5 DN and tandem PEBL) and CD3z shRNA driven off different promoters. FIG.22B shows a graph comparing the level of expression (MFI) of TCR, MHC class I (HLA ABC), MHC class II (HLA DR), and CD58 relative to NTD for each of the different tested promoters. Comparable knockdowns are observed with MND, SFFV and gamma retroviral promoters. FIG.22C shows a graph comparing the relative levels of CD22 (m971) CAR expression on ALLO edited (TCR-) versus ALLO unedited (TCR+) in primary T cells co-transduced with 2 vectors bearing the same MND promoter at various tested MOI ratios. Comparable levels of CAR expression observed in the presence or absence of ALLO transduction demonstrates minimal promoter interference. FIG.22D shows TCR knockdown in CAR+ versus CAR- cells in primary T cells co-transduced with 2 vectors bearing the same MND promoter at various tested MOI ratios. Comparable levels of TCR knockdown observed in the presence or absence of CAR transduction demonstrate minimal promoter interference. 30Mintz Ref. No.: 063384-521001WO

[0181] FIG. 23 shows a graph comparing the level of expression (MFI) of TCR-CD3, MHCclass I, MHC class II and CD58 relative to NTD for each of the tested universal constructs. pSync4750, pSync5121, pSync5128 are constructs that differ in the anti-CD58 and anti-TCR scFv sequences.

[0182] FIGs. 24A show universal vector construct schemas highlighting vector changes toincorporate CD8 inhibitor while maintaining expression of MHC class I and class II, CD58, and TCR-CD3 knockdown components. FIG. 24B shows graphs comparing the level of expression (MFI) of TCR-CD3, MHC class I, MHC class II, and CD58 relative to NTD for each of the tested universal constructs.

[0183] FIG. 25 shows the impact of changing the linker in the CD8 inhibitor from a (G4S)6to a whitlow linker. An extended whitlow linker (with flanking G4S) exhibited the greatest level of whitlow staining in the N-terminal configuration (MHC class I binder – linker – B2M). The bottom figure demonstrates comparable knockdown with either the (G4S)6 linker or the G4S-whitlow-G4S linker. Plotted are TCR-CD3, MHC class I, MHC class II, and CD58 expression (%MFI relative to NTD control) of T cells transduced with vector.

[0184] FIG. 26 shows survival of universal vector transduced T cells in the presence ofallogeneic NK cells. Cells were co-incubated at an E:T ratio of 1:1 for 24 h and analyzed by flow cytometry. Plotted is Mean + / - SD of technical triplicates for 10 NK donors. % graft survival is calculated as %7AAD- (graft cells with allo NK) / %7AAD- (graft only) x 100%.

[0185] FIG.27 shows survival of universal vector transduced T cells in the presence of primedallogeneic host T cells. Cells were co-incubated for 48 h at a 1:1 E:T ratio and analyzed by flow cytometry. Plotted is mean ± SD of technical replicates for 3 HLA-mismatched donors. % Graft survival is calculated as absolute even counts of vector transduced T cells and normalizing in the absence of effector cells.

[0186] FIG. 28 shows the percentage of proliferative host CD8+ T cells in response touniversal vector transduced T cells. Host pan T cells were stained with CellTrace™ Far Red (Life Technologies) and mixed with universal vector transduced T cells at a 1:1 E:T ratio for 7 days in R10 supplemented with 20 IU / mL IL-2. Expression of the CD8 inhibitor attenuated alloreactive CD8+ T proliferation.

[0187] FIG. 29 shows the percentage of TCRαβ negative (%TCR-) cells after TCRαβdepletion. High purity of cells with TCR knockdown can be achieved after magnetic sorting.

[0188] FIG. 30 shows the lack of activation marker expression (CD25, CD69, 4-1BB) onuniversal vector transduced T cells after stimulation with TransACT™ for 72 h. 31Mintz Ref. No.: 063384-521001WO

[0189] FIG. 31 shows the stability of TCR expression of universal vector transduced T cellsafter purification (TCRαβ negative selection). TCR-CD3 expression was measured by MFI relative to control (NTD) or TRAC KO by flow cytometry. T cells were maintained in culture with R10 and 100 IU / mL IL-2.

[0190] FIG. 32 shows the transduction efficiency of a tricistronic, trispecific CAR (CRG-023,pSync3811) with the universal allogeneic vector (pSync5126). Both vectors were delivered simultaneously via co-transduction at varying MOIs. Universal allogeneic vector (ALLO Vector) transduction efficiency was determined by %TCR- and CAR transduction efficiency was determined by anti-Id staining against m971.

[0191] FIG. 33 shows similar long-term killing activity of a CD22 / CD19 / CD20 tricistronicCAR (pSync3811) with or without co-transduction of a universal allogeneic vector (pSync5126). CAR T cells were co-cultured with GFP-expressing Raji cells at a 1:6 E:T ratio and killing was assessed continuously using an Incucyte® S3. Vertical dashed lines represent addition of fresh tumor cells. The horizontal dashed line represents the object count limit of instrument. Mean ± SD of technical triplicate shown.

[0192] FIG. 34A shows the difference in CAR and universal allogeneic vector transductionefficiency between co-transduction and serial transduction. For serial transduction, CAR transduction was performed first followed by addition of the universal allogeneic vector (pSync5126) 24 h later. FIG.34B shows the expression of the individual CARs (upper panel) (CD19 CAR, CD20 CAR, and CD22 CAR) after with and without universal allogeneic vector after co-transduction and serial transduction. The lower panel shows MHC class I, MHC class II, and CD58 expression (%MFI relative to CRG-023). FIG. 34C shows the percent (%) of Tscm, Temra, Tcm, and Tem CD8+ CAR T cells and percent (%) of Tscm, Temra, Tcm, and Tem CD4+ CAR T cells after transduction with CD22 / CD19 / CD20 tricistronic CAR (pSync3811) only or CAR with universal allogeneic vector (pSync5126). Cells were co-transduced or serially transduced as indicated.

[0193] FIG.35A shows a table for a GvHD clinical scoring system used in the Example. FIG.35B shows lack of acute or late-onset GvHD in NSG mice injected with 30 universal allogeneic vector engineered human T cells (pSync4750, pSync5126, pSync4952), unlike untransduced control (UTD) that are TCR-CD3+. Plotted is mean ± SEM (n=5 per group). Top figure shows change in body weight relative to day 0. Middle plot shows GvHD score based on (Naserian et al 2018. Front Immunol). Bottom plot shows Kaplan-Meier survival curves. 32Mintz Ref. No.: 063384-521001WO

[0194] FIG.36 shows expression of CD34 after transduction of the listed constructs in primaryhuman T cells. pSync5161 and pSync5163 include truncated versions of CD34 ectodomain that had the highest degree of CD34 expression. CD34 expression was highest for pSync5161. The bottom plot shows the %CD3- after TCRalpha / beta negative selection or CD34 positive selection. The highest degree of TCR-CD3- purity was generally achieved using TCRalpha / beta negative selection. pSync5163, which has 3 minimal tandem repeats of the QBEND / 10 epitope, was the construct that achieved the highest %CD3- purity by positive selection.

[0195] FIG. 37 shows expression of TCRalpha / beta, MHC class II (HLA-DRDPDQ), andCD58 normalized to NTD control of primary human T cells transduced with universal vectors and purified either by TCRalpha / beta negative selection or combined TCRalpha / beta+HLA- DR negative selection. Combined TCRalpha / beta+HLA-DR negative selection maintains high TCR-CD3- purity and improves MHC class II knockdown in final post-selected cell population.

[0196] FIG. 38A shows the transduction efficiency of a tricistronic, trispecific CARdesignated pSync3811 (targeting CD19, CD20, and CD22) with the universal allogeneic vector (pSync5482). Both vectors were delivered simultaneously via co-transduction 24 hours after activation with TransActTM. (Left) shows % of CAR+ cells as determined by flow cytometry and (Right) shows %TCR- cells prior to TCR depletion. Universal vector transduction efficiency was determined by % percent TCR-CD3 expression, and CAR transduction efficiency was determined by anti-Idiotype (“anti-Id”) staining against m971, which detects the CD22 CAR component of CRG-023. Plotted is the mean ± SEM of two donors.

[0197] FIG. 38B shows the in vitro cytolytic activity of the autologous CRG-023 transducedT cells versus the allogeneic CRG-023 T cells. CAR T cells were co-incubated with GFP- expressing Nalm6 tumor cells at various E:T ratios for 48 hours. %Killing was determined by number of Green Objects relative to the initial time point (0 hour). Plotted is the mean of technical duplicates.

[0198] FIG. 38C shows the IL-2 production of the CRG-023 cells with and without dualtransduction of the universal allo vector (pSync5482). CAR T cells were co-cultured with Nalm6 tumor cells at an E:T ratio of 1:4 for 24 hours and supernatants were collected for ELISA analysis. Plotted is the mean ± SEM of two donors.

[0199] FIG. 39A shows the transduction efficiency of an FMC63 scFv-bearing CD19 CARwith or without co-transduction of the universal allo vector (pSync5482). Both vectors were 33Mintz Ref. No.: 063384-521001WO delivered simultaneously via co-transduction 24 hours after activation with TransActTM. (Left) shows the %CAR+ frequency as determined by flow cytometry and (Right) shows the %TCR- cells prior to TCR depletion. Universal vector transduction efficiency was determined by % percent TCR-CD3 expression, and CAR transduction efficiency was determined by anti-Id staining against FMC63. Plotted is the mean ± SEM of two donors.

[0200] FIG.39B shows the in vitro cytolytic activity of the autologous CAR-019 T cells versusthe allogeneic CAR-019 T cells. CAR T cells were co-incubated with GFP-expressing Nalm6 tumor cells at various E:T ratios for 48 hours. %Killing was determined by number of Green Objects relative to the initial time point (0 hour). Plotted is the mean of technical duplicates.

[0201] FIG. 39C shows the IL-2 production of the CAR-019 cells with and without dualtransduction of the universal allo vector (pSync5482). CAR T cells were co-cultured with Nalm6 tumor cells at an E:T ratio of 1:4 for 24 hours and supernatants are collected for ELISA analysis. Plotted is the mean ± SEM of two donors.

[0202] FIG.40A shows the transduction efficiency of BCMA CAR T cells with or without co-transduction of the universal allo vector (pSync5482). Both vectors were delivered simultaneously via co-transduction 24 hours after activation with TransActTM. (Left) shows the %CAR+ frequency as determined by flow cytometry and (Right) shows the %TCR- cells prior to TCR depletion. Universal vector transduction efficiency was determined by % percent TCR- CD3 expression, and CAR transduction efficiency was determined by staining with soluble BCMA protein. Plotted is the mean ± SEM of two donors.

[0203] FIG. 40B shows the in vitro cytolytic activity of the autologous CAR-BCMA CAR Tcells versus the allogeneic CAR-BCMA T cells. CAR T cells were co-incubated with GFP- expressing tumor lines with varying levels of BCMA surface expression: MM.1S (medium), H929 (high), U266B1 (low). %Killing was determined by number of Green Objects after 48 hours relative to the initial time point (0 hour). Plotted is the mean of technical duplicates.

[0204] FIG. 40C shows the IL-2 production of the CAR-BCMA cells with and without dualtransduction of the universal allo vector (pSync5482). CAR T cells were co-cultured with BCMA-expressing tumor cells at an E:T ratio of 1:4 for 24 hours and supernatants are collected for ELISA analysis. Plotted is the mean ± SEM of two donors.

[0205] FIG. 41A shows the knockdown of TCR expression as determined percentage of TCRMFI for each construct transduced relative to an untransduced control (NTD). A bicistronic iteration (5126) was tested alongside a number of tricistronic universal vectors. Different linkers were also tested tethering the CD8i (CD8 inhibitor) to β2M in the tricistronic constructs: 34Mintz Ref. No.: 063384-521001WO (G4S)6and a G4S-Whitlow-G4S linker (G4S)6Tricis vs. Whitlow Tricis), however given the similar size of the 2 linkers not much effect on TCR knockdown was observed. The final 8 constructs all harbored the Whitlow linker and tested different multiplexed shRNA cassettes as compared to 5308 / 5309 / 5500 / 5501 which all harbored CD3z and CD3e targeting shRNAs.

[0206] FIG. 41B shows the knockdown of additional markers (MHC-I, MHC-II, and CD58)as a percentage of MFI relative to an untransduced control (NTD).

[0207] FIG. 41C shows the knockdown of FasR (CD95) expression as a percentage of MFIrelative to an untransduced control (NTD). All constructs shown harbor a multiplex shRNA cassette with shRNAs targeting CD3z and CD3e while constructs 5428 / 5429 harbor an additional FasR targeting shRNA in this multiplexed cassette.

[0208] FIG. 41D shows the knockdown of TCR expression as determined by percentage ofTCR MFI for each construct transduced relative to a nontransduced control (NTD). A bicistronic iteration (5605) was tested alongside a number of tricistronic universal allo vectors. On the right shows relative expression of MHC-I, MHC-II and CD58 of post-TCR depleted T cells transduced with the universal allo vectors relative to NTD.

[0209] FIG. 42A shows graft survival in a natural killer cell (NK) mixed lymphocyte reaction(MLR) of different modified T cell populations against 3 different NK donors. Percent survival was calculated by T cell numbers in the presence of each NK donor at a 1:1 E:T ratio relative to T cells alone. The dotted line is determined by the mean survival of cells having the T cell Receptor alpha chain (TRAC) knocked out (the gold standard positive control TRAC KO) which has strong survival in the presence of allogenic NK cells due to wildtype MHC (i.e., B2M) expression. In contrast, obliterating both TRAC and MHC-I expression (by β2M KO) leads to strong rejection of T cell grafts (TRAC + B2M KO).

[0210] FIG. 42B shows graft survival in a T cell MLR of different modified graft T cellpopulations against 3 alloreactive T cell donors mismatched at HLA-A2 and other HLA loci. Percent survival was calculated by graft T cell numbers in the presence of each mismatched host T cell donor at a 1:1 E:T ratio relative to the graft T cells alone. The dotted line is determined by the mean survival of the gold standard positive control TRAC+B2M KO which has strong survival in the presence of alloreactive host T cells due to a lack of MHC-I expression.

[0211] FIG. 43A shows TCR expression over time after end-of-process. TCR expression wasdetermined by measuring MFI by flow cytometry relative to NTD control weekly after TCR depletion. 35Mintz Ref. No.: 063384-521001WO

[0212] FIG. 43B shows the residual level of TCR activity of allo vector transduced T cells.Transduced T cells were TCR depleted and maintained in culture for 4 weeks. They were then stimulated with TransActTMfor 72 hours and activation markers (CD25, CD69, CD137) were measured by flow cytometry.

[0213] FIG. 44 shows the proliferative activity of T cells transduced with the universal allovector (pSync5481, pSync5482, pSync5605) against HLA-mismatched allogeneic donor cells.

[0214] FIGs.45A-45C show the in vivo GvHD activity of human graft T cells in an NSG acutexenograft GvHD mouse model.30^106T cells were tail I.V. injected into 8-12 week-old NSG mice that were 1 Gy total body irradiated one day prior. Body weight change, survival, and GvHD score (based on Figure 35A scoring) was tracked. Plotted is mean ± SEM of 5 mice per group.

[0215] FIG. 46A shows the proliferative response of allogeneic host CD8+ (Left) and CD4+(Right) T cells when co-cultured with graft T cells.

[0216] FIG.46B shows the persistence of graft T cells in a primed mixed lymphocyte reactionwith allogeneic T cells.

[0217] FIG. 47A shows the persistence of graft T cells in a mixed lymphocyte reaction withallogeneic NK cells.

[0218] FIG. 47B highlights the diversity of allogeneic NK donors used in the NK MLR (A).The frequency of the activating and inhibitory receptors was measured on the allogeneic NK donors via flow cytometry.

[0219] FIG.48A shows the long-term persistence of graft T cells edited with the universal allovector (pSync5605, pSync5482) in a PBMC mixed lymphocyte reaction with HLA- mismatched host PBMCs.

[0220] FIG. 48B highlights the advantage of universal vector approach compared tobenchmark immune cloaking approaches (B2M KO + HLA-E overexpression; B2M+CIITA KO + CD47 overexpression). DETAILED DESCRIPTION OF THE DISCLOSURE

[0221] The present disclosure relates generally to, among other things, methods for makingand compositions comprising engineered immune cells that can be used in allogeneic immune cell therapies for the treatment of various health conditions, for example cancer and autoimmune diseases. 36Mintz Ref. No.: 063384-521001WO

[0222] With the advent of so-called “second generation” chimeric antigen receptor T cells(“2nd generation CAR T cells”) targeting CD19 for the treatment of hematologic malignancies (R.J. Brentjens et al., “Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15”, Nat. Med. 9:279-286 (2003); M. Kalos et al., “T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia”, Sci. Transl. Med.3(95):95ra73 (2011)), a new field of cancer immunotherapy was born. These autologous (patient-derived) 2nd generation CAR T cells were derived from leukapheresis product obtained from the patient being treated, engineered ex vivo to include a synthetic chimeric receptor comprising (1) an antigen-specific binder (commonly a single chain variable fragment, or “scFv”), (2) a hinge and / or transmembrane domain, and (3) an intracellular domain adding a costimulatory signaling domain to the CD3ζ activating domain used in “first generation” CAR T cells, and reinfused into the same patient.

[0223] While in some cases the impact of these therapies has been transformative (see, e.g.,“Emily Whitehead, First Pediatric Patient to Receive CAR T-Cell Therapy, Celebrates Cure 10 Years Later”, available at https: / / www.chop.edu / news / emily-whitehead-first-pediatric-patient- receive-car-t-cell-therapy-celebrates-cure-10-years (last accessed December 12, 2024)), in many others the limitations of autologous cell therapy have become increasingly clear. Those limitations include, for example, on-target off-tumor activity resulting from targeting tumor antigens that are also expressed on healthy tissue, disease recurrence because of antigen escape, limited persistence or efficacy resulting from loss of co-stimulatory signaling, lack of efficacy against solid tumors, poor trafficking and tumor infiltration, and the inability to overcome an immunosuppressive tumor microenvironment. See, e.g., X. Xu et al., “Mechanisms of relapse after CD19 CAR T-cell therapy for acute lymphoblastic leukemia and its prevention and treatment strategies”, Front. Immunol.10:2664 (2019); and N.N. Shah et al., “Mechanisms of resistance to CAR T cell therapy”, Nat. Rev. Clin. Oncol. 16:372-85 (2019). In addition, autologous CAR T cell therapies are an extremely expensive, bespoke therapy manufactured by a lengthy, complex process that frequently fails to produce sufficient drug product. The manufacturing process is subject to regular failures because the patient T cells used as the starting material for manufacturing are often of poor quality, comprising a large proportion of exhausted T cells that correlate with poor patient outcomes.

[0224] Some of the limitations of autologous CAR T cell therapies have been successfullyaddressed by additional T cell engineering, including, for example, the use of more 37Mintz Ref. No.: 063384-521001WO sophisticated targeting strategies to eliminate on-target, off-tumor toxicities, alternative co- stimulatory signaling domains to improve CAR T cell persistence (e.g., replacing the CD28 costimulatory signaling domain with one derived from 4-1BB, or by targeting multiple antigens to avoid relapse by antigen escape. Others, such as manufacturing failures because of poor quality starting material, have proven more challenging to resolve.

[0225] One art-recognized strategy for improving the quality of the manufacturing startingmaterial is to use T cells derived from a healthy donor to manufacture so-called “allogeneic” (donor-derived) CAR T cells. The high-quality starting material available from a healthy donor theoretically would enable the production of multiple doses of a CAR T cell therapy of consistently high quality from a single leukapheresis sample. The two biggest risks associated with allogeneic CAR T cell therapies are immune rejection of the cell therapy drug product (also known as “Host versus Graft” rejection or “HvG”) and Graft versus Host Disease (“GvHD”) a safety issue where the drug product attacks host tissues. In HvG, the CAR T cell recipient’s (the “host”) immune system recognizes the allogeneic CAR T cell infusion (the “graft”) as “non-self” tissue and mounts an immune response against it culminating in CD8+ T cell-mediated killing and rapid elimination of the drug product. In GvHD, the infused allogeneic CAR T cells (the “graft”) recognize the patient’s (the “host”) healthy cells (other than the CAR antigen-expressing tumor cells) as “non-self” via their endogenous TCR-CD3 and mount an attack against the patient’s cells. This happens because, while CAR T cells have added antigen specificity via the antigen-binding domain of the CAR, they also retain the native antigen specificity associated with their endogenous T cell receptor / CD3 complex (TCR-CD3). This means that CAR T cells can attack both the tumor antigen targeted by the CAR and the antigen targeted by their endogenous TCR-CD3; it is the latter antigen specificity that is the main cause of GvHD in cell therapy.

[0226] To mitigate the risk of both HvG and GvHD, an allogeneic CAR T cell drug productmust be further engineered to minimize or prevent TCR-CD3 activation on the drug product. This complex cell engineering problem was recognized in the cell therapy field at least as early as 2015. See, e.g., Y. Yang et al., “Challenges and Opportunities of Allogeneic Donor-Derived CAR T cells”, Curr. Opin. Hematol. 22(6):509-515 (November 2015)(“To develop donor derived T cells without the effects of TCR-CD3 activation, CAR T cells with CAR-only specificity have been generated by selectively deleting the endogenous TCR-CD3. Using the same techniques, MHC class I could be deleted on donor-derived, off-the-shelf T cells to avoid rejection of transferred cells[]”). One approach to reduce or eliminate the likelihood of TCR- 38Mintz Ref. No.: 063384-521001WO CD3 activation on donor-derived allogeneic T cells is selective deletion of the endogenous TCR-CD3 using any of several genome editing tools including, for example, Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 and related reagents, ARCUS, or base editing. The absence of an endogenous TCR-CD3 reduces or eliminates the possibility of GvHD and the potential distraction of TCR -CD3 signaling from the CAR T cell infusion. Deletion of MHC class I (e.g., β2-microglobulin, B2M) with the same gene editing tools would reduce or eliminate host rejection of transferred CAR T cells (HvG), but in either case significant potential issues remain to be investigated, including: (1) the risk and potential impact of off-target gene edits in the T cell genome; (2) complete absence of MHC class I may elicit a natural killer (“NK”) cell response against the allogeneic T cells; and (3) the functionality of a CAR containing a CD3ζ domain has been shown to be dependent on its ability to dimerize with the endogenous TCR-CD3 in order to activate downstream pathways through interaction with the endogenous TCR-CD3 complex. To date, the vast majority of efforts to address these issues have used risky and clinically unproven gene editing methods to engineer such allogeneic cells.

[0227] Since the initial identification of this problem to be solved, at least thirty-three clinicaltrials testing investigational allogeneic CAR T cells in human cancer patients have been initiated and countless more are in development at the pre-clinical stage, but as of this writing not a single allogeneic CAR T cell drug product has been approved for marketing by the United States Food and Drug Administration (US FDA). See, e.g., C. Lonez and E. Breman, “Allogeneic CAR-T Therapy Technologies: Has the Promise Been Met?”, Cells 13:146-170 (January 2024) (see, e.g., “Table 2. Engineered allogeneic ‘off-the-shelf’ CAR-Ts with published clinical experience”).

[0228] The universal allogeneic vector constructs and compositions provided herein enabledurable, specific reduction of TCR-CD3 expression, MHC class I and MHC class II expression, and expression of related stimulatory and / or co-stimulatory signaling pathways associated with T cell activation and expansion without (1) relying in high risk and clinically unproven gene editing technologies or (2) altering the expression or functional activity of the CAR(s) co- expressed with such constructs and compositions, thereby solving the long-felt art-recognized need for methods of manufacturing allogeneic CAR T cells.

[0229] In particular, described herein are recombinant nucleic acids encoding molecules forreducing TCR-CD3 complex expression, MHC expression, activation of CD8+ T cells, and CD58 expression on the surface of immune cells or populations of immune cells. The 39Mintz Ref. No.: 063384-521001WO recombinant nucleic acids are engineered such that the nucleic acid sequences encoding the molecules are present on the same nucleic acid. The nucleic acids of the disclosure can be engineered and delivered efficiently into immune cells, for example T cells, via a single lentiviral or other virus-derived expression vector. Also provided are methods for generating engineered such immune cells or populations of immune cells with reduced TCR-CD3 complex expression, MHC expression, and CD58 expression on the cell surface; engineered immune cells or populations of immune cells according to the presently described methods, pharmaceutical compositions including the same, as well as methods and kits for the treatment of a health condition in subjects in need thereof.

[0230] As discussed above, the recombinant nucleic acids and engineered immune cells hereinaddress the problems with allogeneic adoptive cell therapies such as treatment with allogeneic T cells in a non-immune matched subject, that can result in Graft versus Host Disease (GvHD) and reduced persistence and efficacy of the adoptive immune cells compared to autologous adoptive cell therapies in the subject.

[0231] The section headings used herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

[0232] Although various features of the disclosure can be described in the context of a singleembodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0233] It is intended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0234] All patent filings, websites, other publications, accession numbers and the like citedabove or below are incorporated by reference in their entirety for all purposes to the same extent 40Mintz Ref. No.: 063384-521001WO as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. DEFINITIONS

[0235] Unless otherwise defined, all terms of art, notations, and other scientific terms orterminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0236] The singular form “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising 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”.

[0237] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeablyherein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule.

[0238] The term “recombinant” or “engineered” nucleic acid molecule, polypeptide, or cell asused herein, refers to a nucleic acid molecule, polypeptide, or cell that has been altered through human intervention.

[0239] The term “operably linked”, as used herein, denotes a physical or functional linkagebetween two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which 41Mintz Ref. No.: 063384-521001WO permits them to operate in their intended fashion. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is a functional link that allows for expression of the polynucleotide of interest. It should be understood that operably linked elements may be contiguous or non-contiguous. In the context of a polypeptide, “operably linked” refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide. In the present disclosure, various domains of the recombinant polypeptides of the disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the recombinant polypeptides in the cell. Operably linked domains of the recombinant polypeptides of the disclosure may be contiguous or non- contiguous (e.g., linked to one another through a heterologous peptide linker).

[0240] The term “percent identity,” as used herein in the context of two or more nucleic acidsor proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the Basic Local Alignment Search Tool (“BLAST”) or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. This definition also refers to, or may be applied to, the complement of a nucleic acid sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis.53705), with the default parameters thereof.

[0241] As used herein, a “subject” or an “individual” includes animals, such as human (e.g.,human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., cancer) and / or one or more symptoms of the health condition. The subject can also be an 42Mintz Ref. No.: 063384-521001WO individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later.

[0242] It is understood that aspects and embodiments of the disclosure described herein include“comprising,” “consisting,” and “consisting essentially of” aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0243] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify aclaim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0244] The terms “dominant-negative mutant proteins” or “dominant-negative mutant proteinsubunits” refers to a type of mutant protein or protein subunit that results in the production of a protein or protein subunit that interferes with the normal function of the wild-type protein or protein subunit. Examples include truncations, deletions, insertions, or substitutions of amino acids in the mutant protein.

[0245] It is appreciated that certain features of the disclosure, which are, for clarity, describedin the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, 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 pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present 43Mintz Ref. No.: 063384-521001WO disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. COMPOSITIONS OF THEDISCLOSURE

[0246] As described in greater detail below, one aspect of the present disclosure relates torecombinant nucleic acids including, but not limited to, multiple nucleotide sequences encoding, for example, short hairpin RNAs (“shRNAs”), antibodies or antibody fragments capable of binding certain proteins of interest that can include organelle localization or retention peptide sequences or tags (e.g., an endoplasmic reticulum, or “ER”, localization sequence or tag), synthetic and / or viral polypeptides that block or disrupt expression and / or activity of native proteins such as CD8 and / or components of the TCR-CD3 complex, and / or dominant negative mutant proteins or protein subunits for reducing the expression of TCR- CD3 complex, MHC class I and MHC class II, and / or CD58 on the surface of an immune cell. Also provided herein are virus-derived (i.e., viral) expression vectors, engineered immune cells or populations of engineered immune cells that have been modified to include such recombinant nucleic acids as disclosed herein. Also provided herein are methods of making and using the engineered immune cells. I. Recombinant Nucleic Acids

[0247] As described in greater detail below, one aspect of the present disclosure relates torecombinant nucleic acid molecules comprising, but not limited to, sequences encoding multiple molecules engineered to reduce TCR-CD3 complex expression, MHC class I and MHC class II expression, CD58 expression on the surface of engineered immune cells from a single nucleic acid, and encoding synthetic and / or viral polypeptides that block or disrupt expression and / or activity of native proteins such as CD8 and / or components of the TCR-CD3 complex. As described in greater detail below, reduction of TCR-CD3 complex expression, MHC class I and MHC class II expression, CD58 expression, disruption of expression and / or activity of native proteins such as CD8 and / or components of the TCR-CD3 complex by expression of synthetic and / or viral polypeptides in engrafted engineered immune cells can reduce incidence of Graft versus Host Disease in human subjects infused with such engineered cells and improve survival of the engineered immune cells in human subjects. As described below, the multicistronic design of the recombinant nucleic acid molecules of the disclosure is advantageous over alternative designs of such recombinant nucleic acids that further include an additional transgene, for example a chimeric antigen receptor (CAR) expression cassette. 44Mintz Ref. No.: 063384-521001WO As shown in the Examples, exemplary recombinant nucleic acids of the disclosure showed improved knockdown of the TCR-CD3 complex, MHC class I and MHC class II, CD58, synthetic and / or viral polypeptides that block or disrupt expression and / or activity of native proteins such as CD8 and / or components of the TCR-CD3 complex compared to a recombinant nucleic acid additionally including a CAR expression cassette (FIGs.1C-1F).

[0248] One aspect of the present disclosure relates to recombinant nucleic acids that include,but are not limited to (1) a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; (2) a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and MHC class II expression on the surface of the immune cell; and (3) a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell. Exemplary molecules that are capable of reducing polypeptide expression on the surface of an immune cell include, but are not limited to, protein expression blockers (PEBL), shRNAs, and dominant negative mutant proteins or protein subunits.

[0249] Another aspect of the present disclosure relates to recombinant nucleic acids thatinclude, but are not limited to (1) a first nucleotide sequence encoding a first molecule capable of reducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; and (2) a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell. In some embodiments, the recombinant nucleic acid is bicistronic.

[0250] Accordingly, some embodiments of the disclosure include, but are not limited to,recombinant nucleic acid sequences encoding a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein or protein subunit that is capable of reducing polypeptide expression in an immune cell, for example of TCR-CD3 complex, MHC class I and MHC class II, and / or CD58.

[0251] In some embodiments, the recombinant nucleic acid further includes, but is not limitedto, at least one or more of an exogenous promoter, a post-transcriptional response element (PRE), and a polyadenylation signal sequence.

[0252] In some embodiments, the recombinant nucleic acid further includes, but is not limitedto, a ribosome skipping sequence disposed between each cistron. In some embodiments, the multicistronic configuration can be tricistronic and a ribosome skipping sequence can be disposed between the first cistron and the second cistron and between the second cistron and the third cistron. In some embodiments, the multicistronic configuration can be bicistronic and 45Mintz Ref. No.: 063384-521001WO a ribosome skipping sequence can be disposed between the first cistron and the second cistron. In some embodiments, the ribosome skipping sequence disposed between the first cistron and the second cistron is different from the ribosome skipping sequence disposed between the second cistron and the third cistron. In some embodiments, the ribosome skipping sequence disposed between the first cistron and the second cistron is the same as the ribosome skipping sequence disposed between the second cistron and the third cistron. A. Reduction of TCR-CD3 Complex Expression on the cell surface

[0253] As described in greater detail below, one aspect of the present disclosure relates torecombinant nucleic acids encoding molecules capable of reducing TCR-CD3 complex expression on the cell surface. Non-limiting exemplary molecules capable of reducing TCR- CD3 complex expression on the cell surface include PEBLs, shRNAs, dominant negative mutant proteins or protein subunits targeting surface expression of TCR-CD3 complex components, and combinations thereof. 1. TCR-CD3 complex

[0254] T cells are characterized by, among other things, expression of the T cell receptorCD3complex (the “TCR-CD3 complex”) on their surface. TCR-CD3 complexes are multisubunit protein complexes that can recognize and bind a cognate Major Histocompatibility Complex (MHC) Class I or Class II presenting an antigenic peptide. Upon binding of the TCR-CD3 complex to a cognate MHC / antigenic peptide pair (coupled with the binding of one or more additional co-stimulatory receptors by their cognate ligands), the TCR-CD3 complex transduces a signal to activate or inhibit the T cell. TCR-CD3 complexes include a TCR dimer in combination with a CD3 protein complex including three dimeric subunits. The TCR dimer includes either a TCR^ subunit and a TCR^ subunit or a TCR^ subunit and a TCR^ subunit. T cells bearing an alpha / beta (^ / ^) TCR heterodimer are referred to as “^ / ^ T cells” or “T^ / ^”. T cells bearing a gamma / delta (^ / ^) TCR heterodimer are referred to as “^ / ^ T cells” or “T^ / ^”. The CD3 protein complex includes homo- and hetero-dimers including different combinations of four protein subunits: a CD3delta (^ or d) subunit, a CD3epsilon (^ or e) subunit, a CD3gamma (^ or g) subunit, and a CD3zeta (^ or z) subunit. The wild-type CD3 complex includes one homodimer of CD3^ / CD3z (“CD3^ / ^” or “CD3z / z”) and two heterodimers, one including a CD3^ / CD3d subunit and a CD3^ / CD3e subunit (“CD3^ / ^” or “CD3d / e”) and one including a CD3^ / CD3g subunit and a CD3^ / CD3e subunit (“CD3^ / ^” or “CD3g / e”). The 46Mintz Ref. No.: 063384-521001WO TCR-CD3 complex is assembled in the endoplasmic reticulum (ER) and all components are required for its cell surface expression.

[0255] Graft versus host disease (GvHD) is a T cell-mediated condition resulting from theinteraction between infused donor T cells and cells of the patient receiving the infusion that can develop in a patient after allogeneic hematopoietic cell transplantation (HCT) or treatment with an infusion of allogeneic CAR T cells. In GvHD, an infused donor T cell (i.e., a donor CAR T cell) can recognize the host cells as foreign and direct an immune response against the host cells. The T cell reaction is mainly driven by the interaction of the TCR-CD3 complex on the grafted T cells and the MHC class I and MHC class II on the host cells.

[0256] To reduce the occurrence of GvHD in patients with allogeneic T cell grafts, the donorT cells can be engineered to reduce TCR-CD3 complex expression on the cell surface. TCR- CD3 complex expression on an immune cell such as a T cell can be reduced, for example, by reducing the expression of one or more components of the TCR-CD3 complex in a donor immune cell. In some embodiments, the TCR-CD3 complex component that has reduced expression on the surface of an engineered immune cell compared to a non-engineered immune cell comprises one or more of a TCR^ / TCRalpha / TCRa, TCR^ / TCRbeta / TCRb, TCR^ / TCRgamma / TCRg, TCR^ / TCRdelta / TCRd, CD3^ / CD3gamma / CD3g, CD3^ / CD3delta / CD3d, CD3^ / CD3epsilon / CD3e, or CD3^ / CD3zeta / CD3z protein subunit. 2. Protein Expression Blockers (PEBLs)

[0257] As described in greater detail below, one aspect of the present disclosure relates to oneor more recombinant nucleic acids that encode, but are not limited to, nucleic acid sequences encoding molecules that are capable of reducing surface expression of the TCR-CD3 complex in immune cells.

[0258] In some embodiments, the recombinant nucleic acids provided herein comprise anucleic acid sequence encodes a protein expression blocker (PEBL) capable of reducing TCR- CD3 complex expression on the surface of an immune cell.

[0259] Protein expression blockers (PEBLs) are proteins that prevent transport of targetedproteins to the cell membrane. A PEBL can include a protein-binding domain operably linked to a protein localization sequence or tag. When expressed in a cell, the protein-binding domain of the PEBL binds to its target protein (e.g., a subunit of the TCR-CD3 complex) and is sequestered in the compartment specified by the protein localization sequence or tag operably linked to the protein-binding domain. In one non-limiting exemplary embodiment, a PEBL 47Mintz Ref. No.: 063384-521001WO comprising an anti-CD3epsilon scFv operably linked to an ER localization tag comprising the amino acid sequence of SEQ ID NO: 11 would bind to CD3epsilon and the CD3epsilon / scFv complex would be sequestered in the endoplasmic reticulum. Non-limiting exemplary protein- binding domains include antibody single chain variable fragments (scFvs), an antigen binding fragment of heavy chain only antibodies (“nanobodies” or “VHHs”), and / or a variable heavy chain domain (VH) and a variable light chain domain (VL). In some embodiments, the scFv can be fully human or fully humanized. In some embodiments, the scFv can be derived from a human antibody. PEBLs can be used to reduce the cell surface expression of various proteins, including subunits of the TCR-CD3 complex, MHC Class I or Class II, and / or CD58 in an immune cell.

[0260] In some embodiments, the scFv is directed to the TCR-CD3 complex. In someembodiments, the scFv is directed to a subunit of the TCR-CD3 complex. In some embodiments, the scFv is directed to the TCRalpha / beta complex. In some embodiments, the scFv is directed to the TCR^ subunit. In some embodiments, the scFv is directed to the TCR^ subunit. In some embodiments, the scFv is directed to the TCR^ subunit. In some embodiments, the scFv is directed to the TCR^ subunit. In some embodiments, the scFv is directed to the CD3gamma subunit. In some embodiments, the scFv is directed to the CD3delta subunit. In some embodiments, the scFv is directed to the CD3epsilon subunit.

[0261] Non-limiting exemplary protein localization sequences or tags include but are notlimited to an endoplasmic reticulum (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. The protein localization sequence or tag can be operably linked to the N-terminus of a protein-binding domain. The protein localization sequence or tag can be operably linked to the C-terminus of a protein-binding domain. The protein localization sequence or tag can be selected from the sequences in Table 1.

[0262] In some embodiments, the protein localization sequence or tag includes an endoplasmicreticulum (ER) localization sequence or tag. In some embodiments, the ER localization sequence includes the amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 9). In some embodiments, the ER localization tag consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ER localization sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 48Mintz Ref. No.: 063384-521001WO 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO: 9. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 9. In some embodiments, the ER localization sequence includes the amino acid sequence KKMP (SEQ ID NO: 10). In some embodiments, the ER localization tag consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ER localization sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 10. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 10.

[0263] In some embodiments, the protein localization sequence or tag includes a Golgiretention sequence or tag. In some embodiments, the Golgi retention sequence includes the amino acid sequence YQRL (SEQ ID NO: 12). In some embodiments, the Golgi retention tag consists of the amino acid sequence YQRL (SEQ ID NO: 12). In some embodiments, Golgi retention sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 12 In some embodiments, the protein localization sequence or tag includes a lysosome retention sequence. In some embodiments, the lysosome retention sequence includes the amino acid sequence KFERQ (SEQ ID NO: 13). In some embodiments, the lysosome retention tag consists of the amino acid sequence KFERQ (SEQ ID NO: 13). In some embodiments, the lysosome retention tag includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 13 In some embodiments, the protein localization sequence or tag includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 a SEQ ID NO: listed in Table 1. 49Mintz Ref. No.: 063384-521001WO Table 1: Exemplary Protein Localization Sequences or Tags

[0264] In some embodiments, a PEBL includes more than one protein binding domain and canprevent the transport of two or more proteins to the cell surface. For example, two or more scFvs can be operably linked in tandem and contain a single protein localization sequence or tag. In some embodiments, the two scFvs are operably linked directly (i.e., without a peptide linker). In some embodiments, the two scFvs are operably linked by a peptide linker. In some embodiments, the two or more scFvs are the same scFv. In some embodiments, the two or more scFvs are different. In some embodiments, the two or more scFvs target the same protein. In some embodiments, the two or more scFvs target different proteins. Exemplary amino acid sequences of tandem PEBLs are listed in Table 11, Table 12, and Table 16. In some embodiments the tandem PEBL comprises a first scFv and a second scFv. In some embodiments, the first scFv is an anti-TCR scFv and the second scFv is an anti-CD58 scFv. In some embodiments, the first scFv is an anti-CD58 scFv and the second scFv is an anti-TCR scFv.

[0265] Exemplary sequences of anti-TCRalpha and anti-TCRbeta scFvs useful in tandemPEBLs are listed in Table 3. Exemplary sequences of anti-CD58 scFvs useful in tandem PEBLs are listed in Table 8, Table 9, and Table 10.

[0266] In some embodiments, a PEBL can be combined with a dominant negative mutantprotein. For example, a PEBL comprising one or more scFvs and a DN mutant protein can be operably linked in tandem and contain a single protein localization sequence or tag. In some 50Mintz Ref. No.: 063384-521001WO embodiments, the PEBL and the DN mutant protein are fused together. In some embodiments, the scFv and the DN mutant protein are fused together with a linker. In some embodiments, a tandem PEBL can be combined with a dominant negative mutant protein. For example, a tandem PEBL comprising one, two, or more scFvs and a DN mutant protein can be operably linked in tandem. In some embodiments, the PEBL comprises an anti-TCR scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL comprises an anti-TCRalpha scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL comprises an anti-TCRbeta scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL comprises an anti-TCRdelta scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL comprises an anti-TCRgamma scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL comprises an anti-CD58 scFv and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCR scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRalpha scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRbeta scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRgamma scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRdelta scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCR scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRalpha scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRbeta scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRgamma scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRdelta scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the DN mutant protein is selected from 51Mintz Ref. No.: 063384-521001WO the DN mutant protein in Table 5. Exemplary sequences of tandem PEBLs are listed in Table 11, Table 12, and Table 16.

[0267] In another aspect, the PEBL further includes, but is not limited to, a ubiquitin ligasedomain. Attachment of a ubiquitin ligase domain leads to ubiquitination of the PEBL, the target protein, and / or a protein that binds the target protein. The ubiquitinated PEBL, target protein, and or protein that binds the target protein (i.e., a TCR complex polypeptide, a CD3 complex polypeptide, or a CD58 polypeptide) is then targeted for ubiquitin-mediated degradation. Non-limiting exemplary embodiments of ubiquitin ligase domains include ubiquitin ligase domains derived from RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase, a U-box E3 ligase family ubiquitin ligase, a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase, a gene related to anergy in lymphocytes (GRAIL) E3 ubiquitin ligase or a carboxy-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase. Additional exemplary ubiquitin ligases including ubiquitin ligase domains capable of use in the PEBL embodiments provided herein include RNF122, RNF133, RNF152, RNF130, RNF148, RNF149, RNF150, MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, and MARCH9. Non-limiting exemplary embodiments of ubiquitin ligase domains of the disclosure are the ubiquitin ligase domains of RNF122, RNF133, RNF152, RNF130, RNF148, RNF149, RNF150, MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, and MARCH9.

[0268] In some embodiments, the ubiquitin ligase domain is derived from a ubiquitin ligaseselected from the group consisting of RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase, a U-box E3 ligase family ubiquitin ligase, a membrane associated ring- CH-type finger (MARCH) family ubiquitin ligase, a gene related to anergy in lymphocytes (GRAIL) E3 ubiquitin ligase, and a carboxy-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase. In some embodiments, the ubiquitin ligase domain is derived from an RNF family ubiquitin ligase. In some embodiments, the RNF family ubiquitin ligase is selected from the group consisting of RNF122, RNF133, RNF152, RNF130, RNF148, RNF149, and RNF150. In some embodiments, the ubiquitin ligase domain is derived from a MARCH family ubiquitin ligase. In some embodiments, the MARCH family ubiquitin ligase is selected from the group consisting of MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, and MARCH9.

[0269] Exemplary Ubiquitin ligase domain sequences that can be used in the recombinantnucleic acids disclosed herein include those in Table 2 below: 52Mintz Ref. No.: 063384-521001WOTable 2: Exemplary Ubiquitin Ligase Domain Sequences53Mintz Ref. No.: 063384-521001WO

[0270] In some embodiments, the PEBL includes an scFv that binds a TCR-CD3 complexcomponent and a protein localization sequence tag. In some embodiments, the scFv binds TCRalpha. In some embodiments, the scFv binds TCRbeta. In some embodiments, the scFv binds TCRgamma. In some embodiments, the scFv binds TCRdelta. In some embodiments, the scFv binds CD3gamma. In some embodiments, the scFv binds CD3delta. In some embodiments, the scFv binds CD3epsilon. In some embodiments, the scFv binds CD3zeta.

[0271] The amino acid and nucleotide sequences of certain exemplary anti-TCRalpha and anti-TCRbeta scFvs, and the corresponding anti-TCRalpha and anti-TCRbeta PEBLs for use in the recombinant nucleic acids disclosed herein are listed in Table 3 below. Throughout all of the Tables herein, it is noted that the amino acid numbering convention is based on UniProt full sequences, which contain signal peptides. For sequences in which signal peptides are not shown, this numbering convention also applies. 54Mintz Ref. No.: 063384-521001WO Table 3: Exemplary anti-TCRalpha or anti-TCRbeta scFvs and PEBLs55Mintz Ref. No.: 063384-521001WO56Mintz Ref. No.: 063384-521001WO57Mintz Ref. No.: 063384-521001WO58Mintz Ref. No.: 063384-521001WO59Mintz Ref. No.: 063384-521001WO60Mintz Ref. No.: 063384-521001WO61Mintz Ref. No.: 063384-521001WO62Mintz Ref. No.: 063384-521001WO63Mintz Ref. No.: 063384-521001WO64Mintz Ref. No.: 063384-521001WO65Mintz Ref. No.: 063384-521001WO66Mintz Ref. No.: 063384-521001WO67Mintz Ref. No.: 063384-521001WO68Mintz Ref. No.: 063384-521001WO69Mintz Ref. No.: 063384-521001WO70Mintz Ref. No.: 063384-521001WO71Mintz Ref. No.: 063384-521001WO72Mintz Ref. No.: 063384-521001WO73Mintz Ref. No.: 063384-521001WO74Mintz Ref. No.: 063384-521001WO75Mintz Ref. No.: 063384-521001WO76Mintz Ref. No.: 063384-521001WO77Mintz Ref. No.: 063384-521001WO78Mintz Ref. No.: 063384-521001WO79Mintz Ref. No.: 063384-521001WO80Mintz Ref. No.: 063384-521001WO81Mintz Ref. No.: 063384-521001WO82Mintz Ref. No.: 063384-521001WO83Mintz Ref. No.: 063384-521001WO84Mintz Ref. No.: 063384-521001WO85Mintz Ref. No.: 063384-521001WO86Mintz Ref. No.: 063384-521001WO87Mintz Ref. No.: 063384-521001WO88Mintz Ref. No.: 063384-521001WO89Mintz Ref. No.: 063384-521001WO90Mintz Ref. No.: 063384-521001WO91Mintz Ref. No.: 063384-521001WO92Mintz Ref. No.: 063384-521001WO93Mintz Ref. No.: 063384-521001WO94Mintz Ref. No.: 063384-521001WO95Mintz Ref. No.: 063384-521001WO96Mintz Ref. No.: 063384-521001WO

[0272] In some embodiments, the coding sequence of the anti-TCRalpha / beta scFv in therecombinant nucleic acids described herein encodes the amino acid sequence of any one or more of SEQ ID NOs: 27-35, 37-46, 48-53, and 55-56. In some embodiments, the recombinant nucleic acids of the disclosure include a recombinant nucleic acid sequence encoding an anti- TCRalpha / beta scFv having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one or more of the amino acid sequences of SEQ ID NOs: 27-35, 37-46, 48-53, and 55-56.

[0273] In some embodiments, the coding sequence of the TCR PEBL in the recombinantnucleic acids described herein encodes the amino acid sequence of any one or more of SEQ ID NOs: 356 or 339. In some embodiments, the recombinant nucleic acids of the disclosure include a recombinant nucleic acid sequence encoding an anti-TCRalpha / beta scFv having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one or more of the amino acid sequences of SEQ ID NOs: 356 or 339. 97Mintz Ref. No.: 063384-521001WO

[0274] Amino acid sequences of certain exemplary anti-CD3epsilon scFvs for use in PEBLembodiments encoded by the recombinant nucleic acids disclosed herein include those in Table 4 below: Table 4: Exemplary anti-CD3epsilon scFvs

[0275] In some embodiments, the recombinant nucleic acids encoding an anti-CD3epsilonscFv disclosed herein include a coding sequence encoding the amino acid sequence of SEQ ID NOs: 80 or 81. In some embodiments, the recombinant nucleic acids encoding an anti- CD3epsilon scFv include a coding sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NOs: 80 or 81. 3. Dominant Negative (DN) Mutant Proteins or Protein Subunits

[0276] In another aspect, the recombinant nucleic acids provided herein comprise a firstnucleotide sequence encoding a dominant negative mutant protein or protein subunit. In some embodiments, the dominant negative mutant protein or protein subunit comprises a dominant negative mutant protein subunit of the TCR-CD3 complex capable of reducing TCR-CD3 complex expression on the surface of an immune cell.

[0277] In some embodiments, the recombinant nucleic acids can include a nucleotide sequenceencoding a dominant negative mutant protein subunit of the TCR-CD3 complex that reduces TCR-CD3 complex expression on the surface of an immune cell.

[0278] Dominant negative (DN) mutant proteins or polypeptides are mutant proteins orpolypeptides that interfere with the normal function of the corresponding wild-type protein or 98Mintz Ref. No.: 063384-521001WO wild-type protein complex. A DN mutant protein can interfere, for example, by competing with the wild-type protein for binding to other cellular components. In some embodiments, the DN mutant protein includes a mutant protein that acts as a “sink” for the wild-type protein, competes with its wild-type counterpart protein for its wild-type binding partners, and / or renders complexes including the wild-type counterpart protein inactive.

[0279] In some embodiments, the nucleotide sequence encodes two or more DN mutantproteins are operably linked together. In some embodiments, the two or more DN mutant proteins are operably linked together in tandem. In some embodiments, the two or more DN mutant proteins can be the same DN mutant protein. In some embodiments, the two or more DN mutant proteins can be different. In some embodiments, the two or more DN mutant proteins can target the same protein or protein complex. In some embodiments, the two or more DN mutant protein can target different proteins or protein complexes.

[0280] In some embodiments, the nucleotide sequence encodes a DN mutant protein thatincludes an additional protein localization sequence or tag. In some embodiments, the additional protein localization sequence or tag includes an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization sequence, a lysosome localization sequence, a plasma membrane localization sequence, a mitochondria localization sequence, a peroxisome localization sequence, a cytosolic localization sequence, and a nuclear localization sequence. In some embodiments, the protein localization sequence or tag is operably linked to the N-terminus of the DN mutant protein. In some embodiments, the protein localization sequence or tag is operably linked to the C-terminus of the DN protein. Exemplary protein localization sequences or tags are described supra.

[0281] In some embodiments, the nucleotide sequence encodes a DN mutant protein that canfurther include a ubiquitin ligase domain. Exemplary ubiquitin ligase domains are described supra.

[0282] In some embodiments, the nucleotide sequence encodes one or more PEBLs and oneor more DN mutant proteins that are operably linked together. In some embodiments, the one or more PEBL and the one or more DN mutant proteins target the same protein. In some embodiments, the one or more PEBL and the one or more DN mutant proteins target different proteins.

[0283] In some embodiments, the nucleotide sequence encodes a dominant negative mutantprotein subunit of the TCR-CD3 complex that associates with a TCR complex component. The TCR component of the TCR-CD3 complex comprises either a TCR^ / ^ dimer or a TCR ^ / ^ 99Mintz Ref. No.: 063384-521001WO dimer. The CD3 component of the TCR-CD3 complex comprises one CD3^ / ^ heterodimer, one CD3^ / ^ heterodimer and one CD3^ / ^ homodimer. Exemplary DN mutant protein subunits of the TCR-CD3 complex comprise, for example, a dominant negative TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta subunit. In some embodiments, the dominant negative mutant protein subunit of the TCR-CD3 complex includes a dominant negative mutant TCR-CD3 complex component. In some embodiments, the recombinant nucleic acid molecules comprise a nucleotide sequence encoding a dominant negative mutant TCR-CD3 complex component that binds a wild-type TCR-CD3 complex component. In some embodiments, the dominant negative mutant TCR-CD3 complex component includes a dominant negative mutant TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta. In some embodiments, the dominant negative mutant TCR-CD3 complex component includes a dominant negative mutant CD3epsilon subunit. In some embodiments, the dominant negative mutant CD3epsilon subunit comprises one or more point mutations in its transmembrane domain, wherein such point mutations reduce or eliminate the ability of the dominant negative mutant CD3epsilon subunit to bind and / or interact with the TCRalpha, TCRbeta, TCRdelta, and / or TCRgamma protein subunits. In some embodiments, the dominant negative mutant TCR-CD3 complex component includes a dominant negative mutant CD3zeta subunit. In some embodiments, the dominant negative mutant CD3zeta subunit comprises one or more point mutations in its transmembrane domain, wherein such point mutations reduce or eliminate the ability of the dominant negative mutant CD3zeta subunit to bind and / or interact with the TCRalpha, TCRbeta, TCRdelta, and / or TCRgamma protein subunits.

[0284] In some embodiments, the dominant negative mutant protein subunit of the TCR-CD3complex component further includes a ubiquitin ligase domain. Exemplary ubiquitin ligase domains are described above.

[0285] In some embodiments, the dominant negative mutant protein subunit includes adominant negative TCRalpha subunit. In some embodiments, the dominant negative mutant protein subunit includes a dominant negative TCRbeta subunit. In some embodiments, the dominant negative mutant protein subunit includes a dominant negative TCRgamma subunit. In some embodiments, the dominant negative mutant protein subunit includes a dominant negative TCRdelta subunit.

[0286] In some embodiments, the dominant negative mutant protein subunit includes adominant negative CD3gamma subunit. In some embodiments, the dominant negative mutant 100Mintz Ref. No.: 063384-521001WO protein subunit includes a dominant negative CD3delta subunit. In some embodiments, the dominant negative mutant protein subunit includes a dominant negative CD3epsilon subunit. In some embodiments, the dominant negative mutant protein subunit includes a dominant negative CD3zeta subunit.

[0287] In some embodiments, the transmembrane domain of a dominant negative CD3zetasubunit includes one or more mutations relative to that of the endogenous or wild-type CD3zeta. The wild-type CD3zeta transmembrane domain comprises the sequence of LCYLLDGILFIYGVILTALFL (SEQ ID NO: 96). In some embodiments, the mutation disrupts the ionic interaction between the transmembrane domain of the dominant negative CD3zeta subunit and other endogenous or wild-type TCR-CD3 complex subunits (e.g., TCRalpha, TCRbeta, TCRdelta, or TCRgamma,). In some embodiments, the mutation in the dominant negative CD3zeta subunit is at the position designated “X” in the amino acid sequence set forth in LCYLLXGILFIYGVILTALFL (SEQ ID NO: 318). In some embodiments, the endogenous CD3zeta transmembrane domain has the sequence of SEQ ID NO: 96. In some embodiments, the dominant negative CD3zeta subunit transmembrane domain has a non-conservative mutation at the position designated “X” in SEQ ID NO: 318. In some embodiments, the endogenous CD3zeta transmembrane domain has a non- conservative mutation comprising an aspartic acid (D) at the position designated ‘‘X’’ in SEQ ID NO: 318. In some embodiments, the endogenous CD3zeta transmembrane domain has a non-conservative mutation comprising an arginine (R) at the position designated ‘‘X’’ in SEQ ID NO: 318. In some embodiments, the dominant negative CD3zeta subunit transmembrane domain has a non-conservative mutation at the position designated “K” in SEQ ID NO: 318. In some embodiments, the dominant negative CD3zeta subunit transmembrane domain has a lysine (K), alanine (A), serine (S), or arginine (R) at the position designated “X” in SEQ ID NO: 318.

[0288] In some embodiments, the transmembrane domain of a dominant negative CD3epsilonsubunit includes one or more mutations relative to that of the endogenous CD3epsilon. In some embodiments, the mutation disrupts the ionic interaction between the transmembrane domain of the dominant negative CD3epsilon subunit and other endogenous TCR-CD3 complex subunits (e.g., TCRalpha, TCRbeta, TCRdelta, or TCRgamma, CD3zeta, CD3gamma, or CD3delta). In some embodiments, the mutation in the dominant negative CD3epsilon subunit is at the position designated “X” in the amino acid sequence set forth in MSVATIVIVXICITGGLLLLVYYWS (SEQ ID NO: 319). In some embodiments, the 101Mintz Ref. No.: 063384-521001WO endogenous wild-type CD3epsilon transmembrane domain includes the sequence of SEQ ID NO: 99. In some embodiments, the endogenous wild-type CD3epsilon transmembrane domain includes an aspartic acid (D) residue at the amino acid designated X in (SEQ ID NO: 319). In some embodiments, the mutant CD3epsilon transmembrane domain includes a non- conservative mutation at the amino acid position designated X in (SEQ ID NO: 319). In some embodiments, the dominant negative CD3epsilon subunit transmembrane domain has a lysine (K) or alanine (A) at the position designated “X” in SEQ ID NO: 318. In some embodiments, the non-conservative dominant negative CD3epsilon subunit transmembrane domain has a lysine (K) at the position designated “X” in SEQ ID NO: 318. In some embodiments, the non- conservative dominant negative CD3epsilon subunit transmembrane domain has an alanine (A) at the position designated “X” in SEQ ID NO: 318.

[0289] Amino acid and nucleic acid sequences encoding certain exemplary dominant negativemutant protein subunits of the TCR-CD3 complex for use in the recombinant nucleic acids described herein are set forth in Table 5 below: Table 5: Exemplary dominant negative mutant protein subunits of the TCR-CD3 complex102Mintz Ref. No.: 063384-521001WO103Mintz Ref. No.: 063384-521001WO104Mintz Ref. No.: 063384-521001WO105Mintz Ref. No.: 063384-521001WO106Mintz Ref. No.: 063384-521001WO107Mintz Ref. No.: 063384-521001WO108Mintz Ref. No.: 063384-521001WO109Mintz Ref. No.: 063384-521001WO110Mintz Ref. No.: 063384-521001WO111Mintz Ref. No.: 063384-521001WO112Mintz Ref. No.: 063384-521001WO113Mintz Ref. No.: 063384-521001WO114Mintz Ref. No.: 063384-521001WO115Mintz Ref. No.: 063384-521001WO116Mintz Ref. No.: 063384-521001WO117Mintz Ref. No.: 063384-521001WO118Mintz Ref. No.: 063384-521001WO119Mintz Ref. No.: 063384-521001WO120Mintz Ref. No.: 063384-521001WO121Mintz Ref. No.: 063384-521001WO122Mintz Ref. No.: 063384-521001WO123Mintz Ref. No.: 063384-521001WO124Mintz Ref. No.: 063384-521001WO125Mintz Ref. No.: 063384-521001WO126Mintz Ref. No.: 063384-521001WO127Mintz Ref. No.: 063384-521001WO128Mintz Ref. No.: 063384-521001WO129Mintz Ref. No.: 063384-521001WO130Mintz Ref. No.: 063384-521001WO131Mintz Ref. No.: 063384-521001WO132Mintz Ref. No.: 063384-521001WO133Mintz Ref. No.: 063384-521001WO134Mintz Ref. No.: 063384-521001WO135Mintz Ref. No.: 063384-521001WO136Mintz Ref. No.: 063384-521001WO137Mintz Ref. No.: 063384-521001WO138Mintz Ref. No.: 063384-521001WO139Mintz Ref. No.: 063384-521001WO140Mintz Ref. No.: 063384-521001WO141Mintz Ref. No.: 063384-521001WO142Mintz Ref. No.: 063384-521001WO143Mintz Ref. No.: 063384-521001WO

[0290] In some embodiments, the recombinant nucleic acid includes a first nucleotidesequence encoding a dominant negative mutant protein subunit of the TCR-CD3 complex including an amino acid sequence selected from the group consisting of SEQ ID NO: 82 to SEQ ID NO: 147, SEQ ID NO: 352, SEQ ID NO: 370, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380 and SEQ ID NO: 505. In some embodiments, the recombinant nucleic acids of the disclosure include a recombinant nucleic acid sequence encoding a dominant negative mutant protein subunit of the TCR-CD3 complex having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one or more of the amino 144Mintz Ref. No.: 063384-521001WO acid sequences of SEQ ID NO: 82 to SEQ ID NO: 147, SEQ ID NO: 352, SEQ ID NO: 370, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, and SEQ ID NO: 505.

[0291] In some embodiments, the dominant negative mutant protein subunit is operably linkedto a protein localization sequence or tag. In some embodiments, the protein localization sequence or tag is operably linked to the N-terminus of the dominant negative mutant protein subunit. In some embodiments, the protein localization sequence or tag is operably linked to the C-terminus of the dominant negative mutant protein subunit. In some embodiments, the protein localization sequence or tag includes an ER localization sequence or tag, a Golgi apparatus (Golgi) retention sequence or tag, a lysosome retention sequence or tag, a plasma membrane retention sequence or tag, a mitochondria retention sequence or tag, a peroxisome retention sequence or tag, a cytosolic retention sequence or tag, or a nuclear retention sequence or tag. 4. Short-hairpin RNA (shRNA)

[0292] In another aspect, the recombinant nucleic acids provided herein comprise a firstnucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell, wherein the nucleotide sequence encodes one or more shRNAs capable of reducing TCR-CD3 complex expression on the surface of an immune cell.

[0293] In some embodiments, the first nucleic acid sequence includes a recombinant nucleicacid encoding one or more shRNAs that reduce TCR-CD3 complex expression on the surface of an immune cell.

[0294] Short hairpin RNAs are RNAs including base-paired stems having single-strandedloops ranging from 4 to 23 or more nucleotides in length, but also may include RNAs with stem loop bulges, micro-RNAs, and small temporal RNAs. RNAs having loops or hairpin loops further encompasses structures where the loops are connected to the stem by linkers such as flexible linkers. Flexible linkers include a wide variety of chemical structures, provided they are of sufficient length to enable effective intramolecular hybridization of the stem elements.

[0295] shRNAs are short RNA sequences that can be encoded in various locations in arecombinant nucleic acid including, for example, before a transgene, after a stop codon of a transgene, or in an intron, for example an EF-1alpha promoter intron. Once an shRNA is transcribed, it is processed by the cellular enzymes Drosha and Dicer into a short interfering 145Mintz Ref. No.: 063384-521001WO RNA (“siRNA”). Such siRNAs are loaded onto the RNA-induced silencing complex (“RISC”) complex where they target specific complementary mRNAs for degradation.

[0296] In some embodiments, the recombinant nucleic acid includes an shRNA targeting aspecific subunit of the TCR (e.g., TCR-α and TCR-β) and / or a specific subunit of CD3 (e.g., CD3^). In some embodiments, the shRNA targets a TCRα polypeptide, a TCRβ polypeptide, a TCR^ polypeptide or a TCR^ polypeptide. In some embodiments, the shRNA targets a CD3^ polypeptide, a CD3^ polypeptide, a CD3^ polypeptide, or a CD3^ polypeptide. In some embodiments, the recombinant nucleic acid includes one or more shRNAs targeting a combination of TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta. In some embodiments, the first recombinant nucleic acid includes an shRNA targeting TCRalpha and an shRNA targeting CD3epsilon, an shRNA targeting TCRalpha and an shRNA targeting CD3zeta, an shRNA targeting TCRbeta and an shRNA targeting CD3epsilon, or an shRNA targeting TCRbeta and an shRNA targeting CD3zeta.

[0297] For example, an exemplary shRNA targeting CD3^ includes the sequencectggaggcttgctgaaggctgtatgctgaacgccaactgataagaggcagttttggccactgactgactgcctcttcagttggcgttcag gacacaaggcctgttactagcactcacatggaacaaatggccca (SEQ ID NO: 113).

[0298] Nucleotide sequences of certain exemplary shRNAs targeting subunits of the TCR-CD3complex for use in the recombinant nucleic acids disclosed herein are set forth in Table 6A and Table 6B below:

[0299] Table 6A: Exemplary shRNAs targeting the TCR-CD3 complex146Mintz Ref. No.: 063384-521001WO Table 6B: Exemplary shRNA constructs targeting the TCR-CD3 complex

[0300] In some embodiments, the sequence of an shRNA targeting the TCR-CD3 complex inthe recombinant nucleic acids described herein comprises a nucleic acid sequence of SEQ ID NOs: 184 to 189. In some embodiments, the recombinant nucleic acids of the disclosure include a nucleic acid sequence comprising an shRNA targeting a subunit of the TCR-CD3 complex having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence of SEQ ID NOs: 184 to 189. In some embodiments, the shRNA includes a sequence including at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of SEQ ID NOs: 184-189. In some embodiments, the CD58 shRNA includes a nucleotide sequence including at most 1, at most 2, or at most 3 mutations in any one of SEQ ID NOs: 184-189. 147Mintz Ref. No.: 063384-521001WO

[0301] In some embodiments, the nucleic acid sequence of an shRNA targeting the TCR-CD3complex in the recombinant nucleic acids described herein comprises a nucleic acid sequence of SEQ ID NOs: 181 to 183, 365, 449, or 802. In some embodiments, the recombinant nucleic acids of the disclosure include a nucleic acid sequence comprising an shRNA targeting a subunit of the TCR-CD3 complex having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence of SEQ ID NOs: 181 to 183, 365, 449, or 802. In some embodiments, the shRNA includes a sequence including at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of SEQ ID NOs: 181 to 183, 365, and 449. In some embodiments, the CD58 shRNA includes a nucleotide sequence including at most 1, at most 2, or at most 3 mutations in any one of SEQ ID NOs: 181 to 183, 365, and 449.

[0302] In some embodiments, the recombinant nucleic acid further includes a fourth nucleotidesequence encoding a fourth molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell.

[0303] In some embodiments, the fourth molecule includes an scFv binding a TCR-CD3complex component of the disclosure, a dominant negative mutant protein subunit of the TCR- CD3 complex of the disclosure, or an shRNA targeting the TCR-CD3 complex of the disclosure.

[0304] In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / betascFv and the fourth nucleotide sequence encodes a CD3zeta shRNA. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a CD3gamma shRNA. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a CD3epsilon shRNA. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a CD3delta shRNA. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a TCRalpha shRNA. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a TCRbeta shRNA.

[0305] In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / betascFv and the fourth nucleotide sequence encodes a CD3epsilon DN. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide 148Mintz Ref. No.: 063384-521001WO sequence encodes a CD3zeta DN. In some embodiments, the first nucleotide sequence encodes an anti-TCRalpha / beta scFv and the fourth nucleotide sequence encodes a CD3delta DN.

[0306] In some embodiments, the first nucleotide sequence encodes a dominant negativemutant protein subunit of the TCR-CD3 complex, and the fourth nucleotide sequence encodes a shRNA targeting the TCR-CD3 complex. B. Reduction of MHC class I and MHC class II Expression on the cell surface

[0307] In another aspect, provided herein are recombinant nucleic acids comprising a secondnucleotide sequence encoding a second molecule capable of reducing MHC class I and MHC class II expression on the surface of an immune cell. Non-limiting examples of molecules capable of reducing MHC class I and MHC class II expression in a cell include PEBLs, dominant negative mutant proteins, or shRNAs targeting MHC class I and MHC class II component expression. PEBLs, dominant negative mutant protein subunits, or shRNAs are described above. 1. Major Histocompatibility Complex (MHC) Class I and Class II

[0308] In another aspect, provided herein are recombinant nucleic acids encoding moleculescapable of reducing MHC class I and MHC class II expression on the surface of an immune cell.

[0309] The major histocompatibility complex (MHC) consists of a linked set of genetic lociencoding proteins involved in antigen presentation to T cells, including the MHC class I and class II glycoproteins that present antigenic peptides to the TCR-CD3 complex. MHC molecules are sometimes referred to interchangeably as human leucocyte antigen (HLA) molecules.

[0310] MHC class I molecules consist of two polypeptides, the α polypeptide and β2microglobulin (B2M) polypeptide. The α polypeptide is polymorphic and includes a transmembrane glycoprotein encoded by the HLA class I molecules including an HLA-A, HLA-B, or HLA-C gene product. Class I MHC molecules bind and present antigenic peptides to T cells. The antigenic peptides are generated mainly from the degradation of cytosolic proteins by the proteasome. MHC class I molecules also serve as an inhibitory ligand for natural killer cells (NKs). MHC class II molecules consist of two polypeptide chains, an α and β chain. The α chain and β chain are polymorphic and both include a transmembrane glycoprotein encoded by HLA-DP, HLA-DM, HLA-DO, HLA-DQ, or HLA-DR. Class II 149Mintz Ref. No.: 063384-521001WO MHC molecules bind and present peptides generated mainly from extracellular pathogens and viruses to T cells.

[0311] In a patient with an allogeneic graft, graft cells are vulnerable to elimination by the hostimmune system. NK and T cells of the host immune system can recognize the graft cells by their non-matching MHC class I or MHC class II molecules and mount a cytotoxic immune response. This host immune response can reduce the lifespan of an allogeneic graft in the patient.

[0312] To reduce the occurrence of host vs graft elimination in patients with allogeneic T cellgrafts, the donor T cells can be engineered to reduce MHC class I and MHC class II expression on the cell surface.

[0313] In some embodiments, the recombinant nucleic acids provided herein encode amolecule capable of reducing MHC class I and MHC class II expression on the surface of an immune cell. In some embodiments, the molecule capable of reducing MHC class I and MHC class II expression on the surface of an immune cell comprises a protein expression blocker (PEBL). In some embodiments, the molecule capable of reducing MHC class I and MHC class II expression on the surface of an immune cell comprises a dominant negative mutant protein. In some embodiments, the molecule capable of reducing MHC class I and MHC class II expression on the surface of an immune cell comprises an shRNA. 2. Dominant Negative (DN) Mutant Proteins

[0314] In another aspect, provided herein are recombinant nucleic acids comprising, but notlimited to, nucleotide sequences encoding dominant negative mutant proteins or protein subunits that reduce MHC class I and MHC class II expression on the surface of an immune cell.

[0315] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequenceencoding a molecule that reduces MHC class I and MHC class II expression on the surface of an immune cell. In some embodiments, the molecule that reduces MHC class I and MHC class II expression on the surface of an immune cell comprises a dominant negative (DN) mutant protein that regulates transcription of certain components of the MHC class I and MHC class II protein complexes. In some embodiments, such DN mutant protein reduces cell surface expression of MHC class I and MHC class II. In some embodiments, the DN mutant protein includes a B2M dominant negative protein. In some embodiments, the DN mutant protein includes an MHC class I alpha chain dominant negative protein. In some embodiments, the DN 150Mintz Ref. No.: 063384-521001WO mutant protein includes an MHC class II alpha chain dominant negative protein. In some embodiments, the DN mutant protein includes an MHC class II beta chain dominant negative protein. In some embodiments, such DN mutant protein includes a DN mutant transcription factor. In some embodiments, the DN mutant transcription factor comprises a truncated version of a wild-type transcription factor that retains the ability to bind its cognate promoter but lacks the ability to drive transcription from that promoter. In some embodiments, the recombinant nucleic acids provided herein comprise a nucleotide sequence encoding a mutated Regulatory factor X 5 (RFX5) (NCBI NM_001025603.2, NP_001020774.1) polypeptide. In some embodiments, the mutated RFX5 polypeptide includes a truncated RFX5 polypeptide (i.e., an RFX5 DN).

[0316] The full-length amino acid sequence of RFX5 is set forth in SEQ ID NO: 190 asfollows: MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDELVEAACALTCDWAERILKRSFSSIVEVARFL LQQHLISARSAHAHVLKAMGLAEEDEHAPRERSSKPKNGLENPEGGAHKKPERLAQ PPKDLEARTGAGPLARGERKKSVVESSAPGANNLQVNALVARLPLLLPRAPRSLIPPI PVSPPILAPRLSSGALKVATLPLSSRAGAPPAAVPIINMILPTVPALPGPGPGPGRAPPG GLTQPRGTENREVGIGGDQGPHDKGVKRTAEVPVSEASGQAPPAKAAKQDIEDTAS DAKRKRGRPRKKSGGSGERNSTPLKSAAAMESAQSSRLPWETWGSGGEGNSAGGA ERPGPMGEAEKGAVLAQGQGDGTVSKGGRGPGSQHTKEAEDKIPLVPSKVSVIKGS RSQKEAFPLAKGEVDTAPQGNKDLKEHVLQSSLSQEHKDPKATPP.

[0317] Amino acid sequences of exemplary truncated RFX5 polypeptides (i.e., RFX5 DN) foruse in the recombinant nucleic acids provided herein are set forth in Table 7 below: Table 7: Exemplary RFX5 polypeptides151Mintz Ref. No.: 063384-521001WO152Mintz Ref. No.: 063384-521001WO153Mintz Ref. No.: 063384-521001WO154Mintz Ref. No.: 063384-521001WO

[0318] In some embodiments, the recombinant nucleic acid comprises a second nucleotidesequence encoding a truncated RFX5 polypeptide having the amino acid sequence of any one of SEQ ID NOs: 190 to 204, SEQ ID NOs: 340, SEQ ID NOs: 456, and SEQ ID NOs: 502. In some embodiments, the recombinant nucleic acid encoding a truncated RFX5 polypeptide includes nucleic acid sequences encoding a truncated RFX5 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NOs: 190 to 204, SEQ ID NOs: 340, SEQ ID NOs: 456, and SEQ ID NOs: 502. 3. Short-hairpin RNA (shRNA)

[0319] In another aspect, the recombinant nucleic acids provided herein comprise a secondnucleotide sequence(s) encoding one or more shRNAs that reduce MHC class I and MHC class II expression on the surface of an immune cell.

[0320] MHC class I and MHC class II expression can be decreased using short hairpin RNAs(shRNAs) to target nucleic acids encoding MHC class I and MHC class II complex proteins, and / or transcription factors / transcriptional activators of MHC class I and MHC class II complex proteins in T cells (e.g., B2M, CIITA, NLRC5, RFX5, RFXANK, RFXAP, viral immunoevasins and the like). In some non-limiting embodiments, shRNAs are designed to target components of the MHC class I and MHC class II complex.

[0321] In some embodiments, the second nucleotide sequence(s) provided herein encode oneor more shRNAs capable of reducing MHC class I and MHC class II expression on the surface of an immune cell. In some embodiments, the recombinant nucleic acids provided herein include nucleotide sequences encoding shRNAs targeting MHC class I and MHC class II polypeptides. In some embodiments, the recombinant nucleic acids provided herein include nucleotide sequences encoding shRNAs targeting the α polypeptide of MHC class I. In some 155Mintz Ref. No.: 063384-521001WO embodiments, the recombinant nucleic acids provided herein include nucleotide sequences encoding shRNAs targeting the β2 microglobulin of MHC class I. An exemplary shRNA targeting B2M includes the sequence: ctggaggcttgctgaaggctgtatgctgaatctttggagtacgctggatgttttggccactgactgacatccagcgctccaaagattcag gacacaaggcctgttactagcactcacatggaacaaatggccca (SEQ ID NO: 205).

[0322] In some embodiments, the recombinant nucleic acids provided herein includenucleotide sequences encoding shRNAs targeting the α chain of MHC class II. In some embodiments, the recombinant nucleic acids provided herein include nucleotide sequences encoding shRNAs targeting the β chain of MHC class II.

[0323] In some embodiments, the recombinant nucleic acids provided herein includenucleotide sequences encoding shRNAs targeting transcription factors and / or transcriptional activators that regulate expression of MHC class I and MHC class II proteins in T cells. In some embodiments, the transcription factor and / or transcriptional activator that regulates expression of MHC class I and MHC class II proteins in T cells is selected from the group including B2M, CIITA, NLRC5, RFX5, RFXANK, and RFXAP.

[0324] In some embodiments, the shRNA is a B2M shRNA. In some embodiments, the B2MshRNA comprises the sequence GACGACTGGAGGCTTGCTTTGGGCTGTATGCTGAATCTTTGGAGTACGCTGGATG TTTTGGCCTCTGACTGACATCCACGTCTCCAAAGATTGGACACAAGGCCCTTTAT CAGCACTCACATGGAACAAATGGCCACCGTGGGAGGATGACAACTGGAG (SEQ ID NO: 557) (eSIBR scaffold, b2m shRNA 3).

[0325] In some embodiments, the shRNA is an RFX5 shRNA. In some embodiments, theRFX5 shRNA comprises the sequence ctggaggcttgctgaaggctgtatgctgAACAGTACCATCTCCCTGACCgttttggccactgactgacGGTCA GGGATGGTACTGTTcaggacacaaggcctgttactagcactcacatggaacaaatggccca (SEQ ID NO: 558) (RFX5 shRNA cassette (mir155 scaffold, RFX5 shRNA 9)). Lower case nucleotides depict mir155 scaffold sequences, capitalized nucleotides depict RFX5 sequences. 4. Expression of US11

[0326] In another aspect, provided herein are recombinant nucleic acids including nucleic acidsequences encoding a viral protein that reduces MHC class I expression on the surface of an immune cell. 156Mintz Ref. No.: 063384-521001WO

[0327] In some embodiments, the recombinant nucleic acid sequence comprises a secondnucleotide sequence encoding a second molecule capable of reducing expression of MHC I / II on the surface of an immune cell. In some embodiments, the second nucleotide molecule encodes a viral immunoevasin. In some embodiments, the viral immunoevasin comprises a US11 polypeptide. US11 is a protein encoded by human cytomegalovirus (HCMV) that upon expression in a cell can degrade the major histocompatibility complex (MHC) class I heavy chains. US11 targets MHC class I chains for destruction via a pathway that involves ubiquitin- dependent retrograde transport, or "dislocation", of the heavy chains from the ER to the cytosol, where the MHC I proteins are degraded by proteasomes.

[0328] In some embodiments, the US11 polypeptide comprises the amino acid sequence ofSEQ ID NO: 419: MNLVMLILALWAPVAGSMPELSLTLFDEPPPLVETEPLPPLSDVSEYRVEYSEARCVL RSGGRLEALWTLRGNLSVPTPTPRVYYQTLEGYADRVPTPVEDVSESLVAKRYWLR DYRVPQRTKLVLFYFSPCHQCQTYYVECEPRCLVPWVPLWSSLEDIERLLFEDRRLM AYYALTIKSAQYTLMMVAVIQVFWGLYVKGWLHRHFPWMFSDQW (SEQ ID NO: 419) or an amino acid sequence having at least 80%, 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% sequence identity of SEQ ID NO: 419.

[0329] In some embodiments, the recombinant nucleic acid includes a nucleotide sequenceencoding a US11 polypeptide comprising the nucleic acid sequence of SEQ ID NO: 420: ATGAACCTGGTCATGCTGATCCTGGCCCTGTGGGCCCCTGTGGCTGGCAGCATGC CTGAGCTGAGCCTCACACTGTTCGACGAGCCACCACCTCTGGTGGAAACCGAGC CCCTGCCTCCTCTGAGCGATGTGAGCGAGTACAGAGTGGAATACAGCGAGGCTA GATGCGTGCTGAGAAGCGGCGGAAGACTGGAAGCCCTGTGGACCCTGAGAGGCA ACCTGTCTGTGCCAACCCCTACACCCAGAGTGTACTACCAGACACTGGAAGGCTA CGCCGACAGAGTGCCCACCCCTGTGGAGGATGTGTCTGAGAGCCTGGTGGCCAA GCGGTACTGGCTGCGGGACTACCGGGTCCCTCAGAGAACCAAGCTGGTTCTGTTT TACTTCTCCCCTTGCCACCAATGCCAGACCTACTATGTGGAATGCGAGCCTAGAT GTCTGGTGCCTTGGGTGCCCCTGTGGAGCAGCCTGGAAGATATCGAGCGGCTGCT GTTTGAGGACAGGCGGCTGATGGCCTACTACGCCCTGACAATCAAGTCCGCCCA GTACACCCTCATGATGGTGGCCGTGATCCAAGTGTTCTGGGGCCTGTATGTGAAA GGCTGGCTGCACAGACACTTCCCCTGGATGTTCAGCGACCAGTGG (SEQ ID NO: 420) or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, 157Mintz Ref. No.: 063384-521001WO at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity of SEQ ID NO: 420. C. Reduction of CD58 Expression on the Cell Surface

[0330] In another aspect, provided herein are recombinant nucleic acids comprising a thirdnucleotide sequence encoding molecules capable of reducing CD58 expression on the surface of an immune cell. Non-limiting exemplary molecules capable of reducing CD58 expression on the surface of an immune cell include anti-CD58 PEBLs, dominant negative mutant proteins, or shRNAs targeting CD58 expression. PEBLs, dominant negative mutant proteins or protein subunits, or shRNAs are described above. 1. CD58

[0331] CD58, or lymphocyte function-associated antigen 3 (LFA-3) (NCBI NP_001138294.1,NM_001144822.2), is a cell adhesion molecule expressed on T cells and antigen presenting cells (APCs), particularly macrophages. CD58 binds to CD2 (LFA-2) on T cells. This co- stimulatory interaction strengthens the adhesion and recognition between the T cells and APCs, facilitating the signal transduction necessary to trigger a robust immune response.

[0332] In a patient with an allogeneic graft, graft cells expressing CD58 are vulnerable toelimination by the host immune system. T cells of the host immune system can recognize CD58 on graft cells and are activated through their CD2 axis to mount a cytotoxic immune response and kill the graft cells. This host immune response reduces the lifespan and efficacy of allogeneic graft cells in a patient.

[0333] SEQ ID NO: 206 comprises the full-length amino acid sequence of human CD58:MVAGSDAGRALGVLSVVCLLHCFGFISCFSQQIYGVVYGNVTFHVPSNVPLKEVLW KKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTM KFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTS IYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHRYALIPIPLAVITTCIVLYM NGILKCDRKPDRTK (SEQ ID NO: 206).

[0334] SEQ ID NO: 207 comprises a full-length cDNA encoding human CD58:gaacttaggg ctgcttgtgg ctgggcactc gcgcagaggc cggcccgacg agccatggtt gctgggagcg acgcggggcg ggccctgggg gtcctcagcg tggtctgcct gctgcactgc tttggtttca tcagctgttt ttcccaacaa atatatggtg ttgtgtatgg gaatgtaact ttccatgtac caagcaatgt gcctttaaaa gaggtcctat ggaaaaaaca aaaggataaa gttgcagaac tggaaaattc tgaattcaga gctttctcat cttttaaaaa tagggtttat ttagacactg tgtcaggtag 158Mintz Ref. No.: 063384-521001WO cctcactatc tacaacttaa catcatcaga tgaagatgag tatgaaatgg aatcgccaaa tattactgat accatgaagt tctttcttta tgtgcttgag tctcttccat ctcccacact aacttgtgca ttgactaatg gaagcattga agtccaatgc atgataccag agcattacaa cagccatcga ggacttataa tgtactcatg ggattgtcct atggagcaat gtaaacgtaa ctcaaccagt atatatttta agatggaaaa tgatcttcca caaaaaatac agtgtactct tagcaatcca ttatttaata caacatcatc aatcattttg acaacctgta tcccaagcag cggtcattca agacacagat atgcacttat acccatacca ttagcagtaa ttacaacatg tattgtgctg tatatgaatg gtattctgaa atgtgacaga aaaccagaca gaaccaagta agtacactgc aggctgtgca ctctgtgcca gaacaacagc agccctgctg tagatgccca ttacttggga agccagattg tactcatctg ttggttgcct tgtacagtaa taagaagtaa catcagcaga tatactaacc atagtgtatt aagtaccaac tgtgtgccat gcactgcact agccactctc tgcatatcgt ttcctggaac cctctcaatg agccttgcag gaggagtatc ataccccttg cttagagaca gcgcatatga gattcataga agtcaagtga gatatcaaag tcacgttgct agtaagtgat gggctgggat ttaaatctag gtctttggac tccaagagtt atactgttaa ccactacaat acactccctc cacctcccat cccagaaaaa gttggatcag ggatagggtt cacttcagga gccagggtca gagctgggcc tgtagtcaca tataaaagat aggacacaaa cacaattagc agcagtgttg gtttaataaa ccgccgta (SEQ ID NO: 207). 2. CD58-specific Protein Expression Blockers (PEBLs)

[0335] In another aspect, provided herein are recombinant nucleic acids including nucleic acidsequences encoding PEBLs that reduce CD58 expression on the surface of an immune cell.

[0336] In some embodiments, the recombinant nucleic acids disclosed herein further include anucleotide sequence encoding a protein expression blocker (PEBL) that reduces CD58 expression on the surface of an immune cell. The PEBL includes an scFv capable of binding CD58 (termed an “anti-CD58 scFv”). The PEBL includes an scFv capable of binding CD58 (termed an “anti-CD58 scFv”) and a protein localization sequence. In some embodiments, the anti-CD58 scFv comprises a murine scFv or a humanized scFv. In some embodiments, the anti- CD58 scFv comprises an scFv derived from a human antibody. In some embodiments the anti- CD58 scFv is a humanized anti-CD58 scFv.

[0337] In some embodiments, the protein localization sequence or tag includes an endoplasmicreticulum (ER) localization sequence or tag. In some embodiments, the ER localization sequence includes the amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 9). In some embodiments, the ER localization tag consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ER localization sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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% sequence identity to SEQ ID NO: 9. In some embodiments, the ER localization tag consists of an amino acid 159Mintz Ref. No.: 063384-521001WO sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 9. In some embodiments, the ER localization sequence includes the amino acid sequence KKMP (SEQ ID NO: 10). In some embodiments, the ER localization tag consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the ER localization sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 10. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 10.

[0338] In some embodiments, the protein localization sequence or tag includes a Golgiretention sequence or tag. In some embodiments, the Golgi retention sequence includes the amino acid sequence YQRL (SEQ ID NO: 12). In some embodiments, the Golgi retention tag consists of the amino acid sequence YQRL (SEQ ID NO: 12). In some embodiments, Golgi retention sequence includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 12 In some embodiments, the protein localization sequence or tag includes a lysosome retention sequence. In some embodiments, the lysosome retention sequence includes the amino acid sequence KFERQ (SEQ ID NO: 13). In some embodiments, the lysosome retention tag consists of the amino acid sequence KFERQ (SEQ ID NO: 13). In some embodiments, the lysosome retention tag includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 13 In some embodiments, the protein localization sequence or tag includes an amino acid sequence with at least 70%, at least 75%, at least 80%, 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 a SEQ ID NO: listed in Table 1.

[0339] Amino acid sequences and / or nucleic acid sequences encoding exemplary anti-CD58scFv and PEBL sequences for use in the recombinant nucleic acids provided herein as set forth in Tables 8, 9, 10, and 11 below: 160Mintz Ref. No.: 063384-521001WO Table 8: Exemplary anti-CD58 scFv, PEBL, and fusion protein sequences161Mintz Ref. No.: 063384-521001WO_ 162Mintz Ref. No.: 063384-521001WO163Mintz Ref. No.: 063384-521001WO164Mintz Ref. No.: 063384-521001WO165Mintz Ref. No.: 063384-521001WO166Mintz Ref. No.: 063384-521001WO167Mintz Ref. No.: 063384-521001WO168Mintz Ref. No.: 063384-521001WO169Mintz Ref. No.: 063384-521001WO170Mintz Ref. No.: 063384-521001WO171Mintz Ref. No.: 063384-521001WO172Mintz Ref. No.: 063384-521001WO173Mintz Ref. No.: 063384-521001WO174Mintz Ref. No.: 063384-521001WO175Mintz Ref. No.: 063384-521001WO176Mintz Ref. No.: 063384-521001WO177Mintz Ref. No.: 063384-521001WO178Mintz Ref. No.: 063384-521001WO179Mintz Ref. No.: 063384-521001WO180Mintz Ref. No.: 063384-521001WO181Mintz Ref. No.: 063384-521001WO182Mintz Ref. No.: 063384-521001WO183Mintz Ref. No.: 063384-521001WO184Mintz Ref. No.: 063384-521001WO185Mintz Ref. No.: 063384-521001WO186Mintz Ref. No.: 063384-521001WO187Mintz Ref. No.: 063384-521001WO

[0340] In some embodiments, the anti-CD58 scFv includes a nucleic acid sequence ofATGGATATCCTGCTGACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGAGC GGGTGTCCTTCAGCTGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTGGT ATCAGCAGCGGACAATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGAGA GCATCAGCGGCATCCCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTCAC CCTGACCATCAACTCCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAGCA AAGCAACAGCTGGCCTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAGGG CAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGGGACA AGTGCAGCTGCAGCAGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGCGTGAA GATCAGCTGCAAGGGCAGCGGCTACACCTTCACCGACTACGCCATCCACTGGGT CAAGCAGTCTCACGCCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTGCACTA CGACAAAACCAACTACAACCAGAAATTCAAGGGCAAGGCCAGCATGACCGTGG ACAAGTCCAGCTCTACAGCCTACATGGAACTGGCTAGACTGACAAGCGAGGACA GCGCTATCTACTATTGTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATAATTG GGGACAGGGCACAACCCTGACCGTGTCCTCTATCTACATCTGGGCCCCACTGGCC GGCACATGCGGCGTGCTGCTGCTGTCCCTGGTGATCACCCTGTATTGCgcggccgcatc gacaGGTTCCAGCGGCGGTGGAGGAGGATCAGGAGGATTGTACAAGTACAAAAGC AGACGCTCTTTTATAGATGAGAAGAAGATGCCG, (SEQ ID NO: 222) or a nucleic acid sequence having at least 75%, at least 80%, 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% sequence identity to SEQ ID NO: 222.

[0341] In some embodiments, the anti-CD58 scFv includes an amino acid sequence ofMDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTIGSPRLLIKYASESISGIPS RFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGSGTKLEIKGSTSGSGKPGSG EGSTKGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGVI SVHYDKTNYNQKFKGKASMTVDKSSSTAYMELARLTSEDSAIYYCARSFYYGRDFD 188Mintz Ref. No.: 063384-521001WO NWGQGTTLTVSSIYIWAPLAGTCGVLLLSLVITLYCAAASTGSSGGGGGSGGLYKYK SRRSFIDEKKMP (SEQ ID NO: 223) or an amino acid sequence having at least 75%, at least 80%, 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% sequence identity to SEQ ID NO: 223.

[0342] In some embodiments, the anti-CD58 scFv includes a nucleic acid sequence ofATGGATATCCTGCTGACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGAGC GGGTGTCCTTCAGCTGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTGGT ATCAGCAGCGGACAATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGAGA GCATCAGCGGCATCCCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTCAC CCTGACCATCAACTCCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAGCA AAGCAACAGCTGGCCTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAGGG CAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGGGACA AGTGCAGCTGCAGCAGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGCGTGAA GATCAGCTGCAAGGGCAGCGGCTACACCTTCACCGACTACGCCATCCACTGGGT CAAGCAGTCTCACGCCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTGCACTA CGACAAAACCAACTACAACCAGAAATTCAAGGGCAAGGCCAGCATGACCGTGG ACAAGTCCAGCTCTACAGCCTACATGGAACTGGCTAGACTGACAAGCGAGGACA GCGCTATCTACTATTGTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATAATTG GGGACAGGGCACAACCCTGACCGTGTCCTCTGGCGGCGGCAGCGGCGGCGGATC TGCCGAAAAGGACGAGCTGATCTACATCTGGGCCCCACTGGCCGGCACATGCGG CGTGCTGCTGCTGTCCCTGGTGATCACCCTGTATTGCgcggccgcatcgacaGGTTCCAGC GGCGGTGGAGGAGGATCAGGAGGATTGTACAAGTACAAAAGCAGACGCTCTTTT ATAGATGAGAAGAAGATGCCG, (SEQ ID NO:224) or a nucleic acid sequence having at least 75%, at least 80%, 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% sequence identity to SEQ ID NO: 224.

[0343] In some embodiments, the anti-CD58 scFv includes an amino acid sequence ofMDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTIGSPRLLIKYASESISGIPS RFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGSGTKLEIKGSTSGSGKPGSG EGSTKGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGVI SVHYDKTNYNQKFKGKASMTVDKSSSTAYMELARLTSEDSAIYYCARSFYYGRDFD NWGQGTTLTVSSGGGSGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCAAASTGS SGGGGGSGGLYKYKSRRSFIDEKKMP, (SEQ ID NO: 225) or an amino acid sequence 189Mintz Ref. No.: 063384-521001WO having at least 75%, at least 80%, 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% sequence identity to SEQ ID NO: 225.

[0344] Amino acid sequences for certain exemplary Variable Heavy (VH) and Variable Light(VL) chains of anti-CD58 scFvs for use in the recombinant nucleic acids provided herein areset forth in Table 9 below: Table 9: Exemplary Variable Heavy (VH) and Variable Light (VL) Chain Sequences for Anti-CD58 Polypeptides with CDRs Underlined190Mintz Ref. No.: 063384-521001WOTable 10: Exemplary humanized anti-CD58 scFv sequences191Mintz Ref. No.: 063384-521001WO192Mintz Ref. No.: 063384-521001WOTable 11: Exemplary Tandem PEBL sequences193Mintz Ref. No.: 063384-521001WO194Mintz Ref. No.: 063384-521001WO195Mintz Ref. No.: 063384-521001WO196Mintz Ref. No.: 063384-521001WO197Mintz Ref. No.: 063384-521001WO198Mintz Ref. No.: 063384-521001WOTable 12: Exemplary Tandem PEBL sequences199Mintz Ref. No.: 063384-521001WO200Mintz Ref. No.: 063384-521001WO201Mintz Ref. No.: 063384-521001WO202Mintz Ref. No.: 063384-521001WO203Mintz Ref. No.: 063384-521001WO204Mintz Ref. No.: 063384-521001WO205Mintz Ref. No.: 063384-521001WO206Mintz Ref. No.: 063384-521001WO207Mintz Ref. No.: 063384-521001WO208Mintz Ref. No.: 063384-521001WO209Mintz Ref. No.: 063384-521001WO210Mintz Ref. No.: 063384-521001WO211Mintz Ref. No.: 063384-521001WO212Mintz Ref. No.: 063384-521001WO213Mintz Ref. No.: 063384-521001WO214Mintz Ref. No.: 063384-521001WO215Mintz Ref. No.: 063384-521001WO216Mintz Ref. No.: 063384-521001WO

[0345] In some embodiments, the recombinant nucleic acids provided herein comprisenucleotide sequences encoding an anti-CD58 PEBL having an amino acid sequence selected from the group consisting of SEQ ID NO: 206, or SEQ ID NOs: 208 to 215. In some embodiments, the anti-CD58 PEBL include nucleotide sequences encoding an anti-CD58 scFv including a nucleic acid sequence encoding an anti-CD58 scFv having at least 80%, at least 217Mintz Ref. No.: 063384-521001WO 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 206, or SEQ ID NOs: 208 to 215. In some embodiments, the anti-CD58 PEBL comprises a nucleotide sequence encoding a humanized anti-CD58 scFv having an amino acid sequence selected from the group consisting of SEQ ID NO: 459, 462, 467, 476, 480, 484, 491, and 696. In some embodiments, the anti-CD58 PEBL comprises a nucleotide sequence encoding a humanized anti-CD58 scFv having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NOs: 459, 462, 467, 476, 480, 484, 491, or 696.

[0346] In some embodiments, the scFv further includes a protein localization sequence or tag.Exemplary protein localization sequences or tags are described supra.

[0347] In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRalpha scFvand an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRbeta scFv and an anti-CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRgamma scFv and an anti- CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-TCRdelta scFv and an anti- CD58 scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCR scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRalpha scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRbeta scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRgamma scFv, and the DN mutant protein is a CD3epsilon DN mutant protein. In some embodiments, the PEBL is a tandem PEBL comprising an anti-CD58 scFv and an anti-TCRdelta scFv, and the DN mutant protein is a CD3epsilon DN mutant protein.

[0348] In some embodiments, the two or more scFvs are operably connected with a linker.Exemplary linker sequences are listed in Table 19.

[0349] In some embodiments, the tandem PEBL further comprises a protein localizationsequence. In some embodiments, the protein localization sequence is selected form Table 1. 218Mintz Ref. No.: 063384-521001WO

[0350] In some embodiments, the DN mutant protein is selected from the DN mutant proteinin Table 5. In some embodiments, the TCRalpha / beta scFv is selected from Table 3. In some embodiments, the CD58 scFv is selected from Table 8, 9, or 10. Exemplary sequences of tandem PEBLs are listed in Table 11, Table 12, and Table 16.

[0351] In some embodiments, the recombinant nucleic acids provided herein comprisenucleotide sequences encoding a tandem PEBL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 258, 393-395, 426, 457, 461, 464, 466, 469, 471, 508, and 619. In some embodiments, the tandem PEBL includes nucleotide sequences encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 258, 393-395, 426, 457, 461, 464, 466, 469, 471, 508, and 619.

[0352] Non-limiting examples of protein localization sequences or tags include but are notlimited to an endoplasmic reticulum (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. The protein localization sequence or tag can be operably linked to the N-terminus of a protein-binding domain. The protein localization sequence or tag can be operably linked to the C-terminus of a protein-binding domain. The protein localization sequence or tag can be selected from the sequences in Table 1.

[0353] In some embodiments, the scFv further includes a ubiquitin ligase domain. Exemplaryubiquitin ligase domains are described above. 3. Short-hairpin RNA (shRNA)

[0354] In another aspect, the recombinant nucleic acids provided herein comprise a thirdnucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of an immune cell. In some embodiments, the third nucleotide sequence comprises nucleic acid sequences encoding shRNAs that reduce CD58 expression on the surface of an immune cell.

[0355] In some embodiments, the recombinant nucleic acids provided herein comprisenucleotide sequences encoding one or more shRNAs capable of reducing CD58 expression on the surface of an immune cell.

[0356] In some embodiments, the CD58 shRNAs further include a mirE scaffold sequence. AmirE scaffold sequence is an optimized microRNA backbone that improves gene knockdown 219Mintz Ref. No.: 063384-521001WO by enhancing the processing of pri-miRNA (Fellmann et al., “An Optimized microRNA Backbone for Effective Single-Copy RNAi,” Cell Reports 5(6):1704-1713 (2013)).

[0357] In some embodiments, the CD58 shRNA includes a sequence including at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of SEQ ID NOs: 234-243. In some embodiments, the CD58 shRNA includes a nucleotide sequence including at most 1, at most 2, or at most 3 mutations in any one of SEQ ID NOs: 234-243.

[0358] In some embodiments, the CD58 shRNA includes a sequence including at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of SEQ ID NOs: 368, 552, 554, 555, or 556. In some embodiments, the CD58 shRNA includes a nucleotide sequence including at most 1, at most 2, or at most 3 mutations in any one of SEQ ID NOs: 368, 552, 554, 555, or 556.

[0359] Nucleotide sequences of certain exemplary shRNAs targeting CD58 for use in therecombinant nucleic acids provided herein are set forth in Table 13 below: Table 13: Exemplary shRNAs targeting the CD58220Mintz Ref. No.: 063384-521001WO

[0360] In some embodiments, the recombinant nucleic acid encoding the CD58 shRNAdescribed herein comprises the sequence of any one of SEQ ID NOs: 234-243. In some embodiments, the recombinant nucleic acids encoding the CD58 shRNA include a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NOs: 234-243.

[0361] In some embodiments, the recombinant nucleic acid encoding the CD58 shRNAdescribed herein includes the sequence of any one of SEQ ID NOs: 368, 552, 554, 555, or 556. In some embodiments, the recombinant nucleic acids encoding the CD58 shRNA include a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NOs: 368, 552, 554, 555, or 556. 4. Viral Proteins

[0362] In another aspect, the recombinant nucleic acids provided herein comprise a thirdnucleotide sequence encoding a molecule capable of reducing CD58 expression on the surface of an immune cell. In some embodiments, the third nucleotide sequence comprises nucleic acid sequences encoding a viral protein that reduces CD58 expression on the surface of an immune cell.

[0363] In some embodiments, the third recombinant nucleic acid sequence encodes a UL148polypeptide. The UL148 polypeptide is an ER-resident glycoprotein encoded by human cytomegalovirus (hCMV) that is involved in hCMV cell tropism.

[0364] In some embodiments, the UL148 polypeptide (strain Toledo) comprises the amino acidsequence of SEQ ID NO: 244: 221Mintz Ref. No.: 063384-521001WO MLRLLFTLVLLALYGPSVDASRDYVHVRLLSYRGDPLVFKHTFSGVRRPFTELGWA ACRDWDSMHCTPFWSTDLEQMTDSVRRYSTVSPGKEVTLQLHGNQTVQPSFLSFTC RLQLEPVVENVGLYVAYVVNDGERPQQFFTPQVDVVRFALYLETLSRIVEPLESGRL TVEFDTPDLALAPDLVSSLFVAGHGETDFYMNWTLRRSQTHYLEEMALQVEILKPR GVRHRAIIHHPKLQPGVGLWIDFCVYRYNARLTRGYVRYTLSPKARLPAKAEGWLV SLDRFIVQYLNTLLITMMAAIWARVLITYLVSRRR (SEQ ID NO: 244) or an amino acid sequence having at least 80%, 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% sequence identity of SEQ ID NO: 244.

[0365] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequenceencoding a UL148 polypeptide having the nucleic acid sequence of SEQ ID NO: 245: ATGAGCTTCCCCTGCAAGTTTGTGGCTTCTTTTCTGCTGATCTTTAACGTGTCTAG CAAAGGAGCCGTGAGCAAGGAAATCACCAACGCCCTGGAGACCTGGGGCGCCCT GGGCCAGGATATCAACCTGGACATCCCTAGCTTCCAGATGAGCGATGATATCGA CGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAAATCGCCCAGTTCAGAA AGGAGAAAGAGACATTCAAGGAAAAGGACACCTACAAGCTGTTCAAGAATGGC ACACTGAAAATCAAACACCTTAAGACAGACGATCAGGACATCTACAAAGTGTCT ATCTACGACACCAAGGGAAAGAACGTTCTGGAAAAAATTTTCGACCTGAAGATC CAGGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGTATCAACACCACCCTG ACCTGCGAGGTGATGAACGGCACCGATCCTGAGCTGAACCTCTACCAGGACGGC AAGCACCTGAAGCTGAGCCAAAGAGTCATCACCCACAAGTGGACAACAAGCCTG TCCGCCAAGTTCAAGTGCACCGCCGGCAACAAGGTGTCCAAGGAGAGCAGCGTG GAACCAGTGTCCTGCCCCGAGAAGGGCCTGGATATCTATCTGATTATCGGAATTT GTGGCGGCGGAAGCCTGCTGATGGTGTTCGTGGCCCTGCTGGTGTTCTACATCAC CAAGCGGAAGAAGCAGAGATCTAGACGGAACGACGAGGAACTGGAAACCCGCG CCCACAGGGTGGCCACCGAGGAAAGAGGCAGAAAGCCACACCAGATCCCCGCCT CAACACCTCAGAATCCCGCCACAAGCCAGCACCCCCCACCTCCTCCTGGCCATAG AAGCCAGGCCCCTAGCCACCGGCCTCCACCTCCCGGTCACAGAGTGCAGCACCA ACCTCAAAAACGGCCCCCCGCTCCAAGCGGCACCCAGGTGCACCAGCAGAAGGG CCCTCCTCTGCCTAGACCTAGAGTCCAGCCTAAGCCTCCTCACGGCGCTGCTGAG AACAGCCTGAGCCCTTCTTCTAAT (SEQ ID NO: 245) or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, 222Mintz Ref. No.: 063384-521001WO at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity of SEQ ID NO: 245.

[0366] In some embodiments, the UL148 polypeptide (strain Toledo) comprises the amino acidsequence of SEQ ID NO: 367: MLRLLFTLVLLALHGQSVGASRDYVHVRLLSYRGDPLVFKHTFSGVRRPFTELGWA ACRDWDSMHCTPFWSTDLEQMTDSVRRYSTVSPGKEVTLQLHGNQTVQPSFLSFTC RLQLEPVVENVGLYVAYVVNDGERPQQFFTPQVDVVRFALYLETLSRIVEPLESGRL AVEFDTPDLALAPDLVSSLFVAGHGETDFYMNWTLRRSQTHYLEEMALQVEILKPR GVRHRAIIHHPKLQPGVGLWIDFCVYRYNARLTRGYVRYTLSPKARLPAKAEGWLV SLDRFIVQYLNTLLITMMAAIWARVLITYLVSRRR (SEQ ID NO: 367) or an amino acid sequence having at least 80%, 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% sequence identity of SEQ ID NO: 367.

[0367] In some embodiments, the recombinant nucleic acid comprises a nucleotide sequenceencoding a UL148 polypeptide having nucleic acid sequence of SEQ ID NO: 369: ATGCTGCGCCTGCTTTTTACATTGGTATTGCTAGCTCTCCATGGACAGAGCGTCG GCGCGAGTCGCGACTACGTGCACGTGCGCTTGCTGAGCTACAGGGGTGATCCTTT GGTGTTCAAGCACACGTTCTCCGGTGTCCGTAGGCCCTTCACCGAGCTGGGCTGG GCAGCCTGCCGAGACTGGGATTCTATGCACTGTACTCCGTTTTGGTCTACAGATC TCGAACAGATGACTGACTCCGTGCGGCGTTATTCGACCGTCTCGCCGGGCAAGG AGGTGACCCTGCAACTACACGGCAACCAGACGGTGCAGCCATCCTTCCTGTCCTT CACCTGCCGGCTGCAGCTGGAGCCCGTGGTGGAGAATGTGGGTCTTTACGTCGCC TACGTGGTCAACGACGGCGAGCGCCCTCAGCAGTTCTTCACCCCTCAGGTCGATG TGGTACGCTTCGCGCTGTACCTGGAGACTCTGTCCCGCATTGTCGAGCCATTAGA ATCCGGCCGCCTGGCCGTGGAGTTCGACACCCCCGACCTGGCGCTCGCTCCCGAC CTGGTATCTTCTCTGTTCGTCGCCGGCCATGGGGAGACCGACTTCTACATGAACT GGACCTTACGCCGCAGCCAGACTCATTACTTGGAGGAGATGGCTCTCCAGGTGG AGATCCTGAAGCCCCGCGGGGTGCGCCACAGAGCTATCATCCACCACCCCAAGC TGCAGCCGGGAGTGGGGCTTTGGATTGACTTTTGCGTGTACCGCTATAACGCGCG CCTGACCCGTGGCTACGTGCGCTACACCCTGTCACCTAAAGCTCGCCTGCCGGCC AAGGCCGAGGGCTGGCTCGTTAGCCTGGACAGGTTTATCGTGCAGTATCTTAACA CGCTGCTCATCACCATGATGGCCGCCATCTGGGCACGAGTGCTGATCACCTACCT AGTGTCCCGGCGGCGT (SEQ ID NO: 369) or a nucleic acid sequence having at least 75%, 223Mintz Ref. No.: 063384-521001WO at least 80%, 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% sequence identity of SEQ ID NO:369.

[0368] In some embodiments, the viral protein further includes a protein localization sequenceor tag. In some embodiments, the protein localization sequence or tag is operably linked to the viral protein. In some embodiments, the protein localization sequence or tag is operably linked to the N-terminus of the viral protein. In some embodiments, the protein localization sequence or tag is operably linked to the C-terminus of the viral protein. In some embodiments, the protein localization sequence or tag is selected from the group consisting of an ER localization sequence or tag, a Golgi apparatus (Golgi) retention sequence or tag, a lysosome retention sequence or tag, a plasma membrane retention sequence or tag, a mitochondria retention sequence or tag, a peroxisome retention sequence or tag, a cytosolic retention sequence or tag, or a nuclear retention sequence or tag.

[0369] In some embodiments, the recombinant nucleic acid encoding a viral protein is operablylinked to a recombinant nucleic acid encoding a dominant negative mutant protein. In some embodiments, the recombinant nucleic acid encoding the UL148 polypeptide is operably linked with a recombinant nucleic acid encoding a dominant negative CD3epsilon polypeptide. In certain embodiments, the recombinant nucleic acid encoding the UL148 polypeptide operably linked to a dominant negative CD3epsilon polypeptide comprises the amino acid sequence MLRLLFTLVLLALHGQSVGASRDYVHVRLLSYRGDPLVFKHTFSGVRRPFTELGWA ACRDWDSMHCTPFWSTDLEQMTDSVRRYSTVSPGKEVTLQLHGNQTVQPSFLSFTC RLQLEPVVENVGLYVAYVVNDGERPQQFFTPQVDVVRFALYLETLSRIVEPLESGRL AVEFDTPDLALAPDLVSSLFVAGHGETDFYMNWTLRRSQTHYLEEMALQVEILKPR GVRHRAIIHHPKLQPGVGLWIDFCVYRYNARLTRGYVRYTLSPKARLPAKAEGWLV SLDRFIVQGGGGSGGGGSGGGGSDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEI LWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLY LRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNRKAKAKPLYKYKSRR SFIDEKKMP (SEQ ID NO: 246) or an amino acid sequence having at least 80%, 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% sequence identity of SEQ ID NO: 246.

[0370] In certain embodiments, the recombinant nucleic acid encoding the UL148 polypeptideoperably linked to a dominant negative CD3epsilon polypeptide comprises the nucleic acid sequence 224Mintz Ref. No.: 063384-521001WO ATGCTGCGCCTGCTTTTTACATTGGTATTGCTAGCTCTCCATGGACAGAGCGTCG GCGCGAGTCGCGACTACGTGCACGTGCGCTTGCTGAGCTACAGGGGTGATCCTTT GGTGTTCAAGCACACGTTCTCCGGTGTCCGTAGGCCCTTCACCGAGCTGGGCTGG GCAGCCTGCCGAGACTGGGATTCTATGCACTGTACTCCGTTTTGGTCTACAGATC TCGAACAGATGACTGACTCCGTGCGGCGTTATTCGACCGTCTCGCCGGGCAAGG AGGTGACCCTGCAACTACACGGCAACCAGACGGTGCAGCCATCCTTCCTGTCCTT CACCTGCCGGCTGCAGCTGGAGCCCGTGGTGGAGAATGTGGGTCTTTACGTCGCC TACGTGGTCAACGACGGCGAGCGCCCTCAGCAGTTCTTCACCCCTCAGGTCGATG TGGTACGCTTCGCGCTGTACCTGGAGACTCTGTCCCGCATTGTCGAGCCATTAGA ATCCGGCCGCCTGGCCGTGGAGTTCGACACCCCCGACCTGGCGCTCGCTCCCGAC CTGGTATCTTCTCTGTTCGTCGCCGGCCATGGGGAGACCGACTTCTACATGAACT GGACCTTACGCCGCAGCCAGACTCATTACTTGGAGGAGATGGCTCTCCAGGTGG AGATCCTGAAGCCCCGCGGGGTGCGCCACAGAGCTATCATCCACCACCCCAAGC TGCAGCCGGGAGTGGGGCTTTGGATTGACTTTTGCGTGTACCGCTATAACGCGCG CCTGACCCGTGGCTACGTGCGCTACACCCTGTCACCTAAAGCTCGCCTGCCGGCC AAGGCCGAGGGCTGGCTCGTTAGCCTGGACAGGTTTATCGTGCAGGGCGGAGGT GGAAGCGGCGGGGGCGGCTCTGGCGGAGGAGGCAGCGACGGCAATGAGGAAAT GGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACCGTGAT CCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACGACAAA AACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGATCACCTG TCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTTACCCTC GGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCAGAGTGT GCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGATTGTGA AAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAAGAACAG AAAGGCTAAAGCCAAGCCCTTGTACAAGTACAAAAGCAGACGCTCTTTTATAGA TGAGAAGAAGATGCCG (SEQ ID NO: 247) or a nucleotide sequence having at least 80%, 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% sequence identity of SEQ ID NO: 247.

[0371] In certain embodiments, the recombinant nucleic acid encoding the UL148 polypeptideoperably linked to a dominant negative CD3epsilon polypeptide comprises the amino acid sequence of DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDED HLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMLRLLFTLVL 225Mintz Ref. No.: 063384-521001WO LALHGQSVGASRDYVHVRLLSYRGDPLVFKHTFSGVRRPFTELGWAACRDWDSMH CTPFWSTDLEQMTDSVRRYSTVSPGKEVTLQLHGNQTVQPSFLSFTCRLQLEPVVEN VGLYVAYVVNDGERPQQFFTPQVDVVRFALYLETLSRIVEPLESGRLAVEFDTPDLA LAPDLVSSLFVAGHGETDFYMNWTLRRSQTHYLEEMALQVEILKPRGVRHRAIIHHP KLQPGVGLWIDFCVYRYNARLTRGYVRYTLSPKARLPAKAEGWLVSLDRFIVQVMS VATIVIVKICITGGLLLLVYYWSSRRR (SEQ ID NO: 147) or an amino acid sequence having at least 80%, 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% sequence identity of SEQ ID NO: 147.

[0372] In certain embodiments, the recombinant nucleic acid encoding the UL148 polypeptideoperably linked to a dominant negative CD3epsilon polypeptide comprises the nucleic acid sequence of GACGGCAATGAGGAAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATC AGCGGCACCACCGTGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGT GGCAGCACAACGACAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGC TCTGATGAGGATCACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGT TATTACGTGTGTTACCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGT ACCTTAGAGCCAGAGTGTGCGAGAACTGCATGGAAATGGATATGCTGCGCCTGC TTTTTACATTGGTATTGCTAGCTCTCCATGGACAGAGCGTCGGCGCGAGTCGCGA CTACGTGCACGTGCGCTTGCTGAGCTACAGGGGTGATCCTTTGGTGTTCAAGCAC ACGTTCTCCGGTGTCCGTAGGCCCTTCACCGAGCTGGGCTGGGCAGCCTGCCGAG ACTGGGATTCTATGCACTGTACTCCGTTTTGGTCTACAGATCTCGAACAGATGAC TGACTCCGTGCGGCGTTATTCGACCGTCTCGCCGGGCAAGGAGGTGACCCTGCAA CTACACGGCAACCAGACGGTGCAGCCATCCTTCCTGTCCTTCACCTGCCGGCTGC AGCTGGAGCCCGTGGTGGAGAATGTGGGTCTTTACGTCGCCTACGTGGTCAACG ACGGCGAGCGCCCTCAGCAGTTCTTCACCCCTCAGGTCGATGTGGTACGCTTCGC GCTGTACCTGGAGACTCTGTCCCGCATTGTCGAGCCATTAGAATCCGGCCGCCTG GCCGTGGAGTTCGACACCCCCGACCTGGCGCTCGCTCCCGACCTGGTATCTTCTC TGTTCGTCGCCGGCCATGGGGAGACCGACTTCTACATGAACTGGACCTTACGCCG CAGCCAGACTCATTACTTGGAGGAGATGGCTCTCCAGGTGGAGATCCTGAAGCC CCGCGGGGTGCGCCACAGAGCTATCATCCACCACCCCAAGCTGCAGCCGGGAGT GGGGCTTTGGATTGACTTTTGCGTGTACCGCTATAACGCGCGCCTGACCCGTGGC TACGTGCGCTACACCCTGTCACCTAAAGCTCGCCTGCCGGCCAAGGCCGAGGGCT 226Mintz Ref. No.: 063384-521001WO GGCTCGTTAGCCTGGACAGGTTTATCGTGCAGGTGATGAGCGTGGCCACCATCGT GATTGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCC TCCCGGCGGCGT (SEQ ID NO: 180) or a nucleotide sequence having at least 80%, 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% sequence identity of SEQ ID NO: 180. D. Reduction of MHC class I – CD8 Interaction

[0373] In another aspect, recombinant nucleic acids provided herein further comprise a fourthnucleotide sequence encoding a fourth molecule capable of reducing the MHC class I –CD8alpha interaction between graft cells and CD8 positive host T cells.

[0374] In HvG the host immune system can recognize graft cells as foreign and attack and killthe graft cells. For example, CD8+ T cells can recognize foreign graft cells by the unmatched MHC class I on their cell surface and eliminate those cells. Blocking or inhibiting the interaction between CD8alpha on a host cell and MHC class I on a graft cell can prevent the recognition and elimination of graft cells in a host by this mechanism.

[0375] In some embodiments, the recombinant nucleic acids provided herein include a fourthnucleotide sequence encoding a CD8alpha blocker or CD8alpha inhibitor. In some embodiments, the CD8alpha blocker or CD8alpha inhibitor comprises an MHC class I binder (i.e., a protein domain capable of binding MHC class I, or a synthetic MHC class I binder).

[0376] In some embodiments, the CD8alpha inhibitor comprises from N to C terminus anMHC class I binder, a linker, and a B2M or fragment thereof. In some embodiments, the CD8alpha inhibitor comprises a CD8alpha extracellular domain or a fragment thereof. In some embodiments, the CD8alpha inhibitor comprises from N to C terminus a CD8alpha extracellular domain and a B2M or fragment thereof. In some embodiments, the CD8alpha inhibitor additionally comprises a linker. In some embodiments, the CD8alpha inhibitor comprises from N to C terminus a CD8alpha extracellular domain or a fragment thereof, a linker, and a B2M or fragment thereof. Exemplary linkers are listed in Table 19.

[0377] In some embodiments, the MHC class I binder comprises a sequence set forth in SEQID NOs: 411 or 412. In some embodiments, the MHC class I binder comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NOs: 411 or 412. 227Mintz Ref. No.: 063384-521001WO

[0378] In some embodiments, the CD8alpha inhibitor comprises a sequence set forth in SEQID NOs: 407, 410, 411, 412, or 499. In some embodiments, the CD8alpha inhibitor comprises a sequence set forth in SEQ ID NOs: 410, 411, or 499 and a B2M. In some embodiments, the CD8alpha inhibitor comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NOs: 407, 410, 411, 412, or 499.

[0379] In some embodiments, the CD8alpha inhibitor comprises a sequence having at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGASWLFQPRGAAASPTFLLYLNQNKP KAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPA SEQ ID NO: 499. In some embodiments, the CD8alpha inhibitor comprises the sequence of SEQ ID NO: 499.

[0380] In some embodiments, the CD8alpha inhibitor is encoded by a nucleic acid sequencecomprising the sequence of TCTCAATTTCGAGTGTCCCCTCTGGACAGAACCTGGAACCTGGGCGAGACAGTGG AACTGAAATGCCAGGTGCTGCTGTCTAATCCTACCAGCGGCGCTAGCTGGCTGTT CCAGCCTAGAGGCGCCGCTGCTTCTCCAACCTTCCTGCTGTATCTGAACCAGAAC AAGCCCAAGGCCGCCGAGGGACTGGACACCCAGCGGTTCAGCGGCAAGCGGCTG GGAGATACCTTCGTGCTCACCCTGAGCGACTTCAGACGCGAAAACGAGGGCTAC TACTTCTGCAGCGCCCTGTCCAACAGCATCATGTACTTTAGCCACTTCGTCCCCGT GTTCCTGCCTGCC (SEQ ID NO: 518).

[0381] In some embodiments, the recombinant nucleic acids provided herein comprise a fourthnucleotide sequence encoding a CD8alpha inhibitor, wherein the CD8alpha inhibitor comprises a sequence selected from the sequences set forth in Table 40A, 40B, and 40C. In some embodiments, the recombinant nucleic acids provided herein comprise a fourth nucleotide sequence encoding a CD8alpha inhibitor, wherein the CD8alpha inhibitor comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences set forth in Table 40A, 40B, and 40C. E. Embedded shRNA 228Mintz Ref. No.: 063384-521001WO

[0382] In another aspect, the recombinant nucleic acids provided herein can include a promoterhaving an intron, wherein the intron comprises one or more embedded shRNAs. In some embodiments, the shRNA is a single shRNA. In some embodiments, the shRNA is a multiplexed shRNA comprising two, three, four, five, or more shRNAs. Exemplary multiplexed shRNAs are described below. An exemplary scheme of multiplexed shRNAs embedded in an intron of a promoter sequence is shown in FIG.17A.

[0383] In some embodiments, the promoter having an intron comprises an EF-1alpha, anhUbC, or an MNDU3 promoter. In some embodiments, a nucleotide sequence encoding one or more shRNAs to reduce one, two, or three of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell comprises one or more shRNA coding sequences embedded in an intron of the promoter. In some embodiments, the promoter can be an EF-1alpha promoter. In some embodiments, the promoter can be a hUbC promoter. In some embodiments, the promoter can be an MNDU3 promoter. In some embodiments, the promoter and the intron sequence are from the same promoter. In some embodiments, the promoter and the intron sequence are from different promoters. In some embodiments, the promoter is an MNDU3 promoter and the intron sequence is from an EF- 1alpha promoter.

[0384] In some embodiments, the shRNA coding sequence embedded in an intron of apromoter sequence comprises a CD3zeta-specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a FasR-specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a B2M-specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a CIITA-specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a CD3epsilon-specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a Trac- specific shRNA. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a CD58-specific shRNA. In some embodiments the shRNA coding sequence embedded in an intron of a promoter sequence comprises one or more shRNAs selected from the shRNAs listed in Table 6. In some embodiments the shRNA coding sequence embedded in an intron of a promoter sequence comprises a shRNA selected from the shRNAs listed in Table 13. In some embodiments the shRNA coding sequence embedded in an intron of a promoter sequence comprises a shRNA selected from the shRNAs listed in Table 229Mintz Ref. No.: 063384-521001WO 15. In some embodiments the shRNA coding sequence embedded in an intron of a promoter sequence comprises a shRNA selected from the shRNAs listed in Table 16. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 6, Table 13, Table 15, or Table 16.

[0385] The shRNA coding sequence embedded in an intron of a promoter sequence can beflanked by additional sequences that can enhance shRNA processing in the cell. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence can include a mirE microRNA scaffold sequence. In some embodiments, the shRNA coding sequence embedded in an intron of a promoter sequence can include a mir155 microRNA scaffold sequence. Exemplary shRNA coding sequences embedded in mirE and mir155 scaffold sequences are listed in Tables 15 and 16.

[0386] The embedded shRNA can be flanked by an intronic region from a promoter. In someembodiments, the embedded shRNA comprises an intronic sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 15, or Table 16.

[0387] Exemplary promoter sequences, microRNA scaffold regions, shRNA coding sequencesembedded in an intron of a promoter sequence and related sequences that are useful in the recombinant nucleic acids disclosed herein include those in Table 15 and Table 16 below. Table 15: Exemplary Sequences for embedded shRNA230Mintz Ref. No.: 063384-521001WOF. Multiplexed shRNA

[0388] In another aspect, the recombinant nucleic acids provided herein can further includemultiplexed shRNAs. Multiplexed shRNAs can comprise 2, 3, 4, 5 or more shRNAs that are expressed from the same promoter. An exemplary scheme of multiplexed shRNAs is shown in FIG.18A.

[0389] In some embodiments, the multiplexed shRNAs comprise 2, 3, 4, 5 or more shRNAstargeting one or more genes. In some embodiments, the recombinant nucleic acids provided herein further comprise multiplexed shRNAs embedded in an intron of a promoter sequence.

[0390] In some embodiments, the multiplexed shRNA embedded in an intron of a promotersequence comprises a CD3zeta-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a FasR-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a B2M-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a CIITA-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a CD3epsilon-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a TRAC-specific shRNA coding sequence. In some embodiments, the multiplexed shRNA embedded in an intron of a promoter sequence comprises a CD58-specific shRNA coding sequence. In some embodiments, the multiplexed 231Mintz Ref. No.: 063384-521001WO shRNA comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 6, Table 13, Table 15, or Table 16. In some embodiments, the multiplexed shRNA comprises a sequence listed in Table 6, Table 13, Table 15, or Table 16. In some embodiments, the multiplexed shRNA comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence according to SEQ ID NOs: 522, 530, 437, 803, 804, or 805. Table 16: Exemplary sequences for multiplexed shRNA232Mintz Ref. No.: 063384-521001WO233Mintz Ref. No.: 063384-521001WO234Mintz Ref. No.: 063384-521001WO235Mintz Ref. No.: 063384-521001WO236Mintz Ref. No.: 063384-521001WO237Mintz Ref. No.: 063384-521001WO238Mintz Ref. No.: 063384-521001WO TATTAACGCTTACAATTT G. Apoptosis Reduction

[0391] In another aspect, the recombinant nucleic acids provided herein further comprise anucleic acid sequence encoding a molecule capable of reducing apoptosis in an immune cell.

[0392] As described above, graft cells in a host subject can be attacked by the host immunecells. Cytotoxic host immune cells such as T cells and NK cells can induce apoptosis in graft cells via injection of Granzyme B (GrzB). SerpinB9 is a Granzyme B inhibitor that can reduce the amount of Granzyme B that is present in a cell thereby reducing apoptosis.

[0393] In some embodiments, the molecule capable of reducing apoptosis includes a SerpinB9.

[0394] In some embodiments, the SerpinB9 comprises an amino acid sequence having at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences in SEQ ID NOs: 421 or 422. In some embodiments, the SerpinB9 comprises a sequence selected from SEQ ID NOs: 421 or 422. In some embodiments, the SerpinB9 comprises the sequence of SEQ ID NO: 421. In some embodiments, the SerpinB9 comprises the sequence of SEQ ID NO: 422. In some embodiments, the SerpinB9 is encoded in a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences in SEQ ID NOs: 423 or 424. In some embodiments, the SerpinB9 comprises a sequence selected from SEQ ID NOs: 423 or 424. Cell Selection Molecules

[0395] In another aspect, the recombinant nucleic acids provided herein can include a nucleicacid sequence encoding a cell selection molecule.

[0396] Presentation of a cell selection molecule on the surface of an engineered cell enablesselection of engineered cells using a selection mechanism. A cell selection molecule should not be naturally expressed on the cell

[0397] Exemplary selection mechanisms include, but are not limited to, tethered antibodies orantibody fragments or cognate ligands of the cell selection molecule. Human CD34 adhesion molecule is of human origin and not expressed on mature immune effector cells, including human T and natural killer (NK) cells and GMP-grade immunomagnetic CD34 enrichment reagents are commercially available and have been used for to enrich CD34+ hematopoietic stem cells. Thus, human CD34 and natural and synthetic epitopes thereof are useful as cell 239Mintz Ref. No.: 063384-521001WO selection molecules for engineered immune cells that naturally do not express CD34, for example T cells.

[0398] In some embodiments, the cell selection molecule includes a CD34 transgene, afragment of a CD34 transgene, or a CD34 epitope. In some embodiments, the CD34 epitope includes a QBEND / 10 epitope (e.g., described in Bister et al., Mol Ther Oncolytics. (2021) 11;23:534–546). In some embodiments, the cell selection molecule includes a linker.

[0399] In some embodiments, the cell selection molecule includes an amino acid sequencewith at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence in Table 17 or Table 18.

[0400] In some embodiments, the cell selection molecule comprises a sequence selected fromSEQ ID NOs: 695, 621, 623, 625, 627, or 629. In some embodiments, the cell selection molecule comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence selected from SEQ ID NOs: 695, 621, 623, 625, 627, or 629.

[0401] In some embodiments, the cell selection molecule is encoded in a nucleic acid sequenceselected from SEQ ID NOs: 638, 639, 640, 641, or 642. In some embodiments, the cell selection molecule is encoded in a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence selected from SEQ ID NOs: 638, 639, 640, 641, or 642.240Mintz Ref. No.: 063384-521001WO241Mintz Ref. No.: 063384-521001WOLinkers

[0402] In another aspect, provided herein are linkers useful in the recombinant nucleic acidsprovided herein.

[0403] In some embodiments, the linker includes a sequence with at least 90%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to a linker sequence in Table 19. In some embodiments, the linker comprises a linker with a sequence set forth in Table 19. Table 19: Exemplary linker sequences242Mintz Ref. No.: 063384-521001WOPromoters

[0404] In another aspect, provided herein are certain expression control sequences useful forexpressing the recombinant nucleic acids provided herein. Non-limiting exemplary embodiments of the recombinant nucleic acids of the disclosure can include one or more of the following features. 243Mintz Ref. No.: 063384-521001WO

[0405] In some embodiments, any of the recombinant nucleic acids provided herein can beoperably linked, e.g., placed under the control of, other structural elements (e.g., promoter sequences) required for expression of such recombinant nucleic acids in host cells, in subjects, or in ex-vivo cell-free expression systems.

[0406] As used herein, the terms “promoter” and “promoter sequence” are usedinterchangeably to refer to a DNA sequence that promotes the expression of a protein coding open reading frame or a nucleotide sequence encoding a functional RNA (e.g., an shRNA). Those skilled in the art understand that different promoters direct gene expression in different tissues or cell types, at different stages of development, or in response to different environmental or physiological conditions.

[0407] In some embodiments, the nucleotide sequences encoding nucleic acids or proteins toreduce one, two, three, or more of TCR-CD3 complex expression, MHC class I and MHC class II expression, and / or CD58 expression on the surface of an immune cell can be operably linked to a single promoter sequence. In some embodiments, each of the nucleotide sequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression, and / or CD58 expression on the surface of an immune cell can be operably linked a promoter sequence.

[0408] In some embodiments, the recombinant nucleic acids provided here, comprising one,two, three, or more nucleotide sequences encoding nucleic acids or proteins to reduce one, two, three, or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can be operably linked to two different promoter sequences. In some embodiments, the first and second nucleic acid nucleotide sequences can be operably linked to a first promoter sequence and the third nucleotide sequence is operably linked to a second promoter sequence. In some embodiments, the second and third nucleotide sequences can be operably linked to a first promoter sequence and the first nucleotide sequence is operably linked to a second promoter sequence. In some embodiments, the first, second, and third nucleotide sequences can be operably linked to the same promoter sequence. In some embodiments, the first and third nucleotide sequences can be operably linked to a first promoter sequence and the second nucleotide sequence is operably linked to a second promoter sequence. In some embodiments, the nucleotide sequences encoding nucleic acids or proteins to reduce one, two, three, or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can be each operably linked to a different promoter sequence. In some 244Mintz Ref. No.: 063384-521001WO embodiments, two or more promoter sequences each can be operably linked to a nucleotide sequence in the same direction. In some embodiments two or more promoter sequences each can be operably linked to a nucleotide sequence in opposite directions. In some embodiments, a first promoter can be operably linked to a nucleotide sequence encoding an shRNA and a second promoter can be operably linked to nucleotide sequences encoding the polypeptides. In some embodiments, the first promoter sequence can be operably linked to a nucleotide sequence encoding a polypeptide and the second promoter sequence can be operably linked to a nucleotide sequence encoding an shRNA.

[0409] In some embodiments, the single promoter sequence is selected from the groupconsisting of a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF- 1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, and an NFkB-responsive promoter. In some embodiments, the single promoter sequence is selected from the group consisting of a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, and an NFkB-responsive promoter and the first, second, and third nucleic acid sequence are under the control of the single promoter. In some embodiments, the single promoter sequence is an MNDU3 promoter. In some embodiments, the single promoter sequence is an EF-1alpha promoter. In some embodiments, the single promoter sequence is a core EF-1alpha promoter. In some embodiments, the single promoter sequence is an hUbC promoter. In some embodiments, the single promoter sequence is a PGK promoter. In some embodiments, the single promoter is a U6 promoter. In some embodiments, the single promoter sequence is a SFFV promoter. In some embodiments, the single promoter sequence is an NFkB-responsive promoter.

[0410] In some embodiments, the first promoter sequence is selected from the group consistingof a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, and an NFkB-responsive promoter. In some embodiments, the second promoter is selected from the group consisting of a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, and an NFkB-responsive promoter. In some embodiments, the third promoter is selected from the group consisting of a MND promoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA 245Mintz Ref. No.: 063384-521001WO promoter, a Gamma Retro 5’ LTR promoter, and an NFkB-responsive promoter. In some embodiments, the first, second, or third promoter can be an MND promoter. In some embodiments, the first, second, or third promoter can be an MNDU3 promoter. In some embodiments, the first, second, or third promoter can be an hUbC promoter. In some embodiments, the first, second, or third promoter can be an EF-1alpha promoter. In some embodiments, the first, second, or third promoter can be a core EF-1alpha promoter. In some embodiments, the first, second, or third promoter can be an SFFV promoter. In some embodiments, the first, second, or third promoter can be an NFkB-responsive promoter. In some embodiments, the first, second, or third promoter can be U6 promoter. In some embodiments, the first, second, or third promoter can be CAG promoter. In some embodiments, the first, second, or third promoter can be CBA promoter. In some embodiments, the first, second, or third promoter can be Gamma Retro 5’ LTR promoter.

[0411] Table 20 provides certain exemplary promoter sequences for use in the recombinantnucleic acids described herein: Table 20: Exemplary Promoter Sequences246Mintz Ref. No.: 063384-521001WO247Mintz Ref. No.: 063384-521001WO248Mintz Ref. No.: 063384-521001WO249Mintz Ref. No.: 063384-521001WO Additional Elements

[0412] In another aspect, the recombinant nucleic acids provided herein further compriseadditional regulatory elements. In some embodiments, the additional regulatory element can be a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence, an insulator sequence, a polyA sequence, and / or an LTR sequence. In some embodiments, the recombinant nucleic acids provided herein further comprise a polyadenylation (PolyA) signal. In some embodiments, the polyadenylation signal includes a simian virus 40 (SV40), α-globin, β-globin, a human growth hormone (hGH), a bovine growth hormone (BGH), a herpes simplex virus type 1, a thymidine kinase (HSV TK), or a synthetic polyadenylation (Synt poly A) polyadenylation signal.

[0413] In some embodiments, the WPRE is a sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence a nucleic acid sequence comprising SEQ ID NO: 450. AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATG TTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATT GCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTT TATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTG CTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG GACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTC GGGGAAGCTGACGTCCTTTCCGGGGCTGCTCGCCTGTGTTGCCACCTGGATTCTG CGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTC CCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAAA CGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO: 450).

[0414] In some embodiments, the insulator is a sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence a nucleic acid sequence comprising SEQ ID NO: 366. Gagctcacggggacagccccctcccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgccc ggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggca cgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctt (SEQ ID NO: 366). 250Mintz Ref. No.: 063384-521001WO

[0415] In some embodiments, the recombinant nucleic acids can include one or more codingsequences for ribosome skipping sequences or autoproteolytic peptide sequences.

[0416] In some embodiments, the recombinant nucleic acid can additionally include one ormore ribosome skipping sequences located between the first, second, and / or third nucleotide sequence. Exemplary ribosome skipping sequences are described supra.

[0417] In some embodiments, the coding sequence for a polypeptide construct in therecombinant nucleic acids described herein can include one or more molecular alterations. Exemplary types of molecular alterations in the coding sequences described herein can be one or more of deletions, substitutions, insertion, duplications, mutations, frameshift variants, splice variants, and combinations of any thereof.

[0418] A coding sequence for a polypeptide construct can be a construct of genetic materialthat includes coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a cell, in vivo and / or ex vivo. The coding sequence for a polypeptide construct can be inserted into a vector for targeting to a desired host cell and / or into a subject. Accordingly, in some embodiments, the term “coding sequence for a polypeptide construct” can be used interchangeably with the term “expression construct.” In some embodiments, a coding sequence for a polypeptide construct can be a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and / or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene. II. Recombinant Nucleic Acid Combinations

[0419] In another aspect, provided herein are recombinant nucleic acids that include, but arenot limited to, two, three, or more nucleotide sequences encoding nucleic acids or proteins capable of reducing cell surface expression of one or more of TCR-CD3 complex expression, MHC class I and class II expression, and / or CD58 expression on the surface of an immune cell.

[0420] In some embodiments, the recombinant nucleic acids can include two or morenucleotide sequences arranged in a bi-cistronic, tri-cistronic, or multi-cistronic configuration expressed via one or more promoter sequences. In some embodiments, the individual nucleotide sequences in the bi-cistronic, tri-cistronic, or multi-cistronic configuration are 251Mintz Ref. No.: 063384-521001WO separated by one or more ribosome skipping sequences disposed between each individual nucleotide sequence.

[0421] The term “ribosome skipping sequence” as used herein refers to certain virus-derivedpeptide sequences that induce ribosomal skipping during translation resulting an apparent cleavage of a polypeptide sequence. Consequently, ribosome skipping sequences are sometimes interchangeably referred to as “self-cleaving” peptides. Exemplary ribosome skipping sequences include, but are not limited to, the F2A peptide derived from foot-and- mouth disease virus, the E2A peptide derived from equine rhinitis A virus, the P2A peptide derived from porcine teschovirus-1, the T2A peptide derived from thosea asigna virus, and the Furin cleavable P2A sequence. In some embodiments, the F2A peptide sequence includes the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 250), the E2A sequence includes the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 251), the T2A sequence includes the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 252), the P2A sequence includes the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 253), and the Furin cleavable P2A sequence includes the amino acid sequence RAKRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 254).

[0422] In some embodiments, the T2A sequence is encoded by the nucleic acid sequenceGAGGGCAGAGGCTCCCTTCTCACGTGCGGAGATGTCGAGGAAAATCCAGGACCC (SEQ ID NO: 249) or GAGGGCAGAGGCAGCCTGCTGACCTGCGGCGATGTGGAGGAGAACCCCGGACCT (SEQ ID NO: 584).

[0423] In some embodiments, the P2A sequence is encoded by the nucleic acid sequenceGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGC CCT (SEQ ID NO: 248).

[0424] In some embodiments, the recombinant nucleic acid includes from 5’ to 3’ end the firstnucleotide sequence; the second nucleotide sequence; and the third nucleotide sequence. In some embodiments, the recombinant nucleic acid includes from 5’ to 3’ end: the first nucleotide sequence; the third nucleotide sequence; and the second nucleotide sequence. In some embodiments, the recombinant nucleic acid includes from 5’ to 3’ end the second nucleotide sequence; the first nucleotide sequence; and the third nucleotide sequence. In some embodiments, the recombinant nucleic acid includes from 5’ to 3’ end the second nucleotide sequence; the third nucleotide sequence; and the first nucleotide sequence. In some 252Mintz Ref. No.: 063384-521001WO embodiments, the recombinant nucleic acid includes from 5’ to 3’ end the third nucleotide sequence; the first nucleotide sequence; and the second nucleotide sequence. In some embodiments, the recombinant nucleic acid includes from 5’ to 3’ end the third nucleotide sequence; the second nucleotide encoding; and the first nucleotide sequence.

[0425] Exemplary recombinant nucleic acids including two, three, or more nucleotidesequences as provided herein are shown in FIG. 1B, FIG. 2A, FIG. 3, FIG. 4A, FIG. 5A, FIG.6A, FIG.7A, FIG.7B, FIG.8, FIG.9, FIG.10, FIG.11A, FIG.11B, FIG.13, FIG. 14A, FIG.15A, FIG.16A, FIG.17A, FIG.18A, FIG.22A, FIG.24A, FIG.42A. FIG.42B, FIG.43A, and FIG.43B.

[0426] In some embodiments, the recombinant nucleic acid can include, but is not limited to,two, three, or more nucleotide sequences encoding nucleic acids or proteins to reduce cell surface expression of one or more of TCR-CD3 complex, MHC class I and MHC class II, and / or CD58 on an immune cell. In some embodiments, the recombinant nucleic acid comprises three nucleotide sequences. In some embodiments, the first nucleotide sequence encodes a first molecule to reduce TCR-CD3 complex expression on the surface of an immune cell, wherein the first molecule comprises a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein subunit of the TCR-CD3 complex, or combinations thereof; the second nucleotide sequence encodes a second molecule to reduce MHC class I and MHC class II expression on the surface of an immune cell, wherein the second nucleotide sequence encodes a protein expression blocker (PEBL), an shRNA, or a dominant negative protein capable of down-regulating transcription of the MHC class I and MHC class II, or combinations thereof; and the third nucleotide sequence encodes a third molecule to reduce CD58 expression on the surface of an immune cell, wherein the third molecule comprises a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein capable of reducing the binding of CD58 to its cognate ligand CD2, or combinations thereof.

[0427] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a PEBL comprising an scFv that binds a TCR-CD3 complex component plus a protein localization sequence or tag, a 2A ribosome skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and a PEBL comprising an scFv that binds CD58 plus a protein localization sequence or tag. In some embodiments, protein localization sequence or 253Mintz Ref. No.: 063384-521001WO tag on the TCR-CD3 PEBL comprises an ER localization sequence selected from the group consisting of [SEQ ID NOs: 1-13]. In some embodiments, the protein localization sequence or tag on the CD58 PEBL comprises an ER localization sequence selected from the group consisting of [SEQ ID NOs: 1-13]. In some embodiments, the recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 255: ATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTCT GCTGATTCCCGATATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCTTCTGTC GGCGATAGAGTGACCATGACATGCAGCGCCACATCTAGCGTGTCCTACATGCAC TGGTATCAGCAGAAACCTGGCAAGGCCCCTAAGCGGTGGATCTACGACACCAGC AAGCTGGCCAGCGGCGTGCCAGCCAGATTCATCGGCAGCGGATCTGGCACAGAG TTCACCCTGACCATCAGCAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGTC AGCAATGGTCCTCCAACCCCCTGACATTTGGCGGAGGCACCAAGGTGGAAATCA AGGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGG GAGAAGTGCAGCTGCTGCAGAGCGGCGGCGGACTGGTGCAACCTGGAGGCAGCC TGAGACTGAGCTGTGCCGCTTCTGGATATAAGTTCACCAGCTACGTGATGCACTG GGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGGCTACATCAACCCCTA CAACGATGTGACCAAGTACAACGAGAAGTTTAAGGGCAGATTCACACTGTCTAG AGATAATAGCAAGAACACCCTCTACCTGCAGATGAACAGCCTGCGGGCCGAGGA CACCGCCGTGTACTACTGCGCCAGAGGCTCCTATTACGATTACGACGGCTTCGTG TACTGGGGCCAGGGCACCCTGGTTACAGTGTCCAGCGCGGCCGCCGGCGGCGGC AGCGGCGGCGGATCTGCCGAAAAGGACGAGCTGATCTACATCTGGGCCCCACTG GCCGGCACATGCGGCGTGCTGCTGCTGTCCCTGGTGATCACCCTGTATTGCGCCG CCGCTGGTGGGACCGAGAACCTGTACTTCCAGTCCGGCAGCACCAAGCACATCC TGTTCCGGCGCCGTCGCAGAGGCTTTAGACAGAAAAAGAAAAAGCGGGACGCCA CCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGCCCTA TGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCTC CAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA 254Mintz Ref. No.: 063384-521001WO TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGA GGGCAGAGGCTCCCTTCTGACCTGCGGAGATGTCGAGGAAAATCCAGGACCCAT GGCCTTGCCCGTGACTGCTCTGCTCCTGCCCCTCGCACTGCTGCTGCACGCCGCG AGGCCCGATATCCTGCTGACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCG AGCGGGTGTCCTTCAGCTGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACT GGTATCAGCAGCGGACAATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTG AGAGCATCAGCGGCATCCCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACT TCACCCTGACCATCAACTCCGTGGAAAGCGAGGACATCGCCGACTACTACTGCC AGCAAAGCAACAGCTGGCCTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCA AGGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGG GACAAGTGCAGCTGCAGCAGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGC GTGAAGATCAGCTGCAAGGGCAGCGGCTACACCTTCACCGACTACGCCATCCAC TGGGTCAAGCAGTCTCACGCCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTG CACTACGACAAAACCAACTACAACCAGAAATTCAAGGGCAAGGCCAGCATGACC GTGGACAAGTCCAGCTCTACAGCCTACATGGAACTGGCTAGACTGACAAGCGAG GACAGCGCTATCTACTATTGTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATA ATTGGGGACAGGGCACAACCCTGACCGTGTCCTCTGCGGCCGCCGGTGGTGGCT CTGGTGGGGGGTCAGCGGAAAAGGATGAACTGATTTATATTTGGGCCCCGCTCG CTGGCACATGCGGGGTCCTGCTCTTGAGCTTGGTCATTACTTTGTATTGTGCCGCT GCGGGCGGTACCGAAAACCTCTATTTCCAAAGTGGGTCCACTAAACATATTTTGT TCAGAAGACGGAGGAGAGGATTTCGCCAGAAGAAGAAAAAGAGAGAC taa (SEQ ID NO: 255). SEQ ID NO: 255 comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 256.

[0428] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell comprising nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 256: MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSTLSASVGDRVTMTCSATSSVSYMHWY QQKPGKAPKRWIYDTSKLASGVPARFIGSGSGTEFTLTISSLQPDDFATYYCQQWSSN PLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGEVQLLQSGGGLVQPGGSLRLSCAASG 255Mintz Ref. No.: 063384-521001WO YKFTSYVMHWVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSRDNSKNTLYL QMNSLRAEDTAVYYCARGSYYDYDGFVYWGQGTLVTVSSAAAGGGSGGGSAEKD ELIYIWAPLAGTCGVLLLSLVITLYCAAAGGTENLYFQSGSTKHILFRRRRRGFRQKK KKRDATNFSLLKQAGDVEENPGPMAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQR LRGTISKAVQNKVEGILQDVQKFSDNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYM YAYRWIRNHLEEHTDTCLPKQSVYDAYRKYCESLACCRPLSTANFGKIIREIFPDIKA RRLGGRGQSKYCYSGIRRKTLVSMPPLPGLDLKGSESPEMGPEVTPAPRDEGRGSLL TCGDVEENPGPMALPVTALLLPLALLLHAARPDILLTQSPAILSVSPGERVSFSCRASQ SIGTSIHWYQQRTIGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYC QQSNSWPFTFGSGTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELVRPGESVKIS CKGSGYTFTDYAIHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMTVDKSSS TAYMELARLTSEDSAIYYCARSFYYGRDFDNWGQGTTLTVSSAAAGGGSGGGSAEK DELIYIWAPLAGTCGVLLLSLVITLYCAAAGGTENLYFQSGSTKHILFRRRRRGFRQK KKKRD (SEQ ID NO: 256)

[0429] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and a fusion protein including an scFv that binds a TCR-CD3 complex component (a TCR-CD3 complex PEBL), an scFv that binds CD58 (a CD58 PEBL), and an ER localization sequence. In some embodiments, such recombinant nucleic acid include a nucleotide sequence of SEQ ID NO: 257: ATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCT CCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA 256Mintz Ref. No.: 063384-521001WO GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGA GGGCAGAGGCTCCCTTCTGACCTGCGGAGATGTCGAGGAAAATCCAGGACCCAT GCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTCTGC TGATTCCCGATATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCTTCTGTCGG CGATAGAGTGACCATGACATGCAGCGCCACATCTAGCGTGTCCTACATGCACTG GTATCAGCAGAAACCTGGCAAGGCCCCTAAGCGGTGGATCTACGACACCAGCAA GCTGGCCAGCGGCGTGCCAGCCAGATTCATCGGCAGCGGATCTGGCACAGAGTT CACCCTGACCATCAGCAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGTCAG CAATGGTCCTCCAACCCCCTGACATTTGGCGGAGGCACCAAGGTGGAAATCAAG GGCAGCACAAGCGGGTCTGGAAAGCCTGGGAGTGGCGAAGGGTCAACCAAAGG AGAAGTGCAGCTGCTGCAGAGCGGCGGCGGACTGGTGCAACCTGGAGGCAGCCT GAGACTGAGCTGTGCCGCTTCTGGATATAAGTTCACCAGCTACGTGATGCACTGG GTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGGCTACATCAACCCCTAC AACGATGTGACCAAGTACAACGAGAAGTTTAAGGGCAGATTCACACTGTCTAGA GATAATAGCAAGAACACCCTCTACCTGCAGATGAACAGCCTGCGGGCCGAGGAC ACCGCCGTGTACTACTGCGCCAGAGGCTCCTATTACGATTACGACGGCTTCGTGT ACTGGGGCCAGGGCACCCTGGTTACAGTGTCCAGCGGCGGAGGTGGAAGCGGCG GGGGCGGCTCTGGCGGAGGAGGCAGCCAAGTGCAGCTGCAGCAGAGCGGACCT GAGCTGGTGCGGCCCGGCGAGAGCGTGAAGATCAGCTGCAAGGGCAGCGGCTAC ACCTTCACCGACTACGCCATCCACTGGGTCAAGCAGTCTCACGCCAAGAGCCTGG AATGGATCGGCGTGATCTCTGTGCACTACGACAAAACCAACTACAACCAGAAAT TCAAGGGCAAGGCCAGCATGACCGTGGACAAGTCCAGCTCTACAGCCTACATGG AACTGGCTAGACTGACAAGCGAGGACAGCGCTATCTACTATTGTGCCCGGAGCT TCTACTACGGCAGAGATTTTGATAATTGGGGACAGGGCACAACCCTGACCGTGTC CTCTGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAA GGGAGATATCCTGCTGACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGA GCGGGTGTCCTTCAGCTGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTG GTATCAGCAGCGGACAATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGA GAGCATCAGCGGCATCCCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTC ACCCTGACCATCAACTCCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAG CAAAGCAACAGCTGGCCTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAG GGCGGCGGCAGCGGCGGCGGATCTGCCGAAAAGGACGAGCTGATCTACATCTGG GCCCCACTGGCCGGCACATGCGGCGTGCTGCTGCTGTCCCTGGTGATCACCCTGT 257Mintz Ref. No.: 063384-521001WO ATTGCgcggccgcatcgacaGGTTCCAGCGGCGGTGGAGGAGGATCAGGAGGATTGTAC AAGTACAAAAGCAGACGCTCTTTTATAGATGAGAAGAAGATGCCGtaa (SEQ ID NO: 257).

[0430] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 258: MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDEGRGSLLTCGDVEENPGPMLLLVTSLLLCELP HPAFLLIPDIQMTQSPSTLSASVGDRVTMTCSATSSVSYMHWYQQKPGKAPKRWIYD TSKLASGVPARFIGSGSGTEFTLTISSLQPDDFATYYCQQWSSNPLTFGGGTKVEIKGS TSGSGKPGSGEGSTKGEVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHWVRQA PGKGLEWVGYINPYNDVTKYNEKFKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYY CARGSYYDYDGFVYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLQQSGPELVRPG ESVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMT VDKSSSTAYMELARLTSEDSAIYYCARSFYYGRDFDNWGQGTTLTVSSGSTSGSGKP GSGEGSTKGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTIGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGSGTKLEIKGGGS GGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCAAASTGSSGGGGGSGGLYKYKSR RSFIDEKKMP (SEQ ID NO: 258). or tag. In some embodiments, the recombinant nucleic acid can include two or more nucleotide sequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 256.

[0431] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end a fusion protein including an scFv that binds CD58, a dominant negative mutant CD3epsilon protein, and a protein localization sequence or tag. In some embodiments, the protein localization 258Mintz Ref. No.: 063384-521001WO sequence or tag can include an ER localization sequence or tag. In some embodiments, the protein localization sequence or tag is selected from the amino acid sequence of any one of SEQ ID NOs.1-13. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 259: ATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCT CCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGC CACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGCCC TATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTC TGCTGATTCCCTACCCCTACGATGTGCCCGACTACGCCgggtccggaGATATCCTGCT GACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGAGCGGGTGTCCTTCAGC TGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTGGTATCAGCAGCGGACA ATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGAGAGCATCAGCGGCATC CCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTCACCCTGACCATCAACT CCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAGCAAAGCAACAGCTGGC CTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAGGGCAGCACATCTGGATC TGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGGGACAAGTGCAGCTGCAGC AGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGCGTGAAGATCAGCTGCAAG GGCAGCGGCTACACCTTCACCGACTACGCCATCCACTGGGTCAAGCAGTCTCACG CCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTGCACTACGACAAAACCAACT ACAACCAGAAATTCAAGGGCAAGGCCAGCATGACCGTGGACAAGTCCAGCTCTA CAGCCTACATGGAACTGGCTAGACTGACAAGCGAGGACAGCGCTATCTACTATT GTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATAATTGGGGACAGGGCACAA CCCTGACCGTGTCCTCTGGCGGCGGCAGCGGCGGCGGATCTGACGGCAATGAGG AAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACCG 259Mintz Ref. No.: 063384-521001WO TGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACGA CAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGATC ACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTTA CCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCAG AGTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGAT TGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAAG AACAGAAAGGCTAAAGCCAAGCCCGGATCAGGATTGTACAAGTACAAAAGCAG ACGCTCTTTTATAGATGAGAAGAAGATGCCGtaa (SEQ ID NO: 259).

[0432] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 260: MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDATNFSLLKQAGDVEENPGPMLLLVTSLLLCEL PHPAFLLIPYPYDVPDYAGSGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQR TIGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGS GTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYA IHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMTVDKSSSTAYMELARLTSE DSAIYYCARSFYYGRDFDNWGQGTTLTVSSGGGSGGGSDGNEEMGGITQTPYKVSIS GTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCY PRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNR KAKAKPGSGLYKYKSRRSFIDEKKMP (SEQ ID NO: 260)

[0433] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein and a fusion protein including an scFv that binds CD58, a dominant negative mutant CD3epsilon protein, and a protein localization sequence or tag. In some embodiments, the protein localization sequence or tag includes an ER localization sequence or tag. In some embodiments, such recombinant nucleic acid includes the nucleotide 260Mintz Ref. No.: 063384-521001WOsequence of SEQ ID NO: 258. In some embodiments, such recombinant nucleic acid furtherincludes es a 2A ribosome skipping sequence disposed 3’ of the ER localization sequence and an scFv that binds a TCR-CD3 complex component. In some embodiments, the protein localization sequence or tag is selected from the amino acid sequence of any one of SEQ ID NOs. 1-13. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 261: ATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCT CCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGC CACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGCCC TATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTC TGCTGATTCCCTACCCCTACGATGTGCCCGACTACGCCgggtccggaGATATCCTGCT GACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGAGCGGGTGTCCTTCAGC TGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTGGTATCAGCAGCGGACA ATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGAGAGCATCAGCGGCATC CCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTCACCCTGACCATCAACT CCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAGCAAAGCAACAGCTGGC CTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAGGGCAGCACATCTGGATC TGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGGGACAAGTGCAGCTGCAGC AGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGCGTGAAGATCAGCTGCAAG GGCAGCGGCTACACCTTCACCGACTACGCCATCCACTGGGTCAAGCAGTCTCACG CCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTGCACTACGACAAAACCAACT ACAACCAGAAATTCAAGGGCAAGGCCAGCATGACCGTGGACAAGTCCAGCTCTA CAGCCTACATGGAACTGGCTAGACTGACAAGCGAGGACAGCGCTATCTACTATT GTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATAATTGGGGACAGGGCACAA 261Mintz Ref. No.: 063384-521001WO CCCTGACCGTGTCCTCTGGCGGCGGCAGCGGCGGCGGATCTGACGGCAATGAGG AAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACCG TGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACGA CAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGATC ACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTTA CCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCAG AGTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGAT TGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAAG AACAGAAAGGCTAAAGCCAAGCCCGCCGCCGCTGGTGGGACCGAGAACCTGTAC TTCCAGTCCGGCAGCACCAAGCACATCCTGTTCCGGCGCCGTCGCAGAGGCTTTA GACAGAAAAAGAAAAAGCGGGACGAGGGCAGAGGCTCCCTTCTGACCTGCGGA GATGTCGAGGAAAATCCAGGACCCATGCTGTTGCTTGTTACAAGCCTCCTCCTGT GCGAATTGCCTCACCCCGCATTTCTCCTGATACCCCAGGTGCAGCTGGTCCAGAG CGGCGCCGAGGTGAAGAAGCCGGGTGCGAGCGTGAAAGTGTCTTGTAAGGCCTC TGGCTACAAGTTTACTTCCTACGTGATGCACTGGGTGCGGCAGGCTCCTGGCCAG AGACTGGAATGGATCGGCTACATCAACCCCTACAACGATGTGACCAAGTACAAT GAGAAGTTCAAGGGACGCGCAACCATCACCAGCGACAAGAGCGCCTCCACCGCC TATATGGAACTGAGCAGCCTCCGCTCCGAGGACACAGCTGTGTACTACTGCGCCA GAGGCAGCTACTACGACTACGATGGGTTCGTGTACTGGGGACAGGGCACCCTGG TCACCGTGTCCTCTGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAG GCTCCACCAAGGGAGAGATCGTGCTGACACAGAGCCCCGCTACACTGAGCCTGA GTCCTGGCGAGCGCGCGACTCTTTCTTGTAGCGCCACCAGCAGCGTCAGCTACAT GCACTGGTACCAGCAGAAACCGGGCCAGGCCCCACGTAGATGGATCTACGACAC CAGCAAGCTGGCCTCTGGCGTGCCCGCACGGTTTAGCGGGAGCGGTTCCGGCAC CGATTACACCCTGACCATCAGCTCCCTGGAGCCTGAGGACTTCGCCGTGTATTAC TGCCAGCAGTGGTCCAGCAACCCTCTGACCTTCGGCGGCGGAACCAAGCTGGAA ATCAAGGCGGCCGCCGGCGGCGGCAGCGGCGGCGGATCTGCCGAAAAGGACGA GCTGATCTACATCTGGGCCCCACTGGCCGGCACATGCGGCGTGCTGCTGCTGTCC CTGGTGATCACCCTGTATTGCtcgacaGGTTCCAGCGGCGGTGGAGGAGGATCAGGA GGATTGTACAAGTACAAAAGCAGACGCTCTTTTATAGATGAGAAGAAGATGCCGt aa (SEQ ID NO: 261).

[0434] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex 262Mintz Ref. No.: 063384-521001WO expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 262: MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDATNFSLLKQAGDVEENPGPMLLLVTSLLLCEL PHPAFLLIPYPYDVPDYAGSGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQR TIGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGS GTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYA IHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMTVDKSSSTAYMELARLTSE DSAIYYCARSFYYGRDFDNWGQGTTLTVSSGGGSGGGSDGNEEMGGITQTPYKVSIS GTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCY PRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNR KAKAKPAAAGGTENLYFQSGSTKHILFRRRRRGFRQKKKKRDEGRGSLLTCGDVEE NPGPMLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYKFTS YVMHWVRQAPGQRLEWIGYINPYNDVTKYNEKFKGRATITSDKSASTAYMELSSLR SEDTAVYYCARGSYYDYDGFVYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGEIVLT QSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFS GSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKLEIKAAAGGGSGGGSAE KDELIYIWAPLAGTCGVLLLSLVITLYCSTGSSGGGGGSGGLYKYKSRRSFIDEKKMP (SEQ ID NO: 262).

[0435] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant CD3epsilon protein, a 2A ribosome skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and an scFv that binds a CD58. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 263: ATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTCT GCTGATTCCCTACCCCTACGATGTGCCCGACTACGCCgggtccggaGACGGCAATGAG GAAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACC 263Mintz Ref. No.: 063384-521001WO GTGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACG ACAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGAT CACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTT ACCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCA GAGTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGA TTGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAA GAACAGAAAGGCTAAAGCCAAGCCCGCCACCAACTTCAGCCTGCTGAAGCAGGC CGGCGACGTGGAAGAGAACCCCGGCCCTATGGCCGAAGATGAGCCCGACGCTAA GAGCCCTAAGACCGGCGGCAGAGCCCCTCCAGGAGGCGCCGAGGCTGGAGAGC CTACAACACTGCTGCAACGGCTGAGAGGCACAATCAGCAAGGCCGTGCAGAACA AGGTGGAAGGCATCCTGCAAGATGTTCAGAAGTTCAGCGACAACGACAAACTGT ACCTGTACCTGCAGCTGCCCTCCGGCCCTACCACCGGCGATAAGAGCAGCGAGC CTAGCACCCTGTCTAATGAAGAATATATGTACGCCTACAGATGGATCAGAAACC ACCTGGAAGAGCACACCGACACCTGTCTGCCCAAACAGAGCGTCTACGACGCCT ATAGAAAGTACTGCGAGAGCCTGGCCTGCTGCAGACCTCTGAGCACCGCTAATTT CGGCAAGATCATCCGGGAAATCTTCCCTGATATCAAGGCCCGGAGACTGGGAGG CAGAGGCCAGAGCAAGTACTGCTACAGCGGCATCCGGAGAAAAACACTGGTTTC TATGCCTCCTCTGCCTGGACTGGACCTGAAGGGCAGCGAGTCCCCTGAGATGGG ACCTGAGGTCACCCCTGCTCCTCGGGACGAGGGCAGAGGCTCCCTTCTGACCTGC GGAGATGTCGAGGAAAATCCAGGACCCATGGCCTTGCCCGTGACTGCTCTGCTCC TGCCCCTCGCACTGCTGCTGCACGCCGCGAGGCCCGATATCCTGCTGACACAGAG CCCTGCTATCCTGAGCGTGTCTCCAGGCGAGCGGGTGTCCTTCAGCTGTAGAGCC AGCCAGAGCATTGGCACCAGCATCCACTGGTATCAGCAGCGGACAATCGGCAGC CCCAGACTGCTGATTAAGTACGCCTCTGAGAGCATCAGCGGCATCCCTAGCAGAT TCAGCGGATCTGGCTCTGGCACAGACTTCACCCTGACCATCAACTCCGTGGAAAG CGAGGACATCGCCGACTACTACTGCCAGCAAAGCAACAGCTGGCCTTTTACCTTC GGCTCCGGAACCAAGCTGGAAATCAAGGGCAGCACATCTGGATCTGGCAAGCCC GGCAGCGGAGAAGGCTCCACCAAGGGACAAGTGCAGCTGCAGCAGAGCGGACC TGAGCTGGTGCGGCCCGGCGAGAGCGTGAAGATCAGCTGCAAGGGCAGCGGCTA CACCTTCACCGACTACGCCATCCACTGGGTCAAGCAGTCTCACGCCAAGAGCCTG GAATGGATCGGCGTGATCTCTGTGCACTACGACAAAACCAACTACAACCAGAAA TTCAAGGGCAAGGCCAGCATGACCGTGGACAAGTCCAGCTCTACAGCCTACATG GAACTGGCTAGACTGACAAGCGAGGACAGCGCTATCTACTATTGTGCCCGGAGC 264Mintz Ref. No.: 063384-521001WO TTCTACTACGGCAGAGATTTTGATAATTGGGGACAGGGCACAACCCTGACCGTGT CCTCTGCGGCCGCCGGTGGTGGCTCTGGTGGGGGGTCAGCGGAAAAGGATGAAC TGATTTATATTTGGGCCCCGCTCGCTGGCACATGCGGGGTCCTGCTCTTGAGCTT GGTCATTACTTTGTATTGTGCCGCTGCGGGCGGTACCGAAAACCTCTATTTCCAA AGTGGGTCCACTAAACATATTTTGTTCAGAAGACGGAGGAGAGGATTTCGCCAG AAGAAGAAAAAGAGAGACtaa (SEQ ID NO: 263)

[0436] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 322: MLLLVTSLLLCELPHPAFLLIPYPYDVPDYAGSGDGNEEMGGITQTPYKVSISGTTVIL TCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSK PEDANFYLYLRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNRKAKAK PATNFSLLKQAGDVEENPGPMAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGT ISKAVQNKVEGILQDVQKFSDNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYR WIRNHLEEHTDTCLPKQSVYDAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLG GRGQSKYCYSGIRRKTLVSMPPLPGLDLKGSESPEMGPEVTPAPRDEGRGSLLTCGD VEENPGPMALPVTALLLPLALLLHAARPDILLTQSPAILSVSPGERVSFSCRASQSIGTS IHWYQQRTIGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNS WPFTFGSGTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELVRPGESVKISCKGSG YTFTDYAIHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMTVDKSSSTAYM ELARLTSEDSAIYYCARSFYYGRDFDNWGQGTTLTVSSAAAGGGSGGGSAEKDELIY IWAPLAGTCGVLLLSLVITLYCAAAGGTENLYFQSGSTKHILFRRRRRGFRQKKKKR D (SEQ ID NO: 322)

[0437] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant CD3zeta protein operably linked to a ubiquitin ligase, a 2A ribosome skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and an scFv that binds a CD58. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 323: 265Mintz Ref. No.: 063384-521001WO atgaagtggaaggccctgttcaccgcagcaatcctgcaggcacagctgccaatcacagaggcccagtctttcggcctgctggacccc aagctgtgctacctgctggatggcatcctgtttatctatggcgtgatcctgaccgccctgttcctgcgcgtgaagttttctcggagcgccg actacagcgccagaagactgcggaacgccagagcccagagcagaaagcagagacagctgaaggccgacgccaagaaagccatc ggcagactgcaactgagaaccctcaagcaaggcgacaaggaaatcggccccgacggggatagctgcgccgtgtgcatcgagctgt acaaacccaacgacctggtgcggattcttacatgcaaccacatctttcacaagacctgtgtggacccctggctgctggaacacagaac atgtcctatgtgcaagtgcgacatcctgaaggccctgggcatcgaggtggacgtCgaagatggatctgtgtccctgcaggtgccagt gtctaatgagatcagcaacagcgccagcagccacgaggaagataacagaagcgagacagccagctctggatacgcctccgtccag ggcactgatgagcctccactggaagagcacgtgcagagcaccaacgagagcctgcagctggttaatcacgaagccaacagcgtgg ccgtggatgtgatcccccatgtggacaaccccacctttgaggaggacgagacacctaaccaggagacagccgtgcgggaaatcaag agcGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCG GCCCTATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAG CCCCTCCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGA GAGGCACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGAT GTTCAGAAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCG GCCCTACCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAAT ATATGTACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCT GTCTGCCCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGG CCTGCTGCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTT CCCTGATATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTA CAGCGGCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGAC CTGAAGGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGG GACGAGGGCAGAGGCTCCCTTCTGACCTGCGGAGATGTCGAGGAAAATCCAGGA CCCATGGCCTTGCCCGTGACTGCTCTGCTCCTGCCCCTCGCACTGCTGCTGCACGC CGCGAGGCCCGATATCCTGCTGACACAGAGCCCTGCTATCCTGAGCGTGTCTCCA GGCGAGCGGGTGTCCTTCAGCTGTAGAGCCAGCCAGAGCATTGGCACCAGCATC CACTGGTATCAGCAGCGGACAATCGGCAGCCCCAGACTGCTGATTAAGTACGCC TCTGAGAGCATCAGCGGCATCCCTAGCAGATTCAGCGGATCTGGCTCTGGCACA GACTTCACCCTGACCATCAACTCCGTGGAAAGCGAGGACATCGCCGACTACTACT GCCAGCAAAGCAACAGCTGGCCTTTTACCTTCGGCTCCGGAACCAAGCTGGAAA TCAAGGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACCA AGGGACAAGTGCAGCTGCAGCAGAGCGGACCTGAGCTGGTGCGGCCCGGCGAG AGCGTGAAGATCAGCTGCAAGGGCAGCGGCTACACCTTCACCGACTACGCCATC CACTGGGTCAAGCAGTCTCACGCCAAGAGCCTGGAATGGATCGGCGTGATCTCT 266Mintz Ref. No.: 063384-521001WO GTGCACTACGACAAAACCAACTACAACCAGAAATTCAAGGGCAAGGCCAGCATG ACCGTGGACAAGTCCAGCTCTACAGCCTACATGGAACTGGCTAGACTGACAAGC GAGGACAGCGCTATCTACTATTGTGCCCGGAGCTTCTACTACGGCAGAGATTTTG ATAATTGGGGACAGGGCACAACCCTGACCGTGTCCTCTGCGGCCGCCGGTGGTG GCTCTGGTGGGGGGTCAGCGGAAAAGGATGAACTGATTTATATTTGGGCCCCGC TCGCTGGCACATGCGGGGTCCTGCTCTTGAGCTTGGTCATTACTTTGTATTGTGCC GCTGCGGGCGGTACCGAAAACCTCTATTTCCAAAGTGGGTCCACTAAACATATTT TGTTCAGAAGACGGAGGAGAGGATTTCGCCAGAAGAAGAAAAAGAGAGACtaa (SEQ ID NO: 323)

[0438] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 324: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSA DYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIEL YKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKALGIEVDVEDGSVSLQV PVSNEISNSASSHEEDNRSETASSGYASVQGTDEPPLEEHVQSTNESLQLVNHEANSV AVDVIPHVDNPTFEEDETPNQETAVREIKSATNFSLLKQAGDVEENPGPMAEDEPDA KSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFSDNDKLYLY LQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVYDAYRKYCE SLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSMPPLPGLDLK GSESPEMGPEVTPAPRDEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDI LLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTIGSPRLLIKYASESISGIPSRFSG SGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGSGTKLEIKGSTSGSGKPGSGEGST KGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGVISVHY DKTNYNQKFKGKASMTVDKSSSTAYMELARLTSEDSAIYYCARSFYYGRDFDNWG QGTTLTVSSAAAGGGSGGGSAEKDELIYIWAPLAGTCGVLLLSLVITLYCAAAGGTE NLYFQSGSTKHILFRRRRRGFRQKKKKRD (SEQ ID NO: 324).

[0439] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a 267Mintz Ref. No.: 063384-521001WO dominant negative mutant CD3epsilon protein, a 2A ribosome skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and a UL148 polypeptide. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 325. ATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTCT GCTGATTCCCTACCCCTACGATGTGCCCGACTACGCCgggtccggaGACGGCAATGAG GAAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACC GTGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACG ACAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGAT CACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTT ACCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCA GAGTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGA TTGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAA GAACAGAAAGGCTAAAGCCAAGCCCGCCACCAACTTCAGCCTGCTGAAGCAGGC CGGCGACGTGGAAGAGAACCCCGGCCCTATGGCCGAAGATGAGCCCGACGCTAA GAGCCCTAAGACCGGCGGCAGAGCCCCTCCAGGAGGCGCCGAGGCTGGAGAGC CTACAACACTGCTGCAACGGCTGAGAGGCACAATCAGCAAGGCCGTGCAGAACA AGGTGGAAGGCATCCTGCAAGATGTTCAGAAGTTCAGCGACAACGACAAACTGT ACCTGTACCTGCAGCTGCCCTCCGGCCCTACCACCGGCGATAAGAGCAGCGAGC CTAGCACCCTGTCTAATGAAGAATATATGTACGCCTACAGATGGATCAGAAACC ACCTGGAAGAGCACACCGACACCTGTCTGCCCAAACAGAGCGTCTACGACGCCT ATAGAAAGTACTGCGAGAGCCTGGCCTGCTGCAGACCTCTGAGCACCGCTAATTT CGGCAAGATCATCCGGGAAATCTTCCCTGATATCAAGGCCCGGAGACTGGGAGG CAGAGGCCAGAGCAAGTACTGCTACAGCGGCATCCGGAGAAAAACACTGGTTTC TATGCCTCCTCTGCCTGGACTGGACCTGAAGGGCAGCGAGTCCCCTGAGATGGG ACCTGAGGTCACCCCTGCTCCTCGGGACGAGGGCAGAGGCTCCCTTCTGACCTGC GGAGATGTCGAGGAAAATCCAGGACCCATGCTGCGCCTGCTTTTTACATTGGTAT TGCTAGCTCTCCATGGACAGAGCGTCGGCGCGAGTCGCGACTACGTGCACGTGC GCTTGCTGAGCTACAGGGGTGATCCTTTGGTGTTCAAGCACACGTTCTCCGGTGT CCGTAGGCCCTTCACCGAGCTGGGCTGGGCAGCCTGCCGAGACTGGGATTCTATG CACTGTACTCCGTTTTGGTCTACAGATCTCGAACAGATGACTGACTCCGTGCGGC GTTATTCGACCGTCTCGCCGGGCAAGGAGGTGACCCTGCAACTACACGGCAACC AGACGGTGCAGCCATCCTTCCTGTCCTTCACCTGCCGGCTGCAGCTGGAGCCCGT 268Mintz Ref. No.: 063384-521001WO GGTGGAGAATGTGGGTCTTTACGTCGCCTACGTGGTCAACGACGGCGAGCGCCC TCAGCAGTTCTTCACCCCTCAGGTCGATGTGGTACGCTTCGCGCTGTACCTGGAG ACTCTGTCCCGCATTGTCGAGCCATTAGAATCCGGCCGCCTGGCCGTGGAGTTCG ACACCCCCGACCTGGCGCTCGCTCCCGACCTGGTATCTTCTCTGTTCGTCGCCGG CCATGGGGAGACCGACTTCTACATGAACTGGACCTTACGCCGCAGCCAGACTCA TTACTTGGAGGAGATGGCTCTCCAGGTGGAGATCCTGAAGCCCCGCGGGGTGCG CCACAGAGCTATCATCCACCACCCCAAGCTGCAGCCGGGAGTGGGGCTTTGGAT TGACTTTTGCGTGTACCGCTATAACGCGCGCCTGACCCGTGGCTACGTGCGCTAC ACCCTGTCACCTAAAGCTCGCCTGCCGGCCAAGGCCGAGGGCTGGCTCGTTAGCC TGGACAGGTTTATCGTGCAGTATCTTAACACGCTGCTCATCACCATGATGGCCGC CATCTGGGCACGAGTGCTGATCACCTACCTAGTGTCCCGGCGGCGTtaa (SEQ ID NO: 325).

[0440] I In some embodiments, the recombinant nucleic acid can include two or morenucleotide sequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 326. MLLLVTSLLLCELPHPAFLLIPYPYDVPDYAGSGDGNEEMGGITQTPYKVSISGTTVIL TCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSK PEDANFYLYLRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNRKAKAK PATNFSLLKQAGDVEENPGPMAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGT ISKAVQNKVEGILQDVQKFSDNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYR WIRNHLEEHTDTCLPKQSVYDAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLG GRGQSKYCYSGIRRKTLVSMPPLPGLDLKGSESPEMGPEVTPAPRDEGRGSLLTCGD VEENPGPMLRLLFTLVLLALHGQSVGASRDYVHVRLLSYRGDPLVFKHTFSGVRRPF TELGWAACRDWDSMHCTPFWSTDLEQMTDSVRRYSTVSPGKEVTLQLHGNQTVQP SFLSFTCRLQLEPVVENVGLYVAYVVNDGERPQQFFTPQVDVVRFALYLETLSRIVEP LESGRLAVEFDTPDLALAPDLVSSLFVAGHGETDFYMNWTLRRSQTHYLEEMALQV EILKPRGVRHRAIIHHPKLQPGVGLWIDFCVYRYNARLTRGYVRYTLSPKARLPAKA EGWLVSLDRFIVQYLNTLLITMMAAIWARVLITYLVSRRR (SEQ ID NO: 326).

[0441] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface 269Mintz Ref. No.: 063384-521001WO of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant CD3zeta protein operably linked to a ubiquitin ligase, a 2A ribosome skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, and a UL148 polypeptide. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 327: atgaagtggaaggccctgttcaccgcagcaatcctgcaggcacagctgccaatcacagaggcccagtctttcggcctgctggacccc aagctgtgctacctgctggatggcatcctgtttatctatggcgtgatcctgaccgccctgttcctgcgcgtgaagttttctcggagcgccg actacagcgccagaagactgcggaacgccagagcccagagcagaaagcagagacagctgaaggccgacgccaagaaagccatc ggcagactgcaactgagaaccctcaagcaaggcgacaaggaaatcggccccgacggggatagctgcgccgtgtgcatcgagctgt acaaacccaacgacctggtgcggattcttacatgcaaccacatctttcacaagacctgtgtggacccctggctgctggaacacagaac atgtcctatgtgcaagtgcgacatcctgaaggccctgggcatcgaggtggacgtCgaagatggatctgtgtccctgcaggtgccagt gtctaatgagatcagcaacagcgccagcagccacgaggaagataacagaagcgagacagccagctctggatacgcctccgtccag ggcactgatgagcctccactggaagagcacgtgcagagcaccaacgagagcctgcagctggttaatcacgaagccaacagcgtgg ccgtggatgtgatcccccatgtggacaaccccacctttgaggaggacgagacacctaaccaggagacagccgtgcgggaaatcaag agcGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCG GCCCTATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAG CCCCTCCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGA GAGGCACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGAT GTTCAGAAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCG GCCCTACCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAAT ATATGTACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCT GTCTGCCCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGG CCTGCTGCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTT CCCTGATATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTA CAGCGGCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGAC CTGAAGGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGG GACGAGGGCAGAGGCTCCCTTCTGACCTGCGGAGATGTCGAGGAAAATCCAGGA CCCATGCTGCGCCTGCTTTTTACATTGGTATTGCTAGCTCTCCATGGACAGAGCGT CGGCGCGAGTCGCGACTACGTGCACGTGCGCTTGCTGAGCTACAGGGGTGATCC TTTGGTGTTCAAGCACACGTTCTCCGGTGTCCGTAGGCCCTTCACCGAGCTGGGC TGGGCAGCCTGCCGAGACTGGGATTCTATGCACTGTACTCCGTTTTGGTCTACAG ATCTCGAACAGATGACTGACTCCGTGCGGCGTTATTCGACCGTCTCGCCGGGCAA GGAGGTGACCCTGCAACTACACGGCAACCAGACGGTGCAGCCATCCTTCCTGTC 270Mintz Ref. No.: 063384-521001WO CTTCACCTGCCGGCTGCAGCTGGAGCCCGTGGTGGAGAATGTGGGTCTTTACGTC GCCTACGTGGTCAACGACGGCGAGCGCCCTCAGCAGTTCTTCACCCCTCAGGTCG ATGTGGTACGCTTCGCGCTGTACCTGGAGACTCTGTCCCGCATTGTCGAGCCATT AGAATCCGGCCGCCTGGCCGTGGAGTTCGACACCCCCGACCTGGCGCTCGCTCCC GACCTGGTATCTTCTCTGTTCGTCGCCGGCCATGGGGAGACCGACTTCTACATGA ACTGGACCTTACGCCGCAGCCAGACTCATTACTTGGAGGAGATGGCTCTCCAGGT GGAGATCCTGAAGCCCCGCGGGGTGCGCCACAGAGCTATCATCCACCACCCCAA GCTGCAGCCGGGAGTGGGGCTTTGGATTGACTTTTGCGTGTACCGCTATAACGCG CGCCTGACCCGTGGCTACGTGCGCTACACCCTGTCACCTAAAGCTCGCCTGCCGG CCAAGGCCGAGGGCTGGCTCGTTAGCCTGGACAGGTTTATCGTGCAGTATCTTAA CACGCTGCTCATCACCATGATGGCCGCCATCTGGGCACGAGTGCTGATCACCTAC CTAGTGTCCCGGCGGCGTtaa (SEQ ID NO: 327)

[0442] In some embodiments, the recombinant nucleic acid can include two or morenucleotide sequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 328: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSA DYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIEL YKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKALGIEVDVEDGSVSLQV PVSNEISNSASSHEEDNRSETASSGYASVQGTDEPPLEEHVQSTNESLQLVNHEANSV AVDVIPHVDNPTFEEDETPNQETAVREIKSATNFSLLKQAGDVEENPGPMAEDEPDA KSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFSDNDKLYLY LQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVYDAYRKYCE SLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSMPPLPGLDLK GSESPEMGPEVTPAPRDEGRGSLLTCGDVEENPGPMLRLLFTLVLLALHGQSVGASR DYVHVRLLSYRGDPLVFKHTFSGVRRPFTELGWAACRDWDSMHCTPFWSTDLEQM TDSVRRYSTVSPGKEVTLQLHGNQTVQPSFLSFTCRLQLEPVVENVGLYVAYVVND GERPQQFFTPQVDVVRFALYLETLSRIVEPLESGRLAVEFDTPDLALAPDLVSSLFVA GHGETDFYMNWTLRRSQTHYLEEMALQVEILKPRGVRHRAIIHHPKLQPGVGLWID FCVYRYNARLTRGYVRYTLSPKARLPAKAEGWLVSLDRFIVQYLNTLLITMMAAIW ARVLITYLVSRRR (SEQ ID NO: 328) 271Mintz Ref. No.: 063384-521001WO

[0443] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a fusion protein including an scFv that binds CD58, a dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 290.

[0444] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequences encoding, from 5’ end to 3’ end, a fusion protein including an scFv that binds CD58, a truncated dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 291 to 297.

[0445] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds CD58, a dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:259. ATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCT CCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA 272Mintz Ref. No.: 063384-521001WO GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGC CACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGCCC TATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTC TGCTGATTCCCTACCCCTACGATGTGCCCGACTACGCCgggtccggaGATATCCTGCT GACACAGAGCCCTGCTATCCTGAGCGTGTCTCCAGGCGAGCGGGTGTCCTTCAGC TGTAGAGCCAGCCAGAGCATTGGCACCAGCATCCACTGGTATCAGCAGCGGACA ATCGGCAGCCCCAGACTGCTGATTAAGTACGCCTCTGAGAGCATCAGCGGCATC CCTAGCAGATTCAGCGGATCTGGCTCTGGCACAGACTTCACCCTGACCATCAACT CCGTGGAAAGCGAGGACATCGCCGACTACTACTGCCAGCAAAGCAACAGCTGGC CTTTTACCTTCGGCTCCGGAACCAAGCTGGAAATCAAGGGCAGCACATCTGGATC TGGCAAGCCCGGCAGCGGAGAAGGCTCCACCAAGGGACAAGTGCAGCTGCAGC AGAGCGGACCTGAGCTGGTGCGGCCCGGCGAGAGCGTGAAGATCAGCTGCAAG GGCAGCGGCTACACCTTCACCGACTACGCCATCCACTGGGTCAAGCAGTCTCACG CCAAGAGCCTGGAATGGATCGGCGTGATCTCTGTGCACTACGACAAAACCAACT ACAACCAGAAATTCAAGGGCAAGGCCAGCATGACCGTGGACAAGTCCAGCTCTA CAGCCTACATGGAACTGGCTAGACTGACAAGCGAGGACAGCGCTATCTACTATT GTGCCCGGAGCTTCTACTACGGCAGAGATTTTGATAATTGGGGACAGGGCACAA CCCTGACCGTGTCCTCTGGCGGCGGCAGCGGCGGCGGATCTGACGGCAATGAGG AAATGGGAGGCATCACACAGACCCCTTACAAGGTGTCCATCAGCGGCACCACCG TGATCCTGACCTGTCCTCAATACCCTGGCTCTGAAATCCTGTGGCAGCACAACGA CAAAAACATCGGAGGAGATGAGGACGATAAGAACATCGGCTCTGATGAGGATC ACCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTTA CCCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCAG AGTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGAT TGTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAAG AACAGAAAGGCTAAAGCCAAGCCCGGATCAGGATTGTACAAGTACAAAAGCAG ACGCTCTTTTATAGATGAGAAGAAGATGCCGtaa (SEQ ID NO:259)

[0446] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:260. MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS 273Mintz Ref. No.: 063384-521001WO DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDATNFSLLKQAGDVEENPGPMLLLVTSLLLCEL PHPAFLLIPYPYDVPDYAGSGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQR TIGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGS GTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELVRPGESVKISCKGSGYTFTDYA IHWVKQSHAKSLEWIGVISVHYDKTNYNQKFKGKASMTVDKSSSTAYMELARLTSE DSAIYYCARSFYYGRDFDNWGQGTTLTVSSGGGSGGGSDGNEEMGGITQTPYKVSIS GTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCY PRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVKICITGGLLLLVYYWSKNR KAKAKPGSGLYKYKSRRSFIDEKKMP (SEQ ID NO:260)

[0447] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds CD58, an scFv that binds TCR-CD3 complex, a truncated dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 331: ATGGCCGAAGATGAGCCCGACGCTAAGAGCCCTAAGACCGGCGGCAGAGCCCCT CCAGGAGGCGCCGAGGCTGGAGAGCCTACAACACTGCTGCAACGGCTGAGAGGC ACAATCAGCAAGGCCGTGCAGAACAAGGTGGAAGGCATCCTGCAAGATGTTCAG AAGTTCAGCGACAACGACAAACTGTACCTGTACCTGCAGCTGCCCTCCGGCCCTA CCACCGGCGATAAGAGCAGCGAGCCTAGCACCCTGTCTAATGAAGAATATATGT ACGCCTACAGATGGATCAGAAACCACCTGGAAGAGCACACCGACACCTGTCTGC CCAAACAGAGCGTCTACGACGCCTATAGAAAGTACTGCGAGAGCCTGGCCTGCT GCAGACCTCTGAGCACCGCTAATTTCGGCAAGATCATCCGGGAAATCTTCCCTGA TATCAAGGCCCGGAGACTGGGAGGCAGAGGCCAGAGCAAGTACTGCTACAGCG GCATCCGGAGAAAAACACTGGTTTCTATGCCTCCTCTGCCTGGACTGGACCTGAA GGGCAGCGAGTCCCCTGAGATGGGACCTGAGGTCACCCCTGCTCCTCGGGACGC CACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCCGGCCC TATGCTGCTGCTCGTGACCAGCCTGCTGTTGTGCGAGCTGCCCCACCCCGCTTTTC 274Mintz Ref. No.: 063384-521001WO TGCTGATTCCCGAGATTGTGCTGACCCAGTCTCCAGACTTCCAGTCCGTCACACC TAAAGAGAAGGTGACCATCACCTGTAGAGCCTCTCAGTCGATTGGCACCAGCAT CCACTGGTATCAGCAGAAGCCCGATCAGTCCCCGAAGCTGCTGATTAAGTACGC CAGCGAGAGCATCAGCGGCATCCCCAGCCGCTTCAGCGGCTCCGGGTCTGGTAC CGACTTCACCCTGACAATCAACTCCCTGGAGGCCGAGGACGCGGCTACCTACTAC TGCCAGCAAAGCAACAGCTGGCCTTTTACCTTCGGCGGCGGAACTAAGCTGGAA ATCAAGGGCAGCACATCTGGATCTGGCAAGCCCGGCAGCGGAGAAGGCTCCACC AAGGGACAGGTGCAGCTGGTCCAGTCTGGCGCCGAGGTCAAGAAGCCTGGCGCA TCTGTCAAAGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCGACTACGCCATCC ACTGGGTGCGGCAGGCTCCCGGACAAGGTCTGGAGTGGATCGGCGTGATCTCCG TGCACTACGATAAGACCAACTACAACCAGAAGTTCAAGGGCAGAGTGACCATGA CCGTGGACACCAGTATTTCCACTGCCTACATGGAACTGAGCAGGCTGAGAAGCG ACGATACAGCCGTGTACTACTGTGCGCGCAGCTTCTATTACGGGCGCGACTTCGA CAACTGGGGCCAGGGCACCACCGTAACAGTGTCCTCTGGCGGAGGTGGAAGCGG CGGGGGCGGCTCTGGCGGAGGAGGCAGCCAGGTGCAGCTGGTCCAGAGCGGCG CCGAGGTGAAGAAGCCGGGTGCGAGCGTGAAAGTGTCTTGTAAGGCCTCTGGCT ACAAGTTTACTTCCTACGTGATGCACTGGGTGCGGCAGGCTCCTGGCCAGAGACT GGAATGGATCGGCTACATCAACCCCTACAACGATGTGACCAAGTACAATGAGAA GTTCAAGGGACGCGCAACCATCACCAGCGACAAGAGCGCCTCCACCGCCTATAT GGAACTGAGCAGCCTCCGCTCCGAGGACACAGCTGTGTACTACTGCGCCAGAGG CAGCTACTACGACTACGATGGGTTCGTGTACTGGGGACAGGGCACCCTGGTCAC CGTGTCCTCTGGCAGCACAAGCGGGTCTGGAAAGCCTGGGAGTGGCGAAGGGTC AACCAAAGGAGAGATCGTGCTGACACAGAGCCCCGCTACACTGAGCCTGAGTCC TGGCGAGCGCGCGACTCTTTCTTGTAGCGCCACCAGCAGCGTCAGCTACATGCAC TGGTACCAGCAGAAACCGGGCCAGGCCCCACGTAGATGGATCTACGACACCAGC AAGCTGGCCTCTGGCGTGCCCGCACGGTTTAGCGGGAGCGGTTCCGGCACCGATT ACACCCTGACCATCAGCTCCCTGGAGCCTGAGGACTTCGCCGTGTATTACTGCCA GCAGTGGTCCAGCAACCCTCTGACCTTCGGCGGCGGAACCAAGCTGGAAATCAA GGGCGGCGGCAGCGGCGGCGGATCTGATAAGAACATCGGCTCTGATGAGGATCA CCTGTCTCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGTTATTACGTGTGTTAC CCTCGGGGCAGCAAGCCTGAGGACGCCAATTTCTACCTGTACCTTAGAGCCAGA GTGTGCGAGAACTGCATGGAAATGGATGTGATGAGCGTGGCCACCATCGTGATT GTGAAAATCTGCATCACCGGCGGCCTGCTCCTGCTGGTGTACTACTGGTCCAAGA 275Mintz Ref. No.: 063384-521001WO ACAGAAAGGCTAAAGCCAAGCCCTTGTACAAGTACAAAAGCAGACGCTCTTTTA TAGATGAGAAGAAGATGCCGtaa (SEQ ID NO: 331)

[0448] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 332: MAEDEPDAKSPKTGGRAPPGGAEAGEPTTLLQRLRGTISKAVQNKVEGILQDVQKFS DNDKLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIRNHLEEHTDTCLPKQSVY DAYRKYCESLACCRPLSTANFGKIIREIFPDIKARRLGGRGQSKYCYSGIRRKTLVSM PPLPGLDLKGSESPEMGPEVTPAPRDATNFSLLKQAGDVEENPGPMLLLVTSLLLCEL PHPAFLLIPEIVLTQSPDFQSVTPKEKVTITCRASQSIGTSIHWYQQKPDQSPKLLIKYA SESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNSWPFTFGGGTKLEIKGSTS GSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAIHWVRQAP GQGLEWIGVISVHYDKTNYNQKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCA RSFYYGRDFDNWGQGTTVTVSSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYKFTSYVMHWVRQAPGQRLEWIGYINPYNDVTKYNEKFKGRATITSDK SASTAYMELSSLRSEDTAVYYCARGSYYDYDGFVYWGQGTLVTVSSGSTSGSGKPG SGEGSTKGEIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGQAPRRWIYD TSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKLEIKG GGSGGGSDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCEN CMEMDVMSVATIVIVKICITGGLLLLVYYWSKNRKAKAKPLYKYKSRRSFIDEKKM P (SEQ ID NO: 332).

[0449] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds a TCR-CD3 complex component, an scFv that binds CD58, and a dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 298. 276Mintz Ref. No.: 063384-521001WO

[0450] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds CD58, an scFv that binds a TCR-CD3 complex component, and a dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 301.

[0451] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds a TCR-CD3 complex component, an scFv that binds CD58, and a truncated dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 299 or 300.

[0452] In some embodiments, the recombinant nucleic acid can include two or more nucleotidesequences encoding nucleic acids or proteins to reduce one or more of TCR-CD3 complex expression, MHC class I and MHC class II expression and / or CD58 expression on the surface of an immune cell can include nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosome skipping sequence, a fusion protein including an scFv that binds CD58, an scFv that binds a TCR-CD3 complex component, and a truncated dominant negative mutant CD3epsilon protein, and an ER localization sequence. In some embodiments, such recombinant nucleic acid includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 302 or 303.

[0453] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins to reduce surface expression of TCR-CD3 complex MHC class I and MHC class II and / or CD58 on immune cells, the nucleotide sequences encoding, from 5’ end to 3’ end, a TCR PEBL sequence, a 2A ribosomal skipping sequence, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, and a CD58 277Mintz Ref. No.: 063384-521001WO PEBL sequence. In some embodiments, the recombinant nucleic acid comprises the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 338.

[0454] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a TCRalpha / CD58 Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 473 (pSync5121).

[0455] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a TCRalpha / CD58 Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 478 (pSync5122).

[0456] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a TCRalpha / CD58 Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 482 (pSync5123).

[0457] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a TCR / CD58 Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 486 (pSync5124).

[0458] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide 278Mintz Ref. No.: 063384-521001WO sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a TCR / CD58 Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 489 (pSync5125).

[0459] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a CD58 / TCR Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 435 (pSync5126).

[0460] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a CD58 / TCR Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 493 (pSync5127).

[0461] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, 2A ribosomal skipping sequence, a CD58 / TCR Tandem PEBL, an ER tag, and a CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 496 (pSync5128).

[0462] In some embodiments, the recombinant nucleic acid provided herein comprises threenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, CD8 / B2M fusion, 2A ribosomal skipping sequence, a CD58 / TCR Tandem scFv, an ER tag, and CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 517 (pSync5260). 279Mintz Ref. No.: 063384-521001WO

[0463] In some embodiments, the recombinant nucleic acid provided herein comprises fournucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 54mer MHC-I binder / B2M fusion, 2A ribosomal skipping sequence, a CD58 / TCR Tandem scFv, an ER tag, and CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 520 (pSync5261).

[0464] In some embodiments, the recombinant nucleic acid provided herein comprises fournucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a CD58 scFv, a dominant negative mutant CD3epsilon protein, an ER tag, and CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 806 (Sync5603).

[0465] In some embodiments, the recombinant nucleic acid provided herein comprises fournucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a CD58 / TCR Tandem scFv, a dominant negative mutant CD3epsilon protein, an ER tag. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 541 (pSync4952).

[0466] In some embodiments, the recombinant nucleic acid provided herein comprises fournucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a CD8 / B2M fusion, a 2A ribosomal skipping sequence, CD58 / TCR Tandem scFv, an ER tag, and CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 541 (pSync5496).

[0467] In some embodiments, the recombinant nucleic acid provided herein comprises fournucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a 54mer MHC-I binder / B2M fusion, a 2A ribosomal skipping 280Mintz Ref. No.: 063384-521001WO sequence, CD58 / TCR Tandem scFv, an ER tag, and CD3zeta shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 541 (pSync5497).

[0468] In some embodiments, the recombinant nucleic acid provided herein comprises fivenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a CD8 / B2M fusion, a 2A ribosomal skipping sequence, CD58 / TCR Tandem scFv, an ER tag, a CD3zeta shRNA, and a B2M shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 545 (pSync5498).

[0469] In some embodiments, the recombinant nucleic acid provided herein comprises fivenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a 54mer MHC-I binder / B2M fusion, a 2A ribosomal skipping sequence, CD58 / TCR Tandem scFv, an ER tag, a CD3zeta shRNA, and a B2M shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 547 (pSync5499).

[0470] In some embodiments, the recombinant nucleic acid provided herein comprises fivenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a CD8 / B2M fusion, 2A ribosomal skipping sequence, a CD58 / TCR Tandem scFv, an ER tag, a CD3zeta shRNA, and a B2M shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 521 (pSync5266).

[0471] In some embodiments, the recombinant nucleic acid provided herein comprises fivenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a CD8 / B2M fusion, 2A ribosomal skipping sequence, a CD58 / TCR Tandem scFv, an ER tag, a CD3zeta shRNA, and a B2M shRNA. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence of SEQ ID NO: 523 (pSync5267). 281Mintz Ref. No.: 063384-521001WO

[0472] In some embodiments, the recombinant nucleic acid provided herein comprises fivenucleotide sequences encoding proteins and nucleic acids to reduce surface expression of MHC class I and MHC class II, TCR-CD3 complex, CD58, and TCR-CD3 complex, the nucleotide sequences encoding, from 5’ end to 3’ end, a dominant negative mutant RFX5 protein, a 2A ribosomal skipping sequence, a CD58 scFv, a dominant negative mutant CD3epsilon protein, an ER tag, and CD3zeta shRNA, CD3epsilon shRNA. In some embodiments,...

Claims

Mintz Ref. No.: 063384-521001WO WHAT IS CLAIMED IS:

1. A recombinant nucleic acid comprising:a) a first nucleotide sequence encoding a first molecule capable of reducingTCR-CD3 complex expression on the surface of an immune cell; b) a second nucleotide sequence encoding a second molecule capable of reducingMHC class I and / or MHC class II expression on the surface of an immune cell; and c) a third nucleotide sequence encoding a third molecule capable of reducingCD58 expression on the surface of an immune cell; wherein the recombinant nucleic acid is multicistronic.

2. A recombinant nucleic acid comprising:a) a first nucleotide sequence encoding a first molecule capable of reducing bothTCR-CD3 complex expression and CD58 expression on the surface of an immune cell; b) a second nucleotide sequence encoding a second molecule capable of reducing MHC class I and / or MHC class II expression on the surface of an immune cell; wherein the recombinant nucleic acid is bicistronic.

3. The recombinant nucleic acid of claim 1 or claim 2, wherein the recombinant nucleicacid comprises at least one or more of an exogenous promoter, a post-transcriptional response element (PRE), and a polyadenylation signal sequence.

4. The recombinant nucleic acid of any one of claims 1 to 3, wherein the recombinantnucleic acid further comprises a ribosome skipping sequence disposed between each cistron.

5. The recombinant nucleic acid of any one of claims 1, 3 and 4, wherein therecombinant configuration is tricistronic and a ribosome skipping sequence is disposed between the first cistron and the second cistron and between the second cistron and the third cistron.

6. The recombinant nucleic acid of any one of claims 1 to 4, wherein the ribosomeskipping sequence disposed between the first cistron and the second cistron is 843Mintz Ref. No.: 063384-521001WO different from the ribosome skipping sequence disposed between the second cistron and the third cistron.

7. The recombinant nucleic acid of any one of claims 1 to 4, wherein the ribosomeskipping sequence disposed between the first cistron and the second cistron is the same as the ribosome skipping sequence disposed between the second cistron and the third cistron.

8. The recombinant nucleic acid of any one of claims 1 to 4, wherein the recombinantconfiguration is bicistronic and a ribosome skipping sequence is disposed between the first cistron and the second cistron.

9. The recombinant nucleic acid of any one of claims 1, and 3 to 8, wherein:(a) the first nucleotide sequence encoding a first molecule capable of reducingTCR-CD3 complex expression on the surface of an immune cell encodes a first molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein subunit of the TCR-CD3 complex capable of reducing assembly of a functional TCR-CD3 complex; (b) the second nucleotide sequence encoding a second molecule capable ofreducing MHC / HLA expression on the surface of an immune cell encodes a second molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative protein capable of regulating transcription of MHC class I and / or MHC class II; and (c) the third nucleic acid encoding a third molecule capable of reducing CD58expression on the surface of an immune cell encodes a third molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein capable of reducing the binding of CD58 to its cognate ligand CD2.

10. The recombinant nucleic acid of any one of claims 1 to 9, wherein the first nucleotidesequence encodes a first molecule comprising an anti-TCR-CD3 PEBL comprising an scFv capable of binding a TCR-CD3 complex component. 844Mintz Ref. No.: 063384-521001WO11. The recombinant nucleic acid of claim 10, wherein the TCR-CD3 complexcomponent comprises a TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta.

12. The recombinant nucleic acid of claim 10, wherein the scFv is capable of bindingCD3zeta.

13. The recombinant nucleic acid of claim 10, wherein the scFv is capable of bindingCD3epsilon.

14. The recombinant nucleic acid of claim 10, wherein the scFv comprises a sequenceselected from the sequences in Table 3 or Table 4.

15. The recombinant nucleic acid of claim 10, wherein the protein localization sequenceor tag of the PEBL is selected from the group consisting of: an endoplasmic reticulum (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.

16. The recombinant nucleic acid of claim 15, wherein the protein localization sequenceor tag comprises an ER localization tag.

17. The recombinant nucleic acid of claim 16, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

18. The recombinant nucleic acid of any one of claims 1 to 9, wherein the first moleculecomprises a dominant negative mutant TCR-CD3 complex component capable of binding an unmutated or wild-type TCR-CD3 complex component. 845Mintz Ref. No.: 063384-521001WO19. The recombinant nucleic acid of claim 18, wherein the dominant negative mutantTCR-CD3 complex component is a dominant negative mutant TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta.

20. The recombinant nucleic acid of claim 18, wherein the dominant negative mutantTCR-CD3 complex component is a dominant negative mutant CD3epsilon.

21. The recombinant nucleic acid of claim 18, wherein the dominant negative mutantTCR-CD3 complex component is a dominant negative mutant CD3zeta.

22. The recombinant nucleic acid of claim 18, wherein the dominant negative mutantTCR-CD3 complex component comprises a sequence selected from the sequences in Table 5.

23. The recombinant nucleic acid of any one of claims 1 to 9, wherein the first moleculecomprises an shRNA targeting a TCR-CD3 complex component.

24. The recombinant nucleic acid of claim 23, wherein the shRNA targets TCRalpha,TCRbeta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta.

25. The recombinant nucleic acid of claim 23, wherein the shRNA targets CD3zeta.

26. The recombinant nucleic acid of claim 23, wherein the shRNA targets CD3epsilon.

27. The recombinant nucleic acid of any one of claims 1 to 26, wherein the secondnucleotide sequence encodes a second molecule capable of inhibiting MHC class I or MHC class II expression.

28. The recombinant nucleic acid of any one of claims 1 to 27, wherein the secondmolecule comprises a mutated RFX5 polypeptide.

29. The recombinant nucleic acid of claim 28, wherein the mutated RFX5 polypeptidecomprises a truncated RFX5 molecule.

30. The recombinant nucleic acid of claim 29, wherein the RFX5 polypeptide comprises asequence selected from the sequences listed in Table 7.

31. The recombinant nucleic acid of any one of claims 1 and 3 to 30, wherein the thirdnucleotide sequence encodes a third molecule comprising an anti-CD58 PEBL comprising an scFv capable of binding CD58 (anti-CD58 scFv). 846Mintz Ref. No.: 063384-521001WO32. The recombinant nucleic acid of claim 31, wherein the anti-CD58 scFv comprises asequence selected from the sequences listed Table 8, Table 9, or Table 10.

33. The recombinant nucleic acid of any one of claims 31 to 32, wherein the proteinlocalization sequence or tag of the PEBL comprises an ER localization tag.

34. The recombinant nucleic acid of claim 33, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

35. The recombinant nucleic acid of any one of claims 1 to 30, wherein the third moleculecomprises an CD58 shRNA.

36. The recombinant nucleic acid of claim 35, wherein the CD58 shRNA comprises asequence selected from the sequences listed in Table 13.

37. The recombinant nucleic acid of any one of claims 1 to 30, wherein the third moleculecomprises an UL148 polypeptide.

38. The recombinant nucleic acid of claim 37, wherein the UL148 polypeptide comprisesthe amino acid sequence of SEQ ID NO: 244.

39. The recombinant nucleic acid of claim 2, wherein the first molecule comprises anucleotide sequence encoding a tandem PEBL comprising an scFv capable of binding a TCR-CD3 complex component and an scFv capable of binding CD58.

40. The recombinant nucleic acid of claim 39, wherein the two scFvs are operably linkedtogether.

41. The recombinant nucleic acid of claim 40, wherein the protein localization sequenceor tag of the PEBL comprises an ER localization tag.

42. The recombinant nucleic acid of claim 41, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), 847Mintz Ref. No.: 063384-521001WO LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

43. The recombinant nucleic acid of claim 2, wherein the first molecule comprises anucleotide sequence encoding a dominant negative mutant TCR-CD3 complex component and a PEBL comprising an scFv capable of binding CD58.

44. The recombinant nucleic acid of claim 43, wherein the dominant negative mutantTCR-CD3 complex component and the scFv are operably linked together.

45. The recombinant nucleic acid of claim 2, wherein the recombinant nucleic acidcomprises from 5’ to 3’ end: (a) the second nucleotide sequence and (b) the first nucleotide sequence.

46. The recombinant nucleic acid of claim 45, wherein the first nucleotide sequencecomprises a nucleotide sequence encoding a dominant negative mutant TCR-CD3 complex component, a PEBL comprising an scFv capable of binding CD58, and a CD3z shRNA.

47. The recombinant nucleic acid of claim 46, wherein the dominant negative mutantTCR-CD3 complex component and the scFv are operably linked together.

48. The recombinant nucleic acid of claim 45 to 47, wherein the second nucleotidesequence comprises a mutated RFX5 polypeptide.

49. The recombinant nucleic acid of claim 48, wherein the mutated RFX5 polypeptidecomprises a truncated RFX5 molecule.

50. The recombinant nucleic acid of claim 49, wherein the RFX5 polypeptide comprises asequence selected from the sequences listed in Table 7.

51. The recombinant nucleic acid of any one of claims 46 to 50, wherein the proteinlocalization sequence or tag of the PEBL comprises an ER localization tag. 848Mintz Ref. No.: 063384-521001WO52. The recombinant nucleic acid of claim 51, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

53. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a mutated RFX5, a T2Aribosome skipping sequence, a fusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon, and a CD3z shRNA.

54. The recombinant nucleic acid of claim 53, wherein the mutated RFX5 polypeptidecomprises a truncated RFX5 molecule.

55. The recombinant nucleic acid of claim 54, wherein the RFX5 polypeptide comprises asequence selected from the sequences listed in Table 7.

56. The recombinant nucleic acid of any one of claims 53 to 55, wherein the proteinlocalization sequence or tag of the PEBL comprises an ER localization tag.

57. The recombinant nucleic acid of claim 56, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

58. The recombinant nucleic acid of claims 53 to 57, wherein the recombinant nucleicacid comprises the sequence of SEQ ID NO: 806.

59. The recombinant nucleic acid of any one of claims 1 to 58, wherein the scFvs arefully humanized. 849Mintz Ref. No.: 063384-521001WO60. The recombinant nucleic acid of any one of claims 1, and 3 to 38, wherein therecombinant nucleic acid comprises from 5’ to 3’ end: (a) the first nucleotide sequence;(b) the second nucleotide sequence; and(c) the third nucleotide sequence; orwherein the nucleic acid comprises from 5’ to 3’ end: a) the first nucleotide sequence;b) the third nucleotide sequence; andc) the second nucleotide sequence; orwherein the nucleic acid comprises from 5’ to 3’ end: a) the second nucleotide sequence;b) the first nucleotide sequence; andc) the third nucleotide sequence; orwherein the nucleic acid comprises from 5’ to 3’ end: a) the second nucleotide sequence;b) the third nucleotide sequence; andc) the first nucleotide sequence; orwherein the nucleic acid comprises from 5’ to 3’ end: a) the third nucleotide sequence;b) the first nucleotide sequence; andc) the second nucleotide sequence; orwherein the nucleic acid comprises from 5’ to 3’ end: a) the third nucleotide sequence;b) the second nucleotide encoding; andc) the first nucleotide sequence.850Mintz Ref. No.: 063384-521001WO61. The recombinant nucleic acid of claim 60, wherein the nucleic acid comprises one ormore ribosome skipping sequences located between the first, second, and / or third nucleotide sequence.

62. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a PEBL comprising anscFv capable of binding a TCR-CD3 complex component, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and PEBL comprising an scFv capable of binding CD58.

63. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a mutated RFX5, a 2Aribosome skipping sequence, and a fusion protein comprising a PEBL comprising an scFv capable of binding a TCR-CD3 complex component, and a PEBL comprising an scFv capable of binding CD58.

64. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a fusion proteincomprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon.

65. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a mutated RFX5 and afusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon.

66. The recombinant nucleic acid of claim 44, further comprising, at the 3’ end, a 2Aribosome skipping sequence and a PEBL comprising an scFv capable of binding a TCR-CD3 complex component.

67. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a dominant negativemutant CD3epsilon, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a PEBL comprising an scFv capable of binding a CD58.

68. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a dominant negativemutant CD3zeta operably linked with a ubiquitin ligase, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a PEBL comprising an scFv capable of binding a CD58. 851Mintz Ref. No.: 063384-521001WO69. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a dominant negativemutant CD3epsilon, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a UL148 polypeptide.

70. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a dominant negativemutant CD3zeta operably linked with a ubiquitin ligase, a 2A ribosome skipping sequence, a mutated RFX5, a 2A ribosome skipping sequence, and a UL148 polypeptide.

71. The recombinant nucleic acid of any one of claims 1 to 70, further comprising anucleic acid sequence encoding US11.

72. The recombinant nucleic acid of any one of claims 1 to 71, further comprising anucleic acid sequence encoding SerpinB9.

73. The recombinant nucleic acid of any one of claims 1 to 72, further comprising anucleic acid sequence encoding a cell selection molecule.

74. The recombinant nucleic acid of claim 73, wherein the cell selection moleculecomprises a CD34 epitope.

75. The recombinant nucleic acid of claim 74, wherein the cell selection moleculecomprises a CD34 epitope selected from Table 18.

76. The recombinant nucleic acid of any one of claims 1 to 75, further comprising apromoter.

77. The recombinant nucleic acid of claim 76, wherein the promoter is an MNDpromoter, an MNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or an NFkB responsive promoter.

78. The recombinant nucleic acid of any one of claims 76 to 77, further comprising asecond promoter.

79. The recombinant nucleic acid of claim 78, wherein each of the promoters is operablylinked to a nucleotide sequence in the same direction. 852Mintz Ref. No.: 063384-521001WO80. The recombinant nucleic acid of claim 78, wherein each of the promoters is operablylinked to a nucleotide sequence in opposite directions.

81. The recombinant nucleic acid of claim 78, wherein one promoter is operably linked tothe nucleotide sequences encoding an shRNA and the other promoter is operably linked to the nucleotide sequences encoding a PEBL or a dominant negative protein.

82. The recombinant nucleic acid of any one of claims 1 to 81, further comprising anshRNA.

83. The recombinant nucleic acid of claim 82, wherein the shRNA is a CD3zeta shRNA.

84. The recombinant nucleic acid of any one of claims 82 to 83, wherein the shRNA islocated in an intron of a promoter.

85. The recombinant nucleic acid of claim 84, wherein the promoter is an EF-1alpha, aMND, MNDU3, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or a hUbC promoter.

86. The recombinant nucleic acid of any one of claims 1 to 85, further comprising aWoodchuck hepatitis virus post-transcriptional regulatory sequence (WPRE) sequence, a polyA sequence, and / or an LTR sequence.

87. The recombinant nucleic acid of any one of claims 1, and 3 to 86, further comprisinga fourth nucleotide sequence encoding a fourth molecule capable of blocking the CD8-MHC class I interaction.

88. The recombinant nucleic acid of claim 87, wherein the fourth molecule capable ofblocking the CD8-MHC class I interaction comprises an MHC class I binder.

89. The recombinant nucleic acid of claim 88, wherein the MHC class I binder comprisesan CD8alpha extracellular domain with a cysteine to serine (C to S) mutation at amino acid position 54 and a serine to asparagine (S to N) mutation at amino acid position 74 in SEQ ID NO: 407.

90. The recombinant nucleic acid of claim 88, wherein the MHC class I binder comprisesSEQ ID NO: 411.

91. The recombinant nucleic acid of claim 88 or claim 89, wherein the MHC class Ibinder additionally comprises a B2M polypeptide or a fragment thereof. 853Mintz Ref. No.: 063384-521001WO92. The recombinant nucleic acid of any one of claims 1 to 91, further comprising a fifthnucleotide sequence encoding one or more shRNAs capable of reducing expression of CD3zeta, FasR, B2M, CIITA, CD3epsilon, TRAC, RFX5, and / or CD58.

93. The recombinant nucleic acid of claim 92, wherein the shRNA is capable of reducingTCR-CD3 complex expression on the surface of the immune cell.

94. The recombinant nucleic acid of any one of claims 1, and 3 to 92, wherein the firstmolecule is a CD3zeta ubiquitin ligase fusion protein and the fifth molecule is a CD3zeta shRNA located in a promoter region.

95. The recombinant nucleic acid of any one of claims 1, and 3 to 82 wherein the first,second, and / or third molecule comprises a ubiquitin ligase fusion protein.

96. A recombinant nucleic acid encoding, from 5’ end to 3’ end, a mutated RFX5, a 2Aribosome skipping sequence, a 54mer MHC class I binder fused to B2M, a 2A ribosome skipping sequence, a fusion protein comprising a PEBL comprising an scFv capable of binding a CD58 and a dominant negative mutant CD3epsilon , a CD3z shRNA, and an RFX5 shRNA.

97. The recombinant nucleic acid of claim 96, wherein the mutated RFX5 comprises atruncated RFX5 molecule.

98. The recombinant nucleic acid of claim 97, wherein the RFX5 comprises a sequenceselected from the sequences listed in Table 7.

99. The recombinant nucleic acid of any one of claims 96 to 98, wherein the proteinlocalization sequence or tag of the PEBL comprises an ER localization tag.

100. The recombinant nucleic acid of claim 99, wherein the ER localization tag is selectedfrom the group consisting of KKAHKSKTH (SEQ ID NO: 1), KKKKRD (SEQ ID NO: 2), PKNRYKKH (SEQ ID NO: 3), KKYL (SEQ ID NO: 4), LLEALTLASARGPLRKRSVPMAKAKPKFSISPDSLS (SEQ ID NO: 5), LLEALTLASARGPLRKRSVPMAKAKPKFSISPKKYL (SEQ ID NO: 6), CFRKLAKTGKKKKRD (SEQ ID NO: 7), KHILFRRRRRGFRQ (SEQ ID NO: 8), LYKYKSRRSFIDEKKMP (SEQ ID NO: 9) KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). 854Mintz Ref. No.: 063384-521001WO101. The recombinant nucleic acid sequence of any one of claims 96 to 100, wherein thenucleic acid sequence comprises the sequence of SEQ ID NO: 811.

102. A vector comprising the recombinant nucleic acid of any one of claims 1 to 101.

103. A lentiviral expression vector comprising the recombinant nucleic acid of any one ofclaims 1 to 101.

104. A virus particle comprising the recombinant nucleic acid of any one of claims 1 to101.

105. The virus particle of claim 104, wherein the virus is a lentivirus.

106. An engineered immune cell comprising the recombinant nucleic acid of any one ofclaims 1-101, the vector of claim 102, or the virus of claims 104 or 105.

107. The engineered immune cell of claim 106, wherein the cell is a T cell.

108. The engineered immune cell of claim 107, wherein the T cell further comprises achimeric antigen receptor (CAR).

109. The engineered immune cell of claim 106, wherein the T cell is a CD8-positive T cell,a CD4-positive T cell, a regulatory T cell (TREG), a cytotoxic T cell (TCTL), a central memory T cell (TCM), an effector memory T cell (TEM), a tissue-resident memory T cell (TRM), a stem cell-like memory T cell (TSCM) or a tumor infiltrating lymphocyte (TIL).

110. The engineered immune cell of claim 106 or 107, wherein the engineered immune cellis a CAR T cell.

111. A population of engineered immune cells comprising the recombinant nucleic acid ofany one of claims 1 to 101, or the vector of claim 102.

112. A composition comprising the recombinant nucleic acid of any one of claims 1 to101, or the vector of claim 102, or the virus of claims 104 or 105, and additionally comprising a nucleic acid encoding a CAR construct.

113. An engineered immune cell comprising the composition of claim 112.

114. A pharmaceutical composition comprising the virus particle of claims 102 or 103 orengineered immune cell of any one of claims 106 to 111 or 113 and a pharmaceutically acceptable excipient. 855Mintz Ref. No.: 063384-521001WO115. A method of producing an engineered immune cell, the method comprising,contacting an immune cell with the recombinant nucleic acid of any one of claims 1 to 101, the vector of claim 102 or 103, the virus of claims 104 or 105, or the composition of claim 92 in vitro.

116. A method of reducing or abolishing TCR-CD3 complex expression on the surface ofan immune cell, the method comprising contacting the immune cell with the recombinant nucleic acid of any one of claims 1 to 101, the vector of claim 102 or 103, the virus of claims 104 or 105, or the composition of claim 90 in vitro.

117. A method of reducing MHC expression on the surface of an immune cell, the methodcomprising contacting the immune cell with the recombinant nucleic acid of any one of [0009] to 101, the vector of claim 102 or 103, the virus of claims 104 or 105, or the composition of claim 112 in vitro.

118. A method of reducing CD58 expression on the surface of an immune cell, the methodcomprising contacting the immune cell with the recombinant nucleic acid of any one of [0009] to 101, the vector of claim 102 or 103, the virus of claims 104 or 105, or the composition of claim 112 in vitro.

119. A method of reducing TCR-CD3 complex expression, MHC expression, and CD58expression on the surface of an immune cell, the method comprising contacting the immune cell with the recombinant nucleic of any one of claims 1 to 101, the vector of claim 102 or 103, the virus of claims 104 or 105, or the composition of claim 112 in vitro.

120. A method of preventing or reducing graft versus host disease in a subject in need ofengineered immune cell, the method comprising administering the engineered immune cell or population of immune cells of any one of claims 106 to 111, or 113 to the subject.

121. A method of treating a subject in need thereof with an engineered immune cell, themethod comprising administering the engineered immune cell or population of immune cells of any one of claims 106 to 111, or 113 to the subject.

122. A method of increasing allogeneic immune cell survival in a subject, the methodcomprising administering the engineered immune cell or population of immune cells of any one of claims 106 to 111, or 113 to the subject. 856Mintz Ref. No.: 063384-521001WO123. A method of reducing immune rejection or aiding in prevention of immune rejectionin a subject in need of an allogeneic graft, the method comprising administering the engineered immune cell or population of immune cells of any one of claims 106 to 111, or 113 to the subject.

124. The method of any one of claims 115 to 123, wherein the immune cell is a cell from abiological sample.

125. The method of claim 124, wherein the biological sample is from a human subject.

126. The method of any one of claims 115 to 123, wherein the immune cell is anallogeneic cell.

127. The method of any one of claims 115 to 123, wherein the engineered immune cell is aT cell.

128. The method of claim 127, wherein the T cell is a CD4+ cell, CD8+ cell, TREG cell,TSCM cell, or TCM cell.

129. The method of any one of claims 115 to 128, wherein the immune cell is a CAR Tcell.

130. An allogeneic system comprising,a first recombinant nucleic acid of any one of claims 1 to 101; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

131. An allogeneic system comprising,a first recombinant nucleic acid comprising: (i) a first nucleotide sequence encoding a first molecule capable ofreducing TCR-CD3 complex expression on the surface of an immune cell; (ii) a second nucleotide sequence encoding a second molecule capable ofreducing MHC expression on the surface of the immune cell; and (iii) a third nucleotide sequence encoding a third molecule capable ofreducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or 857Mintz Ref. No.: 063384-521001WO more Chimeric Antigen Receptors.

132. An allogeneic system comprising,recombinant nucleic acid comprising: (i) a first nucleotide sequence encoding a first molecule capable ofreducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; (ii) b) a second nucleotide sequence encoding a second molecule capableof reducing MHC class I and / or MHC class II expression on the surface of an immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

133. An allogeneic system comprising,a first viral particle comprising a first recombinant nucleic acid comprising: (i) a first nucleotide sequence encoding a first molecule capable ofreducing TCR-CD3 complex expression on the surface of an immune cell; (ii) a second nucleotide sequence encoding a second molecule capable ofreducing MHC expression on the surface of the immune cell; and (iii) a third nucleotide sequence encoding a third molecule capable ofreducing CD58 expression on the surface of the immune cell; and a second viral particle comprising a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

134. An allogeneic system comprising,a first viral particle comprising a first recombinant nucleic acid comprising: (i) a first nucleotide sequence encoding a first molecule capable ofreducing both TCR-CD3 complex expression and CD58 expression on the surface of an immune cell; 858Mintz Ref. No.: 063384-521001WO (ii) b) a second nucleotide sequence encoding a second molecule capableof reducing MHC class I and / or MHC class II expression on the surface of an immune cell; and a second viral particle comprising a second recombinant nucleic acid comprising a nucleotide sequence encoding one or more Chimeric Antigen Receptors.

135. The allogeneic system of claim 133 or 134, wherein the first and second viral particlesare lentiviral particles.

136. The allogeneic system of claim 131 to 135, wherein the first recombinant nucleic acidis multicistronic.

137. The allogeneic system of any one of claims 131 to 136, wherein the first recombinantnucleic acid comprises at least one or more of an exogenous promoter, a post- transcriptional response element (PRE), and a polyadenylation signal sequence.

138. The allogeneic system of any one of claims 131 to 137, wherein the first recombinantnucleic acid further comprises a ribosome skipping sequence disposed between each cistron.

139. The allogeneic system of any one of claims 131 to 138, wherein the first recombinantnucleic acid configuration is tricistronic and a ribosome skipping sequence is disposed between the first cistron and the second cistron and between the second cistron and the third cistron.

140. The allogeneic system of any one of claims 131 to 139, wherein the first recombinantnucleic acid configuration is bicistronic and a ribosome skipping sequence is disposed between the first cistron and the second cistron.

141. The allogeneic system of any one of claims 131 to 140, wherein:(a) the first nucleotide sequence encoding a first molecule capable of reducingTCR-CD3 complex expression on the surface of an immune cell encodes a first molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein subunit of the TCR-CD3 complex capable of reducing assembly of a functional TCR-CD3 complex; (b) the second nucleotide sequence encoding a second molecule capable of859Mintz Ref. No.: 063384-521001WO reducing MHC / HLA expression on the surface of an immune cell encodes a second molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative protein capable of regulating transcription of the MHC / HLA; and (c) the third nucleic acid encoding a third molecule capable of reducing CD58expression on the surface of an immune cell encodes a third molecule comprising a protein expression blocker (PEBL), an shRNA, or a dominant negative mutant protein capable of reducing the binding of CD58 to its cognate ligand CD2.

142. The allogeneic system of any one of claims 131 to 141, wherein the first moleculecomprises an scFv capable of binding a TCR-CD3 complex component.

143. The allogeneic system of any one of claims 131 to 142, wherein the TCR-CD3complex component comprises a TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3epsilon, or CD3zeta.

144. The allogeneic system of claim 143, wherein the scFv is capable of binding CD3zeta.

145. The allogeneic system of claim 143, wherein the scFv is capable of bindingCD3epsilon.

146. The allogeneic system of claim 143, wherein the scFv comprises a sequence selectedfrom the sequences in Table 3 or Table 4.

147. The allogeneic system of any one of claims 144 to 146, wherein the scFv additionallycomprises a protein localization sequence or tag.

148. The allogeneic system of claim 147, wherein the protein localization sequence or tagis selected from the group consisting of: an endoplasmic reticulum (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.

149. The allogeneic system of any one of claims 131 to 148, wherein the first moleculecomprises a dominant negative mutant TCR-CD3 complex component capable of binding an unmutated or wild-type TCR-CD3 complex component.

150. The allogeneic system of claim 149, wherein the dominant negative mutant TCR-CD3860Mintz Ref. No.: 063384-521001WO complex component is a dominant negative mutant TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD3gamma, CD3delta, CD3 epsilon, or CD3zeta.

151. The allogeneic system of claim 149, wherein the dominant negative mutant TCR-CD3complex component is a dominant negative mutant CD3epsilon.

152. The allogeneic system of claim 149, wherein the dominant negative mutant TCR-CD3complex component is a dominant negative mutant CD3zeta.

153. The allogeneic system of claim 149, wherein the dominant negative mutant TCR-CD3complex component comprises a sequence selected from the sequences in Table 5.

154. The allogeneic system of any one of claims 131 to 141, wherein the first moleculecomprises an shRNA targeting a TCR-CD3 complex component.

155. The allogeneic system of claim 154, wherein the shRNA targets TCRalpha, TCRbeta,CD3gamma, CD3delta, CD3 epsilon, or CD3zeta.

156. The allogeneic system of claim 154, wherein the shRNA targets CD3zeta.

157. The allogeneic system of claim 154, wherein the shRNA targets CD3epsilon.

158. The allogeneic system of any one of claims 131-156, additionally comprising a fourthnucleotide sequence encoding a fourth molecule capable of reducing TCR-CD3 complex expression on the surface of the immune cell.

159. The allogeneic system of claim 158, wherein the first molecule is a CD3zeta shRNAlocated in a promoter region and the fourth molecule is a CD3zeta ubiquitin ligase fusion protein.

160. The allogeneic system of any one of claims 131-159, wherein the first, second, and / orthird molecule comprises a ubiquitin ligase fusion protein.

161. The allogeneic system of any one of claims 131 to 160, wherein the second moleculeis capable of inhibiting MHC class I or MHC class II expression.

162. The allogeneic system of any one of claims 131 to 160, wherein the second moleculeis a mutated RFX5 polypeptide.

163. The allogeneic system of claim 162, wherein the mutated RFX5 polypeptide is atruncated RFX5 molecule.

164. The allogeneic system of claim 162, wherein the mutated RFX5 polypeptide861Mintz Ref. No.: 063384-521001WO comprises a sequence selected from the sequences listed in Table 7.

165. The allogeneic system of any one of claims 131 to 164, wherein the third moleculecomprises an anti-CD58 scFv capable of binding a CD58.

166. The allogeneic system of claim 165, wherein the anti-CD58 scFv comprises asequence selected from the sequences listed Table 8, Table 9, or Table 10.

167. The allogeneic system of any one of claims 131 to 164, wherein the third moleculecomprises an CD58 shRNA.

168. The allogeneic system of claim 167, wherein the CD58 shRNA comprises a sequenceselected from the sequences listed in Table 13.

169. The allogeneic system of any one of claims 131 to 164, wherein the third moleculecomprises a UL148 polypeptide.

170. The allogeneic system of any one of claims 131 to 164, wherein the third moleculecomprises the amino acid sequence of SEQ ID NO: 244.

171. The allogeneic system of any one of claims 131 to 170, wherein the first molecule andthe third molecule are each an scFv.

172. The allogeneic system of claim 171, wherein the two scFvs are operably linkedtogether.

173. The allogeneic system of claim 171, wherein the first scFv is capable of binding aTCR-CD3 complex component and the second scFv is capable of binding CD58.

174. The allogeneic system of any one of claims 172 to 173, wherein the operably linkedscFvs further comprise an ER localization sequence or tag.

175. The allogeneic system of claim 174, wherein the ER localization sequence is at the C-terminus.

176. The allogeneic system of any one of claims 149 to 153, wherein the first molecule is adominant negative mutant TCR-CD3 complex component and the third molecule is an scFv capable of binding CD58.

177. The allogeneic system of claim 176, wherein the dominant negative mutant TCR-CD3complex component and the scFv are operably linked together.

178. The allogeneic system of any one of claims 131 to 177, wherein the scFvs are fully862Mintz Ref. No.: 063384-521001WO humanized.

179. The allogeneic system of any one of claims 131 to 178, wherein the first recombinantnucleic acid comprises the recombinant nucleic acid of any one of claims 1 to 75.

180. The allogeneic system of any one of claims 131 to 179, wherein the first recombinantnucleic acid further comprises a promoter.

181. The allogeneic system of claim 180, wherein the promoter is a MND promoter, anMNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, a CAG promoter, a CBA promoter, a Gamma Retro 5’ LTR promoter, or an NFkB responsive promoter.

182. The allogeneic system of claim 180, further comprising a second promoter.

183. The allogeneic system of claim 182, wherein each of the promoters is operably linkedto a nucleotide sequence in the same direction.

184. The allogeneic system of claim 182, wherein each of the promoters is operably linkedto a nucleotide sequence in opposite directions.

185. The allogeneic system of claim 182, wherein one promoter is operably linked toexpress shRNA and another promoter is operably linked to express the polypeptide molecules.

186. The allogeneic system of any one of claims 131 to 185, wherein the first recombinantnucleic acid further comprises an shRNA.

187. The allogeneic system of any one of claims 131 to 185, wherein the first recombinantnucleic acid further comprises a CD3zeta shRNA.

188. The allogeneic system of claim 187, wherein the shRNA is located in an intronicregion of a promoter.

189. The allogeneic system of claim 187, wherein the promoter is an EF-1alpha or a hUbCpromoter.

190. The allogeneic system of any one of claims 131 to 189, wherein the first recombinantnucleic acid further comprises a WPRE sequence, a polyA sequence, and / or an LTR sequence.

191. The allogeneic system of any one of claims 131 to 189, wherein the first recombinant863Mintz Ref. No.: 063384-521001WO nucleic acid further comprises a fourth molecule comprising an MHC class I binder.

192. The allogeneic system of claim 191, wherein the MHC class I binder comprises anCD8alpha extracellular domain with amino acid mutations S74N and C54S at amino acid positions 74 and 54 in SEQ ID NO: 407.

193. The allogeneic system of claim 191, wherein the MHC class I binder comprises SEQID NO: 411.

194. The recombinant nucleic acid of claim 191 or claim 192, wherein the MHC class Ibinder additionally comprises a B2M polypeptide.

195. The allogeneic system of any one of claims 131 to 194, wherein the one or moreCARs encoded by the second recombinant nucleic acid comprises a CD22-specific CAR.

196. The allogeneic system of claim 195, wherein the CD22-specific CAR comprises aCD22-specific binding domain, a transmembrane domain, and an intracellular domain.

197. The allogeneic system of any one of claims 195 to 196, wherein the CD22-specificCAR comprises a CD22-specific binding domain, a spacer, a hinge domain, a transmembrane domain, a peptide linker, and an intracellular domain.

198. The allogeneic system of any one of claims 195 to 196, wherein the CD22-specificbinding domain comprises an antibody that binds CD22 or an antigen-binding fragment thereof.

199. The allogeneic system of any one of claims 195 to 196, wherein the CD22-specificbinding domain comprises an antibody that binds human CD22.

200. The allogeneic system of any one of claims 195 to 196, wherein the CD22-specificbinding domain comprises an antigen-binding fragment of an antibody that binds human CD22.

201. The allogeneic system of claim 200, wherein the antigen-binding fragment of anantibody that binds human CD22 is a single chain variable fragment (scFv) that binds CD22.

202. The allogeneic system of claim 201, wherein the scFv that binds CD22 has the864Mintz Ref. No.: 063384-521001WO sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGR TYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAE DFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 559).

203. The allogeneic system of claim 202, wherein the scFv that binds CD22 comprises asequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 559.

204. The allogeneic system of any one of claims 195 to 203, wherein the CD22-specificCAR comprises a CD8α hinge domain comprising the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 560) and a CD8α transmembrane domain comprising the sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 694) and optionally a peptide linker having the sequence of LYC.

205. The allogeneic system of any one of claims 195 to 204, wherein the CD22-specificCAR comprises a CD8α hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 560 and a CD8α transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 694.

206. The allogeneic system of any one of claims 195 to 205, wherein the CD8αtransmembrane domain further comprises a spacer having the sequence of LYC.

207. The allogeneic system of any one of claims 195 to 205, wherein the CD22-specificCAR comprises an intracellular domain comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.

208. The allogeneic system of claim 207, wherein the primary T cell activating domaincomprising an ITAM comprises a CD3ζ intracellular signaling domain.

209. The allogeneic system of claim 207, wherein the CD3ζ intracellular signaling domaincomprises the sequence of 865Mintz Ref. No.: 063384-521001WO RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 562).

210. The allogeneic system of claim 209, wherein the CD3ζ intracellular signaling domaincomprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562.

211. The allogeneic system of claim 207, wherein the costimulatory signaling domaincomprises a 4-1BB / CD137 signaling domain.

212. The allogeneic system of claim 211, wherein the 4-1BB / CD137 signaling domaincomprises the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 561).

213. The allogeneic system of claim 212, wherein the 4-1BB / CD137 costimulatorysignaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 561.

214. The allogeneic system of any one of claims 195 to 213, wherein the CD22-specificCAR comprises the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGR TYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAE DFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 646).

215. The allogeneic system of any one of claims 195 to 214, wherein the CD22-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 646.

216. The allogeneic system of any one of claims 195 to 213, wherein the CD22-specific866Mintz Ref. No.: 063384-521001WO CAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSL QLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVP SRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 342).

217. The allogeneic system of any one of claims 195 to 213, wherein the CD22-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 342.

218. The allogeneic system of any one of claims 195 to 217, wherein the one or moreCARs encoded by the second recombinant nucleic acid further comprises a CD19- specific CAR and a CD20-specific CAR.

219. The allogeneic system of claim 218, wherein the CD19-specific CAR comprises aCD19-specific binding domain, a transmembrane domain, and an intracellular domain.

220. The allogeneic system of any one of claims 218 to 219, wherein the CD19-specificCAR comprises a CD19-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain.

221. The allogeneic system of any one of claims 218 to 220, wherein the CD19-specificbinding domain comprises an antibody that binds CD19 or an antigen-binding fragment thereof.

222. The allogeneic system of claim 221, wherein the CD19-specific binding domaincomprises an antibody that binds human CD19.

223. The allogeneic system of claim 221, wherein the CD19-specific binding domaincomprises an antigen-binding fragment of an antibody that binds human CD19. 867Mintz Ref. No.: 063384-521001WO224. The allogeneic system of claim 223, wherein the antigen-binding fragment of anantibody that binds human CD19 is a single chain variable fragment (scFv) that binds CD19.

225. The allogeneic system of claim 223, wherein the scFv capable of binding CD19 hasthe sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRI DPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDIL TGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVS AAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 564).

226. The allogeneic system of claim 223, wherein the scFv capable of binding CD19comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 564.

227. The allogeneic system of any one of claims 218 to 224, wherein the CD19-specificCAR comprises a CD28 hinge domain comprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 565) and a CD28 transmembrane domain comprising the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 566).

228. The allogeneic system of any one of claims 218 to 225, wherein the CD19-specificCAR comprises a CD28 hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 565 and a CD28 transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 566.

229. The allogeneic system of any one of claims 218 to 226, wherein the CD19-specificCAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.

230. The allogeneic system of claim 227, wherein the primary T cell activating domaincomprising an ITAM comprises a CD3ζ intracellular signaling domain. 868Mintz Ref. No.: 063384-521001WO231. The allogeneic system of claim 228, wherein the CD3ζ intracellular signaling domaincomprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 562).

232. The allogeneic system of claim 230, wherein the CD3ζ intracellular signaling domaincomprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562.

233. The allogeneic system of claim 229, wherein the costimulatory signaling domaincomprises a CD28 signaling domain.

234. The allogeneic system of claim 233, wherein the CD28 signaling domain comprisesthe sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 567).

235. The allogeneic system of claim 233, wherein the CD28 costimulatory signalingdomain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 567.

236. The allogeneic system of any one of claims 218 to 235, wherein the CD19-specificCAR comprises the sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRI DPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDIL TGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVS AAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMY PPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 647).

237. The allogeneic system of any one of claims 218 to 235, wherein the CD19-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO:

647. 869Mintz Ref. No.: 063384-521001WO238. The allogeneic system of any one of claims 218 to 235, wherein the CD19-specificCAR comprises the sequence of MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTN YWIAWVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQ WSSLKASDTAMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGG GGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGT APKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLS GNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 563).

239. The allogeneic system of any one of claims 218 to 235, wherein the CD19-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 563.

240. The allogeneic system of any one of claims 218 to 239, wherein the CD20-specificCAR comprises a CD20-specific binding domain, a transmembrane domain, and an intracellular domain.

241. The allogeneic system of any one of claims 218 to 240, wherein the CD20-specificbinding domain comprises an antibody that binds CD20 or an antigen-binding fragment thereof.

242. The allogeneic system of claim 241, wherein the CD20-specific binding domaincomprises an antibody that binds human CD20.

243. The allogeneic system of claim 241, wherein the CD20-specific binding domaincomprises an antigen-binding fragment of an antibody that binds human CD20.

244. The allogeneic system of claim 243, wherein the antigen-binding fragment of anantibody that binds human CD20 is a single chain variable fragment (scFv) that binds CD20.

245. The allogeneic system of claim 244, wherein the scFv capable of binding CD20 hasthe sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKL 870Mintz Ref. No.: 063384-521001WO LIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFG QGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCTASGFTF GDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 569).

246. The allogeneic system of claim 245, wherein the scFv capable of binding CD20comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 569.

247. The allogeneic system of any one of claims 218 to 246, wherein the CD20-specificCAR comprises a CD28 hinge domain comprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 565) and a CD28 transmembrane domain comprising the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 566).

248. The allogeneic system of any one of claims 218 to 247, wherein the CD20-specificCAR comprises a CD28 hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 565 and a CD28 transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 566.

249. The allogeneic system of any one of claims 218 to 248, wherein the CD20-specificCAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.

250. The allogeneic system of claim 249, wherein the primary T cell activating domaincomprising an ITAM comprises a CD3ζ intracellular signaling domain.

251. The allogeneic system of claim 250, wherein the CD3ζ intracellular signaling domaincomprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 562).

252. The allogeneic system of claim 250, wherein the CD3ζ intracellular signaling domain871Mintz Ref. No.: 063384-521001WO comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 562.

253. The allogeneic system of claim 249, wherein the costimulatory signaling domaincomprises a CD2 signaling domain.

254. The allogeneic system of claim 253, wherein the CD2 signaling domain comprises thesequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGH RSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGA AENSLSPSSN (SEQ ID NO: 570).

255. The allogeneic system of claim 253, wherein the CD2 costimulatory signaling domaincomprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 570.

256. The allogeneic system of any one of claims 218 to 255, wherein the CD20-specificCAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSS NNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQA EDVAVYYCQQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGG GVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTG YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARKSYYGSGSPDVF DIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEELETRAHRVATEE RGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPA PSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 568).

257. The allogeneic system of any one of claims 218 to 255, wherein the CD20-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 568.

258. The allogeneic system of any one of claims 218 to 255, wherein the CD20-specificCAR comprises the sequence of 872Mintz Ref. No.: 063384-521001WO DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFG QGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCTASGFTF GDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPP PYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHP PPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQP KPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 648).

259. The allogeneic system of any one of claims 218 to 255, wherein the CD20-specificCAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 648.

260. The allogeneic system of any one of claims 218 to 259, wherein the secondrecombinant nucleic acid comprises a polynucleotide sequence encoding the CD19- specific CAR, a polynucleotide sequence encoding the CD20-specific CAR, and a polynucleotide sequence encoding the CD22-specific CAR, wherein each CAR- encoding polynucleotide sequence is separated from the other CAR-encoding polynucleotide sequences by a sequence encoding a viral ribosome skipping peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide.

261. The allogeneic system of any one of claims 218 to 259, wherein the secondrecombinant nucleic acid comprises from 5’ to 3’, a sequence encoding the CD22- specific CAR; a sequence encoding a viral P2A ribosome skipping peptide; a sequence encoding the CD19-specific CAR; a sequence encoding a viral T2A ribosome skipping peptide; and a sequence encoding the CD20-specific CAR.

262. The allogeneic system of any one of claims 131 to 261, wherein the one or moreCARs encoded by the second recombinant nucleic acid is specific for a tumor antigen.

263. The allogeneic system of claim 262, wherein the tumor antigen is selected from thegroup consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), 873Mintz Ref. No.: 063384-521001WO beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43- 9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, GPC3, TGFBR2, and GD2, or any combinations thereof.

264. The allogeneic system of claim 262, wherein tumor antigen is a solid tumor antigen.

265. The allogeneic system of claim 262, wherein the tumor antigen is BCMA, TGFBR2,and / or GPC3.

266. The allogeneic system of any one of claims 131 to 265, wherein the secondrecombinant nucleic acid encoding the one or more CARs further comprises a promoter.

267. The allogeneic system of claim 266, wherein the promoter is a MND promoter, anMNDU3 promoter, an EF-1alpha promoter, a core EF-1alpha promoter, a gamma retro promoter, an hUbC promoter, a PGK promoter, a U6 promoter, a SFFV promoter, or an NFkB responsive promoter.

268. The allogeneic system of any one of claims 131 to 267, wherein the one or moreCARs encoded by the second recombinant nucleic acid is a bicistronic CAR.

269. The allogeneic system of any one of claims 131 to 268, wherein the first recombinantnucleic acid further comprises a fourth nucleotide sequence encoding a fourth 874Mintz Ref. No.: 063384-521001WO molecule capable of blocking the CD8-MHC class I interaction.

270. An allogeneic system comprising,a first recombinant nucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a first nucleotide sequence encoding an anti-CD19 CAR; a second nucleotide sequence encoding an anti-CD20 CAR; and a third nucleotide sequence encoding an anti-CD22 CAR.

271. An allogeneic system comprising,a first recombinant nucleic acid comprising: a first nucleotide sequence encoding a first molecule capable of reducing TCR-CD3 complex expression on the surface of an immune cell; a second nucleotide sequence encoding a second molecule capable of reducing MHC expression on the surface of the immune cell; and a third nucleotide sequence encoding a third molecule capable of reducing CD58 expression on the surface of the immune cell; and a second recombinant nucleic acid comprising a nucleotide sequence encoding an anti- CD22 CAR.

272. An immune cell comprising the allogeneic system of any one of claims 104-271.875

Images