Car-t cells secreting a CD2-binding multispecific antibody

Abstract

This disclosure provides CAR T cells secreting multispecific antibodies, multispecific antibodies that engage CAR-T cells to cancer cells, and methods of use thereof in treating cancer.

Description

[0001] CAR-T CELLS SECRETING A CD2-BINDING MULTISPECIFIC ANTIBODY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 715,891, filed November 4, 2024, entitled “CAR-T CELLS SECRETING A CD2-BINDING MUTISPECIFIC ANTIBODY” the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (M105370055WO00-SEQ-ARM.xml; Size: 86,649 bytes; and Date of Creation: November 4, 2025) are herein incorporated by reference in their entirety.

[0006] GOVERNMENT FUNDING

[0007] This invention was made with government support under Grant No. 5R01CA238268- 05 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment. CAR T cells express a CAR that directs the T cell to attack and kill cancer cells. Specially, the CAR has antigen binding domain that binds to a cancer cell antigen. Despite clinical success in treating some cancer types, CAR-T cell efficacy can be lost when the target cell decreases or eliminates expression of the target antigen.

[0010] SUMMARY

[0011] This disclosure includes CAR-T cells that secrete a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof. CD2 is typically expressed on T cells and is a T cell co-stimulatory ligand important for T cell target binding and T cell activation. In other words, binding of CD2 by the multispecific antibody can increase CAR- T cell activation, which results in increased cancer killing. The multispecific antibody may further comprise a cancer antigen binding antibody or antibody fragment thereof (e.g., binding CD20 or CD79b). The cancer antigen binding antibody or antibody fragment thereof

[0012] 1

[0013] #14564947v1 can provide a complementary strategy for targeting the CAR-T cell to the cancer cell (e.g., when the cancer cell expresses an antigen that is bound by the cancer antigen binding antibody or antibody fragment thereof). In some embodiments, the multispecific antibody further comprises an anti-CD3 binding antibody or antibody fragment. T cells express CD3. CD3, when activated (e.g., bound by an antibody) can activate the T cell, which can result increased cancer cell killing. As explained in detail in the Examples, CD 19 binding CAR T cells expressing a multispecific antibodies comprising an anti-CD2 binding antibody or antibody fragment, a CD20 antibody or antibody fragment or CD79b antibody or antibody fragment, and an anti-CD3 binding antibody or antibody fragment improve treatment of B cell malignancies with decreased CD 19 expression compared to a CD 19 only CAR-T cell.

[0014] In some embodiments, this disclosure provides a T cell comprising: a first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof; and a second polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the T cell secretes the multispecific antibody.

[0015] In some embodiments, the multispecific antibody comprises an anti-CD3 binding antibody or antibody fragment thereof and / or a cancer antigen binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody comprises, from N- terminal to C-Terminal, the cancer antigen binding antibody or antibody fragment thereof, the anti-CD2 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody comprises, from N-terminal to C-Terminal, the anti-CD2 binding antibody or antibody fragment thereof, the cancer antigen binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody comprises a linker between one or more of the antibodies or antibody fragments. In some embodiments, the linker is a glycine linker. In some embodiments, the glycine linker is a GGGGS (SEQ ID NO: 16) linker. In some embodiments, the GGGGS (SEQ ID NO: 16) linker is a (GGGGS)3 (SEQ ID NO: 70) linker. In some embodiments, the anti-CD2 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a variable light chain (VL) CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6. In some embodiments, the anti-CD2 antibody or antibody fragment thereof comprises a VH or SEQ ID NO: 7 and a VL or SEQ ID NO: 8.

[0016] In some embodiments, the anti-CD2 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD3 antibody or 2

[0017] #14564947v1 antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 10; a VH CDR2 of SEQ ID NO: 11; a VH CDR3 of SEQ ID NO: 12; a variable light chain (VL) CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 14; and a VL CDR3 of SEQ ID NO: 15. In some embodiments, the anti-CD3 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 18.

[0018] In some embodiments, the cancer antigen binding antibody or antibody fragment thereof binds to CD20 or CD79b. In some embodiments, the multispecific antibody is a bispecific antibody or trispecific antibody. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof binds to CD20. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 19; a VH CDR2 of SEQ ID NO: 20; a VH CDR3 of SEQ ID NO: 21.

[0019] In some embodiments, the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof binds to CD79b.

[0020] In some embodiments, the cancer antigen binding antibody or antibody fragment thereof comprises a VH or SEQ ID ON: 34 and a VL or SEQ ID NO: 35. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the multispecific antibody comprises an IgK leader, optionally the IgK leader is on the N-terminal of the multispecific antibody. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 61-68. In some embodiments, the multispecific antibody comprises an amino acid of any one of SEQ ID NOs: 61-68.

[0021] In some embodiments, the CAR comprises: an antigen-binding domain comprising an anti-CD19 antibody or antibody fragment thereof; a transmembrane domain; and an intracellular signaling domain.

[0022] In some embodiments, the anti-CD19 antibody comprises a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44.

[0023] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the intracellular signaling domain comprises a 4- IBB intracellular domain and / or a CD3zeta intracellular signaling domain. In some embodiments, 3

[0024] #14564947v1 the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 48. In some embodiments, the first polynucleotide and the second polynucleotide are in the same polynucleotide. In some embodiments, a linker is located between the first polynucleotide and / or the second polynucleotide. In some embodiments, the linker is a polynucleotide encoding a cleavable peptide. In some embodiments, the cleavable peptide is 2A peptide. In some embodiments, the cleavable peptide is P2A, T2A, E2A or F2A peptide. In some embodiments, the multispecific protein and the CAR are expressed as a polyprotein. In some embodiments, the polyprotein comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 55-60. In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 55-60. In some embodiments, the linker is an internal ribosome entry site (IRES).

[0025] In some embodiments, this disclosure provides a multispecific antibody comprising: an anti-CD79b binding antibody or antibody fragment thereof, or an anti-CD20 binding antibody or antibody fragment thereof; and an anti-CD2 binding antibody or antibody fragment thereof.

[0026] In some embodiments, the multispecific antibody comprises an anti-CD3 binding antibody or antibody fragment thereof.

[0027] In some embodiments, the anti-CD2 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a variable light chain (VL) CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6.

[0028] In some embodiments, the anti-CD3 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 10; a VH CDR2 of SEQ ID NO: 11; a VH CDR3 of SEQ ID NO: 12; a variable light chain (VL) CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 14; and a VL CDR3 of SEQ ID NO: 15.

[0029] In some embodiments, the anti-CD20 binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 19; a VH CDR2 of SEQ ID NO: 20; and a VH CDR3 of SEQ ID NO: 21.

[0030] In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) of SEQ ID NO: 34 and a variable light chain (VL) of SEQ ID NO: 35. In some embodiments, the multispecific antibody comprises, from N- terminal to C-Terminal, the anti-CD79b binding antibody or antibody fragment thereof or the 4

[0031] #14564947v1 anti-CD20 binding antibody or antibody fragment thereof, the anti-CD2 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

[0032] In some embodiments, the multispecific antibody comprises, from N-terminal to C- Terminal, the anti-CD2 binding antibody or antibody fragment thereof, the anti-CD79b binding antibody or antibody fragment thereof or the anti-CD20 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody comprises a leader sequence, optionally an IgK leader sequence. In some embodiments, the IgK leader sequence in on the N-terminal of the multispecific antibody. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 61-68. In some embodiments, the multispecific antibody comprises an amino acid of any one of SEQ ID NOs: 61-68.

[0033] In some embodiments, this disclosure provides a polyprotein encoding the multispecific antibody, a cleavable linker, and a CAR. In some embodiments, the CAR comprises: an antigen-binding domain comprising an anti-CD19 antibody or antibody fragment thereof; a CD8 hinge / transmembrane domain; and a 4 IBB intracellular signaling domain and a CD3-zeta intracellular signaling domain. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 45. In some embodiments, this disclosure provides a polynucleotide encoding the multispecific antibody or the polyprotein. In some embodiments, this disclosure provides a cell comprising the polynucleotide. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, this disclosure provides a method of treating a B-cell malignancy in a subject, the method comprising administering the T cell or the cell to the subject. In some embodiments, the B-cell malignancy is lymphoma or leukemia.

[0034] In some embodiments, the lymphoma is Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma or Lymphoplasmacytic lymphoma. In some embodiments, the cell is Hairy cell leukemia, B- cell acute lymphoblastic leukemia (B-ALL), Chronic lymphocytic leukemia (CLL), or Prolymphocytic leukemia (PLL). In some embodiments, the B-cell malignancy expresses CD19. In some embodiments, the B-cell malignancy expresses CD20 and / or CD79b.

[0035] In some embodiments, this disclosure provide a method of delivering a multispecific antibody to a cell expressing a target antigen in a subject, the method comprising

[0036] 5

[0037] #14564947v1 administering to the subject a T cell described herein, wherein the CAR of the T cell comprises an antigen-binding domain that binds to the target antigen of the cell of the subject. In some embodiments, the method of delivering comprises delivering to a B-cell.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIGs 1A-1E show the design of trispecific immune effector cell engager (TRIEC)- secreting CAR T cells. FIG. 1 A is a diagram of vector design and length. FIG. IB depicts a schematic of TRIEC structures. FIG. 1C are predicted protein structures (AlphaFold v3) of CD2 scFv and binding to CD2 protein (left), predicted binding of endogenous CD58 and CD2 scFv on CD2 protein (right). FIG. ID shows the predicted protein structure (Alphafold v3) of TRIEC molecule (left) and predicted interactions of TRIEC scFvs with target ligands (right). FIG. IE shows detection of binding to target antigens of TRIEC and TRIEC controls. Target cells were incubated with TRIEC-containing or null supernatant and binding of TRIEC. Mean fluorescence intensity (MFI) of anti-G4S linker APC targeting linker component of TRIEC was assessed via flow cytometry.

[0040] FIGs. 2A-2E show CD19 low with CD58 knockout creates resistance to CAR T cells in B-lymphocyte malignancies. FIG. 2A show a luciferase assay of 19BBz CAR-T cells coculture with Jeko-1 CBG-GFP CD19 WT and CD19-low cells incubated with CD58 blocking antibody. FIG. 2B shows a Incuctye Live-CellTM killing assay of 19BBz CAR-T cells cocultured 1:2 with Nalm6 CBG-GFP cell lines. FIG. 2C show cytokines from in vitro coculture of 19BBz CAR-T cells with Nalm6 CD191ow + / - CD58KO cells. FIG. 2D is a schematic representation of experimental design: NSG mice were injected with respective IX 106Jeko-1 CD19WT, CD191ow, or CD191ow / CD58KO. Seven days later mice received 1E6 19BBz CAR-T cells, tumor burden was periodically assessed by BLI. FIG. 2E shows the average radiance (p / s / cm2 / sr) FLUX over time of whole mice receiving different initial Jeko- 1 conditions.

[0041] FIGs. 3A-3D show CD79b TRIEC enhances UTD and CAR-T cell cytotoxicity and proliferation in the presence to leukemia / lymphoma cells in vitro. FIG. 3A shows a co-culture of untransduced primary human T-cells (UTD) with Jeko-1 CBG-GFP+ cells, incubated in supernatant conditions and assessed for cytotoxicity via luciferase assay. Conditions performed in triplicate with statistical comparison of means using t-test. FIG. 3B shows an assessment of CD69 expression of UTD or CAR-T cells after 24 hours of culture with (+) and without (-) stimulation of CD19+ / CD79b+ Jeko-1 cells. FIG. 3C shows cell trace violent proliferation dye staining of CAR-T cells in monoculture over time. FIG. 3D is a Incucyte 6

[0042] #14564947v1 LiveCell assay of CAR-T / UTD conditions against parental, CD191ow / CD58KO, and CD19K0 tumor lines. All conditions performed in triplicate at 1:2 E:T. Cytotoxicity represented by green area normalized to hour 0. Statistical analysis between 19BBz and 19BBz + 79b_CD2_CD3 conditions performed using two-way ANOVA.

[0043] FIGs. 4A-4E show design and in vivo activity of CD79b-targeted TRIEC. FIG. 4A is a schematic depicting in vivo experiment: NSG mice were injected with IX 10E6 Jeko-1 CD191ow / CD58KO cells or Jekol CD19 WT / CD58 WT cells. Seven days later mice received 1E6 CAR-T cells, tumor burden was periodically assessed by BLI. Cartoon depicts components of control CAR TRIEC (79b_EGFR_CD3) and active TRIEC (CD79b_CD2_CD3) constructs utilized for in vivo experiments. FIG. 4B shows the average radiance (p / s / cm2 / sr) FLUX over time of whole mice receiving 1E6 CD19-41BBz CAR T cells in respective Jekol conditions. FIG. 4C shows the average radiance (p / s / cm2 / sr) FLUX over time of whole mice receiving 1E6 CD19-41BBz + TRIEC / control TRIEC CAR T cells in respective Jekol CD191owCD58KO infused mice. FIG. 4D, Schematic depicting in vivo experiment: NSG mice were injected with 1.0 X 10E6 Jeko-1 CD191ow / CD58KO cells. Seven days later mice received 1.5E6 CAR-T cells, tumor burden was periodically assessed by BLI. FIG. 4E shows the average radiance (p / s / cm2 / sr) FLUX over time of whole mice receiving 1.5E6 CD19-41BBz + TRIEC / control TRIEC CAR T cells in respective Jekol CD191owCD58KO infused mice.

[0044] FIGs. 5A-5D show in vitro and in vivo activity of CD20-targeted TRIEC. FIG. 5A is a schematic of CD20 TRIEC molecule employing a CD20 nanobody targeting B-cells. FIG. 5B is an Incucyte LiveCell assay of UTD + CD20 TRIEC supernatant, and CD 19 CAR + CD20 TRIEC supernatant conditions against parental (top) and CD191ow / CD58KO (bottom) Jeko-1 cells. All conditions performed in triplicate at 1:2 E:T. Cytotoxicity represented by green area normalized to hour 0. FIG. 5C is a schematic depicting in vivo experiment: NSG mice were injected with IX 10E6 Jeko-1 CD191ow / CD58KO cells. Seven days later mice received 2E6 CAR-T cells, tumor burden was periodically assessed by BLI. FIG. 5D shows the average radiance (p / s / cm2 / sr) FLUX over time of whole mice receiving different CAR T cell conditions.

[0045] FIGs. 6A-6B show CAR constructs and a schematic of a CAR-T cell treatment schedule. FIG. 6A shows CAR constructs comprising a CAR operably linked to a constitutively active promoter and a TRIEC operably linked to an inducible promoter. FIG. 6B shows a treatment schedule for determining cancer killing activity of the T cells comprising the CAR constructs of FIG. 6A.

[0046] 7

[0047] #14564947v1 FIGs. 7A-7B show a method of administering a CAR-T cell and TRIEC separately. FIG. 7A shows the CAR constructs of the CAR-T cell to be administered. FIG. 7B shows a treatment plan for administering the CAR-T cells and the TRIEC s.

[0048] DETAIEED DESCRIPTION

[0049] In some aspects, this disclosure provides a T cell comprising: a first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody further comprises an anti-CD3 binding antibody or antibody fragment thereof. In some embodiments, the multispecific antibody further comprises a cancer antigen binding antibody or antibody fragment thereof antibody or antibody fragment thereof. In some embodiments, the T cell further comprises a second polynucleotide encoding a chimeric antigen receptor (CAR).

[0050] In some embodiments, the T cell is a mammalian T cell. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a mouse T cell. In some embodiments, the T cells is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, the T cell is obtained from a subject e.g., a subject having a B cell malignancy and engineered to comprises the first and / or second polynucleotide.

[0051] The term "polynucleotide" is used herein interchangeably with "nucleic acid molecule" to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this disclosure refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double- stranded forms (and complements of each single- stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein the term, “heterologous nucleic acid molecule” refers to a nucleic acid molecule that does not naturally

[0052] 8

[0053] #14564947v1 exist within a given cell. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

[0054] Some embodiments relate to amino acid sequences or nucleotide sequences (e.g., polynucleic acids) having some percentage identity to a reference amino acid sequence or reference nucleotide sequence, respectively. As used herein, the term “identity” refers to the degree to which two or more sequences (e.g., amino acid sequences, nucleotide sequences) are related, as determined by the number of matches between each sequence. “Percent (%) identity” refers to the percentage of residues or nucleotides in a first amino acid or first nucleic acid sequence that are identical to the residues in a second amino acid sequence or second nucleic acid sequence. The skilled artisan will understand that determining percent identity may require a step of aligning a first and second sequence and / or introducing gaps. Several algorithms for aligning sequences and / or determining percent identity between sequences are known in the art. For example, EMBOSS NEEDLE (e.g., as described in Madeira F. et al., Nucleic Acids Research. 2024 Jul;52(Wl):W521-W525. PMID: 38597606) can be used to align sequences and determine percent identity between sequences.

[0055] Multispecific Antibodies

[0056] In some embodiments, this disclosure provide a first polynucleotide encoding a multispecific antibody. A multispecific antibody refers to a protein or peptide that can bind to two or more targets. The targets may be protein targets (e.g., CD3, CD2, CD20, CD79b, etc.) or non-protein targets (e.g., complex sugars or lipids). In some embodiments, the protein targets are proteins expressed by a T cell and / or other immune cell (e.g., CD2 or CD3). In some embodiments, the protein targets are proteins expressed by a target cancer (e.g., CD20 or CD79b). In some embodiments, the target binding domains of the multispecific antibody comprise antibodies (e.g., monoclonal antibodies) and / or antibody fragments thereof (e.g. an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a fragment variable (fv), a single chain fragment variable (scFv), or a VHH). In some embodiments, the multispecific antibody is a bispecific antibody (i.e., comprises two different target binding domains). In some embodiments, the multispecific antibody is a trispecific antibody (i.e., comprises three different target binding domains.

[0057] In some embodiments, one or more target binding domains of the antibody are joined to one another via a linker. As used herein, "linker" refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains / regions of CAR and / or multispecific antibody as described herein. In some embodiment, linkers can 9

[0058] #14564947v1 include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Linker sequences may be from 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids in length, and include any suitable linkers known in the art. For instance, linker sequences may include, but are not limited to, glycine / serine (GS) linkers. In some embodiments, the linker is a (GS)n(SEQ ID NO: 17) linker where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is a (GGS)n(SEQ ID NO: 26) linker where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is a (GGGS)n(SEQ ID NO: 28) linker where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is a (GGGGS)n (SEQ ID NO: 29) linker where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is a glycine repeat linker. In some embodiments, the bispecific antibody comprises from n-terminal to c-terminal a first target binding domain, a linker (e.g., (GGGGSjs (SEQ ID NO: 70)) and a second target binding domain. In some embodiments, the trispecific antibody comprises from n-terminal to c-terminal a first target binding domain, a first linker (e.g., (GGGGSjs (SEQ ID NO: 70)), a second target binding domain, a second linker (e.g., (GGGGS)s (SEQ ID NO: 70)), and a third target binding domain. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 70.

[0059] In some embodiments, the multispecific antibody comprises a leader (e.g., a signal peptide). In some embodiments, the leader refers to a peptide at the N-terminus of a newly synthesized protein that serves to direct a nascent protein into the endoplasmic reticulum. In some embodiments, the leader is a CD8 signal peptide (e.g., SEQ ID NO: 46) or IgK signal peptide (SEQ ID NO: 47).

[0060] In some embodiments, the T cell secretes the multispecific antibody (e.g., this may be performed by attaching a leader to the multispecific antibody e.g., as described herein).

[0061] In some embodiments, the multispecific antibody comprises an anti-CD2 binding antibody or antibody fragment thereof. CD2 (e.g., UniProt: P06729) is a cell adhesion molecule found on the surface of some immune cells (e.g., T cells). CD2 can contribute to activation of T cells. In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof is a full length antibody. In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof is an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a fragment variable (fv), a single chain fragment variable (scFv), or a VHH). In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof is Siplizumab or an antibody fragment thereof.

[0062] 10

[0063] #14564947v1 In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof comprises a heavy chain variable region complementary determining region 1 (VH CDR1) of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a light chain variable region complementary determining region 1 (VL CDR1) of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5, of VL CDR2 of SEQ ID NO: 6. In some embodiments, the anti- CD2 binding antibody or antibody fragment thereof comprises a VH of SEQ ID NO: 7 and / or a VL of SEQ ID NO: 8. In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof comprises a VH of SEQ ID NO: 7 and / or a VL of SEQ ID NO: 8 and one or more mutations in the framework region of the VH and / or VL domain. In some embodiments, the anti-CD2 binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 9.

[0064] In some embodiments, the multispecific antibody comprises an anti-CD2 binding antibody or antibody fragment thereof and a cancer antigen binding antibody or antibody fragment thereof. In some embodiments, the cancer antigen binding antibody or antibody fragment thereof is an anti-CD79b binding antibody or antibody fragment thereof. CD79b (e.g., UniProt: P40259) is expressed by B cells and plays a role in B cell activation.

[0065] In some embodiments, the anti-CD79b binding antibody or antibody fragment is a full length antibody. In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof is an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a fragment variable (fv), a single chain fragment variable (scFv), or a VHH.

[0066] In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof comprises a VH of SEQ ID NO: 34 and / or a VL of SEQ ID NO: 35. In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof comprises a VH of SEQ ID NO: 34 and / or a VL of SEQ ID NO: 35 and one or more mutations in the framework region of the VH and / or VL domain. In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 36. In some embodiments, the anti-CD79b binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to

[0067] 1 1

[0068] #14564947v1 SEQ ID NO: 36, wherein the CDR regions of the antibody or antibody fragment are not mutated.

[0069] In some embodiments, the cancer antigen binding antibody or antibody fragment thereof is an anti-CD20 binding antibody or antibody fragment thereof. CD20 (e.g., UniProt: Pl 1836) is expressed by B cells and plays a role in regulating calcium influx.

[0070] In some embodiments, the anti-CD20 binding antibody or antibody fragment is a full length antibody. In some embodiments, the anti-CD20 binding antibody or antibody fragment thereof is an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a fragment variable (fv), a single chain fragment variable (scFv), or a VHH). In some embodiments, the anti- CD20 binding antibody is a camelid antibody.

[0071] In some embodiments, the anti-CD20 binding antibody or antibody fragment thereof comprises a heavy chain variable region complementary determining region 1 (VH CDR1) of SEQ ID NO: 19, a VH CDR2 of SEQ ID NO: 20, a VH CDR3 of SEQ ID NO: 21. In some embodiments, the anti-CD20 binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 27. In some embodiments, the anti-CD20 binding antibody or antibody fragment thereof comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 27, and does not comprise a mutation in a CDR.

[0072] In some embodiments, the multispecific antibody comprises an anti-CD2 binding antibody or antibody fragment thereof and an anti-CD3 binding antibody or fragment thereof. In some embodiments, the multispecific antibody comprises an anti-CD2 binding antibody or antibody fragment thereof, an anti-CD3 binding antibody or fragment thereof, and a cancer antigen binding antibody or antibody fragment thereof (e.g. a CD79b or CD20 binding antibody or antibody fragment thereof). In some embodiments, the multispecific antibody comprises an anti-CD2 binding antibody or antibody fragment thereof and a cancer antigen binding antibody or antibody fragment thereof (e.g. a CD79b or CD20 binding antibody or antibody fragment thereof).

[0073] CD3 is a protein complex and T cell receptor that has a role in activating T cells. Binding of CD3 by an antibody can result in T cell activation.

[0074] In some embodiments, the anti-CD3 binding antibody or antibody fragment thereof is a full length antibody. In some embodiments, the anti-CD3 binding antibody or antibody

[0075] 12

[0076] #14564947v1 fragment thereof is an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a fragment variable (fv), a single chain fragment variable (scFv), or a VHH.

[0077] In some embodiments, the anti-CD3 binding antibody or antibody fragment thereof comprises a heavy chain variable region complementary determining region 1 (VH CDR1) of SEQ ID NO: 10, a VH CDR2 of SEQ ID NO: 11, a VH CDR3 of SEQ ID NO: 12, a light chain variable region complementary determining region 1 (VL CDR1) of SEQ ID NO: 13, VL CDR2 of SEQ ID NO: 14, and a VL CDR3 of SEQ ID NO: 15. In some embodiments, the anti-CD3 binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 18. In some embodiments, the anti-CD3 binding antibody or antibody fragment thereof comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 18, and does not comprise a mutation in a CDR.

[0078] Table 1: Exemplary bispecific antibody orientations

[0079] N-terminal C- terminal

[0080] CD2 binder refers to an anti-CD2 antibody or antibody fragment thereof CD20 binder refers to an anti-CD20 antibody or antibody fragment thereof CD79b binder refers to an anti-CD79b antibody or antibody fragment thereof

[0081] Table 2: Exemplary bispecific antibody orientations with linkers

[0082] N-terminal C- terminal

[0083] CD2 binder refers to an anti-CD2 antibody or antibody fragment thereof

[0084] CD20 binder refers to an anti-CD20 antibody or antibody fragment thereof

[0085] CD79b binder refers to an anti-CD79b antibody or antibody fragment thereof

[0086] Linker may be any suitable linker including those described herein. Typically, the linker is a non-cleavable linker.

[0087] Table 3: Exemplary trispecific antibody orientations

[0088] 13

[0089] #14564947v1 N-terminal C- terminal

[0090] CD2 binder refers to an anti-CD2 antibody or antibody fragment thereof CD20 binder refers to an anti-CD20 antibody or antibody fragment thereof CD79b binder refers to an anti-CD79b antibody or antibody fragment thereof

[0091] Table 4: Exemplary trispecific antibody orientations with linkers

[0092] N-terminal C- terminal CD2 binder refers to an anti-CD2 antibody or antibody fragment thereof

[0093] CD20 binder refers to an anti-CD20 antibody or antibody fragment thereof

[0094] CD79b binder refers to an anti-CD79b antibody or antibody fragment thereof

[0095] Linker- 1 and Linker-2 may be any suitable linker including those described herein. Linker- 1 and linker-2 may be the same linker or different linkers. Typically linker- 1 and linker-2 are non-cleavable linkers.

[0096] In some embodiments, the T cell comprises a first polynucleotide encoding a multispecific antibody (e.g., as described herein) and a second polynucleotide encoding a chimeric antigen receptor. In some embodiments, the multispecific antibody comprise a tag

[0097] #14564947v1 (e.g., a his tag or flag tag). In some embodiments, the tag is on the c-terminal of the multispecific antibody. In some embodiments, the his tag is HHHHHH (SEQ ID NO: 25).

[0098] In some embodiments, the multispecific antibody comprises an amino acid sequence of any one of SEQ ID NOs: 61-68 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to any one of SEQ ID NOs: 61-68. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to any one of SEQ ID NOs: 61-68 and does not comprise a mutation in a CDR of the multispecific antibody.

[0099] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 61 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 61. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 61 and does not comprise a mutation in a CDR of the multispecific antibody.

[0100] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 62 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 62. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 62 and does not comprise a mutation in a CDR of the multispecific antibody.

[0101] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 63 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 63. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 63 and does not comprise a mutation in a CDR of the multispecific antibody.

[0102] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 64 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 64. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 15

[0103] #14564947v1 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 64 and does not comprise a mutation in a CDR of the multispecific antibody.

[0104] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 65 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 65. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 65 and does not comprise a mutation in a CDR of the multispecific antibody.

[0105] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 66 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 66. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 66 and does not comprise a mutation in a CDR of the multispecific antibody.

[0106] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 67 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 67. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 67 and does not comprise a mutation in a CDR of the multispecific antibody.

[0107] In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 68 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 68. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 68 and does not comprise a mutation in a CDR of the multispecific antibody.

[0108] In some embodiments, a multispecific antibody comprises a CD20 binding domain, an EGFR binding domain and a mesothelin binding domain. In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 71 or having at 16

[0109] #14564947v1 least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 71. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 71 and does not comprise a mutation in a CDR of the multispecific antibody.

[0110] In some embodiments, a multispecific antibody comprises a CD20 binding domain, an EGFR binding domain and a CD3 binding domain. In some embodiments, the multispecific antibody comprises an amino acid sequence of SEQ ID NO: 72 or having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 72. In some embodiments, the multispecific antibody comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 72 and does not comprise a mutation in a CDR of the multispecific antibody.

[0111] In some embodiments, a multispecific antibody comprises an EGFR binding domain. In some embodiments, an EGFR binding domain comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 74 and does not comprise a mutation in a CDR of the multispecific antibody. In some embodiments, an anti-EGFR binding antibody or antibody fragment thereof comprises a heavy chain variable region complementary determining region 1 (VH CDR1) of SEQ ID NO: 82, a VH CDR2 of SEQ ID NO: 83, a VH CDR3 of SEQ ID NO: 84, a light chain variable region complementary determining region 1 (VL CDR1) of SEQ ID NO: 85, VL CDR2 of SEQ ID NO: 86, and a VL CDR3 of SEQ ID NO: 87.

[0112] In some embodiments, a multispecific antibody comprises an mesothelin binding domain (e.g., an SSI antibody or fragment thereof). In some embodiments, an mesothelin binding domain comprises an amino acid sequence having at least 85% identity (e.g., at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 75 and does not comprise a mutation in a CDR of the multispecific antibody. In some embodiments, an anti-mesothelin binding antibody or antibody fragment thereof comprises a heavy chain variable region complementary determining region 1 (VH CDR1) of SEQ ID NO: 76, a VH CDR2 of SEQ ID NO: 77, a VH CDR3 of SEQ ID NO: 78, a light chain variable region complementary determining region 1 (VL CDR1) of SEQ ID NO: 79, VL CDR2 of SEQ ID NO: 80, and a VL CDR3 of SEQ ID NO: 81.

[0113] Chimeric Antigen Receptors (CARs)

[0114] 17

[0115] #14564947v1 The terms "chimeric antigen receptor" or "CAR" or "CARs", as used herein, refer to engineered T cell receptors, which graft a ligand or antigen specificity onto immune cells. In some embodiments, the CAR is a bispecific CAR. CARs can be specific for one antigen or multiple antigens, depending on the configuration of the antigen binding domain.

[0116] In some embodiments, a CAR comprises an antigen binding domain that specifically binds a target, e.g., a polypeptide, expressed on the surface of a cell to be targeted for a T cell response, onto a construct including a transmembrane domain and intracellular domain(s) of a T cell receptor molecule.

[0117] As used herein, a "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. In some embodiments, when expressed in a T cell, CARs have the ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. In some embodiments, the non- MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0118] In some embodiments, a cell described herein (e.g., a T cell) is an isolated cell. As used herein, an “isolated” cell refers to a cell that has been extracted from a subject (e.g., via whole blood collection, an apheresis system, a leukapheresis system, a subject-connected closed loop system, etc.). In some embodiments an isolated cell (e.g., an isolated T cell) is a cell that has been extracted from the subject and engineered (e.g., engineered to express a CAR polypeptide provided herein).

[0119] As can be determined by those of skill in the art, various functionally similar or equivalent components of these CARs can be swapped or substituted with one another, as well as other similar or functionally equivalent components known in the art or listed herein.

[0120] Any cell-surface moiety can be targeted by a CAR. Often, the target will be a cellsurface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. In some embodiments, the antigen-binding domains binds CD19. CD19 may be used as a marker for cells associated with various cancers (e.g., over-expressed in various B-cell cancers), and CARs and CAR-T cells that bind to CD 19 or a portion thereof may be used to treat subjects having, e.g., cancers associated with CD 19 expression (e.g., B cell malignancies).

[0121] Antigen-Binding Domain

[0122] 18

[0123] #14564947v1 In some embodiments, a CAR comprises an extracellular domain. In some embodiments, the extracellular domain comprising an antigen-binding domain. As used herein, the term "antigen-binding domain" refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding to a target (e.g., a cancer antigen). In some embodiments, the CARs described herein comprise an antigen-binding domain. The antigenbinding domain will specifically bind to its binding partner, i.e., the target. As non-limiting examples, the antigen-binding domain can include an antibody or antibody fragment thereof (e.g., an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a VVH, a fragment variable (fv) or a single chain fragment variable (scFv), VHH), or a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule that binds specifically to a portion of a protein and / or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand:cognate partner binding can result in the alteration of the ligandbearing receptor, or activate a physiological response, for example, the activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a conserved function across at least two species.

[0124] In some embodiments, the disclosure provides a CAR comprising an antigen-binding domain that binds to CD 19.

[0125] In some embodiments, the first antigen-binding domain comprises an anti-CD19 antibody or antibody fragment thereof. In some embodiments, the anti-CD19 antibody or antibody fragment thereof is an antigen-binding fragment (Fab), a Fab’, a F(ab’)2, a VVH, a fragment variable (fv), a single chain fragment variable (scFv), or VHH. In some embodiments, the anti-CD19 antibody or antibody fragment thereof comprises a VH comprising an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 43 and the VL comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 44. In some embodiments, the anti-CD19 antibody or antibody fragment thereof comprises a VH comprising an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 43 and the VL comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 44, wherein the CDRs of the VH and the VL and not mutated. In some embodiments, the anti-CD19 antibody or antibody fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 43 and the VL comprises an amino acid 19

[0126] #14564947v1 sequence of SEQ ID NO: 44. In some embodiments, the anti-CD19 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 45.

[0127] In some embodiments, the CAR comprises an amino acid sequence having at least 85% e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NOs: 48. In some embodiments, a CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 48.

[0128] Hinge and Transmembrane Domains

[0129] In some embodiments, a CAR further comprises a transmembrane domain, or a hinge / transmembrane domain, which joins the antigen binding domain(s) to the intracellular signaling domain. The binding domain of a bispecific CAR is, in some embodiments, followed by one or more "hinge domains," which plays a role in positioning the antigen binding domain(s) away from the effector cell surface to enable proper cell / cell contact, antigen-binding (by the antigen binding domain(s)) and activation. A bispecific CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semisynthetic, or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the bispecific CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4- IBB, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, a bispecific CAR comprises a polynucleotide encoding CD8a hinge / transmembrane domain. In some embodiments, a bispecific CAR comprises a polynucleotide encoding a 4- IBB intracellular domain.

[0130] In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8). In some embodiments, the hinge domain includes a CD8a hinge region.

[0131] As used herein, "transmembrane domain" (TM domain) refers to the portion of a CAR that fuses the antigen binding domain, in some embodiments via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and intracellular signaling domain). The transmembrane domain is a generally hydrophobic region of a CAR, which crosses the 20

[0132] #14564947v1 plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used herein, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of a CAR.

[0133] As used in relation to a transmembrane domain of a protein or polypeptide, "fragment thereof" refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0134] In some embodiments, the transmembrane domain or fragment thereof of a bispecific CAR described herein includes a transmembrane domain selected from the transmembrane domain of an alpha (a), beta (0) or zeta (Q chain of a T cell receptor, CD2, CD28, CD3 epsilon (e), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R0, IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0135] As used herein, a “hinge / transmembrane domain” refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4- IBB. In some embodiments, the hinge / transmembrane domain of a bispecific CAR or fragment thereof is derived from or includes the hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 49, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha, CD8a, or CD8a) and beta (CD8beta, CD8b, or CD80) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including 21

[0136] #14564947v1 naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.

[0137] Homologs and / or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD8 sequence.

[0138] In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO: 49; or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 49.

[0139] Intracellular Signaling Domains

[0140] In some embodiments, a CAR described herein comprises the intracellular domain of one or more co-stimulatory molecule or co-stimulatory domain. As used herein, the term "costimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fe receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4- IBB, CD27, CD28, or 0X40. Additional illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the co- stimulatory domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation induced co-stimulatory molecule and is an important regulator of immune responses.

[0141] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4- IBB is expressed on activated T lymphocytes. 4-1BB sequences are known for a number of species, e.g., human 4-1 BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BB can refer to the 4-1BB of, e.g., dog, cat, cow, horse, pig, and the like.

[0142] 22

[0143] #14564947v1 Homologs and / or orthologs of human 4- IBB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4-1 BB sequence. In some embodiments, the co-stimulatory domain comprises a 4- IBB co-stimulatory domain comprising an amino acid sequence of SEQ ID NO: 50, or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 50.

[0144] In some embodiments, CAR comprise a polynucleotide encoding a CD3<^ intracellular signaling domain.

[0145] The properties of the intracellular signaling domain(s) of a bispecific CAR can vary as known in the art and as disclosed herein, but the chimeric target / antigen binding domains(s) render the receptor sensitive to signaling activation when the chimeric target / antigen binding domain(s) binds the target / antigen on the surface of a targeted cell.

[0146] With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3<^ signals upon antigen-binding by the antigen binding domain(s). So-called "second-generation" CARs include those that provide both costimulation (e.g., CD28 or CD137) and activation (CD3 domains, and so-called "third- generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3 . In some embodiments, a bispecific CAR is selected to have high affinity or avidity for the targets / antigens - for example, antigen binding domains will generally have higher affinity and / or avidity for the target antigen than would a naturally occurring T cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific T cell targeting by CAR-T cells.

[0147] CARs as described herein may include an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a bispecific CAR that participates in transducing the message of effective bispecific CAR binding to a target antigen or target antigens into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the bispecific CAR-bound target cell, or other cellular responses elicited following antigen-binding to the antigen-binding domain(s) of the bispecific CAR. In various examples, the intracellular signaling domain is from CD3i^ (see, e.g., below). Additional nonlimiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing

[0148] 23

[0149] #14564947v1 intracellular signaling domains that are of particular use in the technology include those derived from 4-1BB (e.g., as described above), CD28, CD27, TCR zeta ( , FcR gamma (y), FcR beta (P), CD3 gamma (y), CD3 theta (0), CD3 sigma (o), CD3 eta (r|), CD3 epsilon (E), CD3 zeta (Q, CD22, CD79a, CD79b, and CD66d.

[0150] CD3 is a T cell co-receptor that facilitates T lymphocyte activation when simultaneously engaged with the appropriate co- stimulation (e.g., binding of a co- stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammalian CD3 consists of a CD3y chain, a CD38 chain, and two CD3E chains.

[0151] These chains associate with a molecule known as the T cell receptor (TCR) and the CD3<^ to generate an activation signal in T lymphocytes. A complete TCR complex includes a TCR, CD3i^, and the complete CD3 complex.

[0152] In some embodiments, a bispecific CAR described herein includes an intracellular signaling domain that includes an Immunoreceptor Tyrosine-based Activation Motif or IT AM from CD3i^, including variants of CD3i^ such as IT AM-mutated CD3i^, CD3r|, or CD30. In some embodiments of any aspect, the IT AM includes three motifs of IT AM of CD3i^ (ITAM3). In some embodiments of any aspect, the three motifs of IT AM of CD3i^ are not mutated and, therefore, include native or wild-type sequences. In some embodiments, the CD3<^ sequence includes the sequence of a CD3<^ as set forth in the sequences provided herein, e.g., a CD3i^ sequence of SEQ ID NO: 51 or variants thereof.

[0153] For example, a CAR described herein includes the intracellular signaling domain of CD3<^. In some embodiments, the CD3<^ intracellular signaling domain corresponds to an amino acid sequence of SEQ ID NO: 51 or includes a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence of SEQ ID NO: 51.

[0154] In some embodiments, the intracellular domain is the intracellular domain of a 4- IBB. In some embodiments, the 4- IBB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO: 50 or includes at least 75%, at least 80%, at least 85%, 35 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 50. Individual CAR and bispecific CAR, and other construct components as described herein can be used with one another and swapped in and out of various constructs described herein, as can be determined

[0155] 24

[0156] #14564947v1 by those of skill in the art. Each of these components can include or consist of any of the corresponding sequences set forth herein, or variants thereof.

[0157] A more detailed description of CARs and CAR-T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012.

[0158] Signal Peptide / Leader

[0159] In some embodiments, a CAR as described herein includes a signal peptide. Signal peptides, also called leader sequences, can be derived from any protein that has an extracellular domain or is secreted. A CAR as described herein may include any leader sequence known in the art. In some embodiments, a CAR includes a CD8 leader sequence, e.g., a CD8 leader sequence comprising the amino acid sequence of SEQ ID NO: 46 or including an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 46. In some embodiments, a CAR includes an IgK leader sequence, e.g., an IgK leader sequence comprising the amino acid sequence of SEQ ID NO: 47 or including an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 47.

[0160] In further embodiments, a CAR described herein may optionally exclude one of the signal peptides described herein, e.g., a CD8 signal peptide of SEQ ID NO: 46 or an IgK signal peptide of SEQ ID NO: 47.

[0161] In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, a CD8 hinge / TM domain, a 4- IBB intracellular domain, and a CD3-zeta intracellular domain.

[0162] Multispecific Antibody - CAR Constructs and Polyproteins

[0163] In some embodiments, the first polynucleotide encoding the multispecific antibody (e.g., as described herein) and the second polynucleotide encoding the CAR (e.g., as described herein) are the same polynucleotide. In some embodiments, the first polynucleotide encoding the multispecific antibody (e.g., as described herein) and the second polynucleotide encoding the CAR (e.g., as described herein) are the different polynucleotides.

[0164] 25

[0165] #14564947v1 In some embodiments, this disclosure described a polyprotein comprising a multispecific antibody and a CAR. In some embodiments, the polyprotein comprises, from N-terminal to C-terminal a multispecific antibody and a CAR. In some embodiments, the polyprotein comprises, from N-terminal to C-terminal a CAR and a multispecific antibody. In some embodiments, the polyprotein comprises a cleavable peptide between the CAR and the multispecific antibody. In some embodiments, the cleavable peptide is a 2A peptide. In some embodiments, the 2A peptide is P2A, T2A, E2A or F2A 2A peptide. In some embodiments, the polyprotein comprises a CAR, a cleavable linker, and a multispecific protein. In some embodiments, the polyprotein comprises a multispecific protein, a cleavable linker, and a CAR.

[0166] In some embodiments, the first polynucleotide encoding the multispecific antibody is operably linked to a promoter (e.g., a first promoter). In some embodiments, the second polynucleotide encoding the CAR is operably linked to a promoter (e.g., a second promoter). I some embodiments, the first polynucleotide encoding the multispecific antibody and the second polynucleotide encoding the CAR are operably linked to the same promoter. I some embodiments, the first polynucleotide encoding the multispecific antibody and the second polynucleotide encoding the CAR are operably linked to different promoters.

[0167] A promoter described herein (e.g., a first promoter and a second promoter) may be any suitable promoter. As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a CAR or a multispecific antibody, where the polynucleotide molecules are so arranged that the promoter can direct a RNA polymerase to transcribe the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0168] In some embodiments, a promoter is a constitutively active promoter. In some embodiments, a U6 promoter is from a non-human species. In some embodiments, a promoter (e.g., the first promoter and / or the second promoter) is selected from the group consisting of a CMV promoter, an EFla promoter, an EF la-short promoter, a CAG promoter, a PGK promoter, Hl promoter, or a U6 promoter. In some embodiments, the U6 promoter is from a human U6 promoter. In some embodiments, the U6 promoter is from cow, mouse, rat, pig, yeast, dog, cat, drosophila, or C. elegans. In some embodiments, the promoter is a Hl promoter. In some embodiments, the promoter is a tissue-specific promoter (e.g., the HP1, 26

[0169] #14564947v1 CD 14, CD43, CD45, C68, elastase, endoglin, fibronectin, Fit, GFAP, GPIIb, ICAM-2, mlFN- beta, Mb, NphsI, OG-2, SP-B, SYN1, or WASP gene promoter). In some embodiments, a promoter is an inducible promoter (e.g., a tet or lac promoter).

[0170] Without being bound to theory, constitutive expression of a TRIEC by a CAR-T cell may result in consistent activation of the CAR-T cell (even before the CAR-T cell is contacted with cancer cells), which can result in CAR-T cell exhaustion and decreased CAR- T cell cancer killing efficiency. This is surprising because prior work has shown that bispecific T cell engager expression by CAR-T cells does not result in the increased level of CAR-T cell exhaustion observed in TRIEC induced exhaustion. It is expected that TRIEC- induced CAR-T cell exhaustion can be reduced by controlling the timing of TRIEC expression by the CAR-T cell (e.g., using an inducible promoter operably linked to the polynucleotide encoding the TREIC). For example, TRIEC expression may be controlled by a promoter that is induced by CAR-T cell activation. Thus, in some embodiments, such a CAR-T cell would express the TRIEC when the CAR-T cell is activated by binding to a cancer cell (e.g., via the CAR of the CAR-T cell).

[0171] In some embodiments, the inducible promoter is a promoter that is induced by T cell activation (e.g., a Nuclear Factor of Activated T-cells (NFAT) promoter or a NR4A-based promoter). NFAT promoters and NR4A-based promoters are described in the art, e.g., as described in Guo et al., ACS Synth Biol. 2022 Apr 15;ll(4):1440-1453; PMID: 35316028. In some embodiments, a promotor is an NFAT promoter. In some embodiments, an NFAT promoter comprises a nucleic acid sequence of SEQ ID NO: 73. In some embodiments, a promotor is an NR4A-based promoter

[0172] In some embodiments, this disclosure provides a polynucleic acid comprising the first polynucleotide encoding the multispecific antibody and the second polynucleotide encoding the CAR. In some embodiments, the first polynucleotide is operably linked to a first promoter (e.g., an inducible promoter) and the second polynucleotide is operably linked to a second promoter (e.g., a constitutively active promoter). In some embodiments, the first promoter and the second promoter are in a divergent orientation in the polynucleic acid. In some embodiments, the first promoter and the second promoter are in a convergent orientation in the polynucleic acid. In some embodiments, the first promoter and the second promoter are in the same orientation in the polynucleic acid.

[0173] In some embodiments, this disclosure provides a polynucleic acid comprising the first polynucleotide encoding the multispecific antibody and the second polynucleotide encoding the CAR. In some embodiments, the first polynucleotide is operably linked to a first 27

[0174] #14564947v1 promoter (e.g., a constitutively active promoter) and the second polynucleotide is operably linked to a second promoter (e.g., a constitutively active promoter). In some embodiments, the first polynucleotide is operably linked to a first promoter (e.g., a constitutively active promoter) and the second polynucleotide is operably linked to a second promoter (e.g., an inducible promoter). In some embodiments, the first polynucleotide is operably linked to a first promoter (e.g., an inducible promoter) and the second polynucleotide is operably linked to a second promoter (e.g., an inducible promoter).

[0175] In some embodiments, the first polynucleotide comprises an inducible promoter (e.g., NFAT promoter or NR4A-based promoter) and the second polynucleotide comprises a constitutively active promoter (e.g., a EFla promoter). In some embodiments, the first polynucleotide is operably linked to an inducible promoter (e.g., NFAT promoter or NR4A- based promoter) and the second polynucleotide is operably linked to a constitutively active promoter (e.g., a EFla promoter).

[0176] In some embodiments, this disclosure provide a cell comprising a nucleic acid encoding the first polynucleotide and the second polynucleotide. The cell may be any suitable cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a bacterial cell (e.g., an E. coli).

[0177] In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 55, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 55.

[0178] In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 56, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 56.

[0179] In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 57, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 57.

[0180] In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 58, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 58.

[0181] In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 59, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 59.

[0182] 28

[0183] #14564947v1 In some embodiments, the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence to any one of SEQ ID NOs: 60.

[0184] Methods of Treatment

[0185] In some embodiments, this disclosure provides a method of treating a cancer (e.g., a B-cell malignancy) in a subject, the method comprising administering a cell described herein (e.g., a T cell) or administering a multispecific antibody described herein. For example, administering a T cell comprising the first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof; and a second polynucleotide encoding a chimeric antigen receptor (CAR). For example, administering a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof. In some embodiments, a method of treating a cancer in a subject (e.g., a B-Cell malignancy) comprising, administering (a) a CAR-T cell (e.g., a CD19 CAR-T cell) that does not express a TRIEC; and (b) a TRIEC to the subject. In some embodiments, the CAR-T cell and the TRIEC are administered serially (e.g., within a 24 hour time period of one another). In some embodiments, the CAR-T cell and the TRIEC are administering concurrently. In some embodiments, the CAR-T cell is administered before the TRIEC. In some embodiments, the CAR-T cell is administered after the TRIEC.

[0186] In some embodiments, the B-cell malignancy is lymphoma or leukemia.

[0187] In some embodiments, the lymphoma is Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma or Lymphoplasmacytic lymphoma. In some embodiments, the cell is Hairy cell leukemia, B- cell acute lymphoblastic leukemia (B-ALL), Chronic lymphocytic leukemia (CLL), or Prolymphocytic leukemia (PLL). In some embodiments, the B-cell malignancy expresses CD19. In some embodiments, the B-cell malignancy expresses CD20 and / or CD79b.

[0188] As used herein, a “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., 29

[0189] #14564947v1 chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female.

[0190] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., diagnosed with CD 19, CD20, and / or CD79b expressing cancer and / or a B cell malignancy) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.

[0191] As used herein, “treating a subject having a B cell malignancy” is ameliorating any condition or symptom associated with the B cell malignancy. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of a tumor.

[0192] Modes of Administration

[0193] Modes of administration (e.g., of the T cell) can include, for example intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient transarterially, intratumorally, intranodally, intraperitoneally, intrathecally, intramedullary, or orally. In some embodiments, the compositions of CAR-T cells may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0194] In some embodiments, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate the cells of interest, e.g., T cells. These T cell isolates can be expanded by contact with an artificial APC (aAPC), e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs, and treated such that one or more bispecific CAR constructs of the technology may be introduced, thereby creating a CAR-T cell.

[0195] 30

[0196] #14564947v1 Sequences

[0197] 31

[0198] #14564947v1

[0199] 32

[0200] #14564947v1

[0201] 33

[0202] #14564947v1

[0203] 34

[0204] #14564947v1

[0205] 35

[0206] #14564947v1

[0207] 36

[0208] #14564947v1

[0209] 37

[0210] #14564947v1

[0211] 38

[0212] #14564947v1

[0213] ADDITIONAL EMBODIMENTS

[0214] 1. A polynucleic acid comprising: a first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof; and a second polynucleotide encoding a chimeric antigen receptor (CAR).

[0215] 2. The polynucleic acid of embodiment 1, wherein the multispecific antibody comprises an anti-CD3 binding antibody or antibody fragment thereof and / or a cancer antigen binding antibody or antibody fragment thereof.

[0216] 3. The polynucleic acid of embodiment 2, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the cancer antigen binding antibody or antibody fragment thereof, the anti-CD2 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

[0217] 4. The polynucleic acid of embodiment 2, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the anti-CD2 binding antibody or antibody fragment thereof, the cancer antigen binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

[0218] 5. The polynucleic acid of any one of embodiment 1-4, wherein the multispecific antibody comprises a linker between one or more of the antibodies or antibody fragments.

[0219] 39

[0220] #14564947v1 6. The polynucleic acid of embodiment 5, wherein the linker is a glycine linker.

[0221] 7. The polynucleic acid of embodiment 6, wherein the glycine linker is a GGGGS (SEQ ID NO: 16) linker.

[0222] 8. The polynucleic acid of embodiment 7, wherein the GGGGS (SEQ ID NO: 16) linker is a (GGGGS)3(SEQ ID NO: 70) linker.

[0223] 9. The polynucleic acid of any one of embodiments 1-8, wherein the anti-CD2 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a variable light chain (VL) CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6.

[0224] 10. The polynucleic acid of embodiment 9, wherein the anti-CD2 antibody or antibody fragment thereof comprises a VH or SEQ ID ON: 7 and a VL or SEQ ID NO: 8.

[0225] 11. The polynucleic acid of embodiment 10, wherein the anti-CD2 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 9.

[0226] 12. The polynucleic acid of any one of embodiments 2-11, wherein the anti-CD3 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 10; a VH CDR2 of SEQ ID NO: 11; a VH CDR3 of SEQ ID NO: 12; a variable light chain (VL) CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 14; and a VL CDR3 of SEQ ID NO: 15.

[0227] 40

[0228] #14564947v1 13. The polynucleic acid of embodiment 12, wherein the anti-CD3 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 18.

[0229] 14. The polynucleic acid of any one of embodiments 2-13, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD20 or CD79b.

[0230] 15. The polynucleic acid of any one of embodiments 1-14, wherein the multispecific antibody is a bispecific antibody or trispecific antibody.

[0231] 16. The polynucleic acid of embodiment 14 or 15, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD20.

[0232] 17. The polynucleic acid of embodiment 16, wherein the cancer antigen binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 19; a VH CDR2 of SEQ ID NO: 20; a VH CDR3 of SEQ ID NO: 21;

[0233] 18. The polynucleic acid of embodiment 17, wherein the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 27.

[0234] 19. The polynucleic acid of embodiment 17 or embodiment 18, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD79b.

[0235] 20. The polynucleic acid of embodiment 19, wherein the cancer antigen binding antibody or antibody fragment thereof comprises a VH or SEQ ID ON: 34 and a VL or SEQ ID NO: 35.

[0236] 21. The polynucleic acid of embodiment 19, wherein the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 36.

[0237] 41

[0238] #14564947v1 22. The polynucleic acid of any one of embodiments 1-21, wherein the multispecific antibody comprises an IgK leader, optionally the IgK leader is on the N-terminal of the multispecific antibody.

[0239] 23. The polynucleic acid of embodiment 1, wherein the multispecific antibody comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 61-68.

[0240] 24. The polynucleic acid of embodiment 1, wherein the multispecific antibody comprises an amino acid of any one of SEQ ID NOs: 61-68.

[0241] 25. The polynucleic acid of any one of embodiments 1-24, wherein the CAR comprises: an antigen-binding domain comprising an anti-CD19 antibody or antibody fragment thereof; a transmembrane domain; and an intracellular signaling domain.

[0242] 26. The polynucleic acid of embodiment 25, wherein the anti-CD19 antibody comprises: a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44.

[0243] 27. The polynucleic acid of embodiment 25 or embodiment 26, wherein the transmembrane domain comprises a CD8 transmembrane domain.

[0244] 28. The polynucleic acid of any one of embodiments 25-27, wherein the intracellular signaling domain comprises a 4- IBB intracellular domain and / or a CD3zeta intracellular signaling domain.

[0245] 29. The polynucleic acid of any one of embodiments 1-28, wherein the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 48.

[0246] 30. The polynucleic acid of any one of embodiments 1-29, wherein the first polynucleotide and the second polynucleotide are in the same polynucleotide.

[0247] 31. The polynucleic acid of embodiment 30, wherein a linker is located between the first polynucleotide and / or the second polynucleotide.

[0248] 42

[0249] #14564947v1 32. The polynucleic acid of embodiment 31, wherein the linker is a polynucleotide encoding a cleavable peptide.

[0250] 33. The polynucleic acid of embodiment 32, wherein the cleavable peptide is 2A peptide.

[0251] 34. The polynucleic acid of embodiment 33, wherein the cleavable peptide is P2A, T2A, E2A or F2A.

[0252] 35. The polynucleic acid of any one of embodiments 30-34, wherein the multispecific protein and the CAR are expressed as a polyprotein.

[0253] 36. The polynucleic acid of embodiment 35, wherein the polyprotein comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 55-60.

[0254] 37. The polynucleic acid of embodiment 35, wherein the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 55-60.

[0255] 38. The polynucleic acid of embodiment 31, wherein the linker is an internal ribosome entry site (IRES).

[0256] 39. The polynucleic acid of any one of embodiments 1-38, wherein the first polynucleotide is operably linked to a first promoter.

[0257] 40. The polynucleic acid of embodiment 39, wherein the first promoter is an inducible promoter.

[0258] 41. The polynucleic acid of embodiment 40, wherein the inducible promoter is activated by polynucleic acid signaling.

[0259] 42. The polynucleic acid of embodiment 39 or embodiment 40, wherein the inducible promoter is an NFAT promoter.

[0260] 43

[0261] #14564947v1 43. The polynucleic acid of embodiment 39, wherein the first promoter is a constitutive promoter.

[0262] 44. The polynucleic acid of any one of embodiments 1-40, wherein the second polynucleotide is operably linked to a second promoter.

[0263] 45. The polynucleic acid of embodiment 44, wherein the second promoter is an inducible promoter.

[0264] 46. The polynucleic acid of embodiment 44, wherein the inducible promoter is activated by polynucleic acid signaling.

[0265] 47. The polynucleic acid of embodiment 45 or embodiment 46, wherein the inducible promoter is an NFAT promoter.

[0266] 48. The polynucleic acid of embodiment 47, wherein the second promoter is a constitutive promoter.

[0267] 49. The polynucleic acid of any one of embodiments 39-48, wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter.

[0268] 50. The polynucleic acid of any one of embodiment 1-38, wherein the first polynucleotide is operably linked to a first promoter that is an NFAT promoter and the second polynucleotide is operably linked to a second promoter that is a constitutively active promoter.

[0269] 51. The polynucleotide of any one of claims 39-49, wherein the first promoter and the second promoter are in a divergent orientation on the polynucleic acid.

[0270] EXAMPLES

[0271] Background: Loss or down-regulation of CD19 on B-cell lymphomas is a well described resistance mechanism to CD19-directed chimeric antigen receptor (CAR) T-cell therapies. Loss of CD58, ligand for CD2 on T-cells, has recently been identified as another mechanism of resistance to CD19-directed CAR-T cells. CD2 is a costimulatory receptor on T-cells, present in the immune synapse and important for T-cell activation and adhesion to

[0272] 44

[0273] #14564947v1 antigen-presenting cells. A novel CD 19 CAR design incorporating a secreted trispecific immune effector cell engager (TRIEC) consisting of three single chain variable fragments (scFv) targeting CD79b, CD2, and CD3 connected by glycine(4) serine linkers was tested as a way to address these challenges (FIGs. 1A-1D).

[0274] Methods: Tumor models of resistant malignant B-cell lines were made through modulation or knockout of CD19 expression, with or without concomitant knockout of CD58 in Jeko-1 (Mantle Cell Eymphoma), Nalm6 (Acute Lymphoblastic Leukemia), and Raji (Burkitt lymphoma) cell lines. Primary human T cells were transduced with a lentivirus encoding for second generation CD 19 CAR-T and TRIEC molecule, CD 19 CAR-T alone, or CD19 CAR-T with null TRIEC absent CD2 binding (CD2 scFv replaced with EGFR scFv).

[0275] Results: CD19-directed second generation CAR T cells demonstrated reduced cytotoxicity and lower levels of cytotoxic cytokines when exposed to CD191ow, CD191ow / CD58KO, and CD19K0 B-cell tumor lines compared to CD19 / CD58 unmodified parental B-cell lines in vitro (FIG. 2A-2C, and progressive decrement in tumor clearance of CD191ow, CD191ow / CD58KO, and CD19K0 B-cell tumors in vivo (FIGs. 2D-2E and 4A- 5D). TRIEC molecules showed specific binding of CD79b, CD2 (TRIEC only), EGFR (null TRIEC only) and CD3 antigen targets as assessed by anti-glycine(4) serine linker antibody. Untransduced T-cells co-cultured with Jeko-1 cells demonstrated increased cytolysis in the presence of TRIEC-containing supernatant vs null supernatant (p<0.0001) (3A-3D). CD19 CAR, CD 19 CAR + null TRIEC, and CD 19 CAR + TRIEC constructs demonstrated equivalent activation and proliferation in the absence of tumor. CD 19 CAR / TRIEC constructs demonstrated superior in vitro cytotoxicity against parental (p=.004), CD191ow / 58KO (p=.0011), and CD19K0 (p=.0002) Jeko-1 and Raji 191ow / 58KO (p<.0001) cell lines compared to CD 19 CAR and CD 19 CAR / null TRIEC constructs. Similar in vivo results were shown using an anti-CD19 CAR + a CD2, CD3, and CD20 TRIEC (FIGs. 5A-5D).

[0276] Conclusion: Harnessing CD2 signaling through the incorporation of a secreted trispecific CD79b / CD2 / CD3 TRIEC and CD20 / CD2 / CD3 TRIEC enhanced killing of CAR- resistant lymphoma models. CD 19 CARs secreting TRIEC s were effective against CD 19 low / negative and CD58KO B-cell lines. Incorporation of CD2 binding into the TRIEC molecule increased cytotoxicity without eliciting nonspecific activation or unstimulated proliferation of CAR-T cells in the absence of a tumor. Results show that incorporation of CD2 signaling into CAR-T cells circumvents tumor-intrinsic resistance mechanisms to current CD19-directed CAR-T therapies.

[0277] 45

[0278] #14564947v1 Example 2: Further In vivo Experiments using CD19 CAR-T and TRIEC expressing T cells The experiment shown in FIG. 4 was repeated (i.e., treating Jekol cell derived cancer in mice using CAR-T cells expressing a CD19 CAR and a TRIEC). The effects of administering a CAR-T cell comprising a CD19 CAR and CD79b-CD2-CD3 TRIEC were compared to a control CAR-T cell comprising a CD 19 CAR and a CD79b-EGFR-CD3 TRIEC (EGFR is not expressed by the Jekol cells). Unlike FIG. 4, The results showed no difference in treatment between the CAR-T cell comprising a CD19 CAR and CD79b-CD2- CD3 TRIEC and the control. This experiment was repeated a third time and again showed no difference between the T cell comprising CD19 CAR and CD79b-CD2-CD3 TRIEC and the control. Interestingly, in in vitro experiments, T cells comprising a CD 19 CAR and CD79b- CD2-CD3 TRIEC consistently have better cancer cell killing activity than the control T cells. In vitro experiments are much shorter in duration than in vivo experiments. Overall, these results suggest that prolonged exposure of a CAR-T cells to a TRIEC in vivo experiments results in prolonged activation of the CAR-T cell and in turn CAR T cell exhaustion, which reduces CAR-T cell cytotoxicity; whereas, the CAR-T cells do not become exhausted in the shorter in vitro experiments because exposure to the TRIEC is for a shorter period of time.

[0279] Example 3: Using inducible promoters to control TRIEC CAR-T cell expression

[0280] CAR and TRIEC expressing constructs were designed as shown in FIGs. 6A-6B. These constructs place the CAR (a CD 19 CAR) under the control of a constitutively active promoter (EFla) and the TRIEC (a CD3-CD2-CD20 TRIEC or a CD3-EGFR-CD20 TRIEC) under the control of an inducible promoter. The inducible promoter is an NFAT promoter that is induced upon CAR-T cell activation. Thus, expression of the TRIEC occurs when the CAR-T cells are activated (e.g., the CAR of the CAR T cell binds a tumor antigen). This is expected to reduce CAR-T cell exhaustion from TRIEC expression and thus increase CAR-T cell cancer killing efficacy.

[0281] Example 4: Exogenous Delivery of the TRIEC during CAR-T cell therapy

[0282] Instead of being expressing by the CAR-T cell, the TRIEC is administered separately during CAR-T cell therapy (e.g., see FIGs. 7A-7B). The TRIEC can be administered (e.g., with an osmotic pump) serially or currently with the CAR-T cell therapy. Separate administration of the TRIEC and the CAR-T cell is expected to decrease CAR-T cell exhaustion because the CAR-T cell is not constitutively expressing the TRIEC.

[0283] 46

[0284] #14564947v1

Claims

CLAIMS1. A T cell comprising : a first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof; and a second polynucleotide encoding a chimeric antigen receptor (CAR).

2. The T cell of claim 1, wherein the T cell secretes the multispecific antibody.

3. The T cell of claim 1 or claim 2, wherein the multispecific antibody comprises an anti-CD3 binding antibody or antibody fragment thereof and / or a cancer antigen binding antibody or antibody fragment thereof.

4. The T cell of claim 3, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the cancer antigen binding antibody or antibody fragment thereof, the anti- CD2 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

5. The T cell of claim 3, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the anti-CD2 binding antibody or antibody fragment thereof, the cancer antigen binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

6. The T cell of any one of claim 1-5, wherein the multispecific antibody comprises a linker between one or more of the antibodies or antibody fragments.

7. The T cell of claim 6, wherein the linker is a glycine linker.

8. The T cell of claim 7, wherein the glycine linker is a GGGGS (SEQ ID NO: 16) linker.

9. The T cell of claim 8, wherein the GGGGS (SEQ ID NO: 16) linker is a (GGGGS)3(SEQ ID NO: 70) linker.47#14564947v110. The T cell of any one of claims 1-9, wherein the anti-CD2 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a variable light chain (VL) CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6.

11. The T cell of claim 10, wherein the anti-CD2 antibody or antibody fragment thereof comprises a VH or SEQ ID ON: 7 and a VL or SEQ ID NO: 8.

12. The T cell of claim 11, wherein the anti-CD2 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 9.

13. The T cell of any one of claims 2-12, wherein the anti-CD3 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 10; a VH CDR2 of SEQ ID NO: 11; a VH CDR3 of SEQ ID NO: 12; a variable light chain (VL) CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 14; and a VL CDR3 of SEQ ID NO: 15.

14. The T cell of claim 13, wherein the anti-CD3 antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 18.

15. The T cell of any one of claims 2-14, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD20 or CD79b.

16. The T cell of any one of claims 1-15, wherein the multispecific antibody is a bispecific antibody or trispecific antibody.48#14564947v117. The T cell of claim 15 or 16, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD20.

18. The T cell of claim 17, wherein the cancer antigen binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 19; a VH CDR2 of SEQ ID NO: 20; a VH CDR3 of SEQ ID NO: 21;19. The T cell of claim 18, wherein the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 27.

20. The T cell of claim 15 or claim 16, wherein the cancer antigen binding antibody or antibody fragment thereof binds to CD79b.

21. The T cell of claim 20, wherein the cancer antigen binding antibody or antibody fragment thereof comprises a VH or SEQ ID ON: 34 and a VL or SEQ ID NO: 35.

22. The T cell of claim 20, wherein the cancer antigen binding antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 36.

23. The T cell of any one of claims 1-22, wherein the multispecific antibody comprises an IgK leader, optionally the IgK leader is on the N-terminal of the multispecific antibody.

24. The T cell of claim 1, wherein the multispecific antibody comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 61-68.

25. The T cell of claim 1, wherein the multispecific antibody comprises an amino acid of any one of SEQ ID NOs: 61-68.

26. The T cell of any one of claims 1-25, wherein the CAR comprises:49#14564947v1an antigen-binding domain comprising an anti-CD19 antibody or antibody fragment thereof; a transmembrane domain; and an intracellular signaling domain.

27. The T cell of claim 26, wherein the anti-CD19 antibody comprises: a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44.

28. The T cell of claim 26 or claim 27, wherein the transmembrane domain comprises a CD8 transmembrane domain.

29. The T cell of any one of claims 26-28, wherein the intracellular signaling domain comprises a 4- IBB intracellular domain and / or a CD3zeta intracellular signaling domain.

30. The T cell of any one of claims 1-29, wherein the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 48.

31. The T cell of any one of claims 1-30, wherein the first polynucleotide and the second polynucleotide are in the same polynucleotide.

32. The T cell of claim 31, wherein a linker is located between the first polynucleotide and / or the second polynucleotide.

33. The T cell of claim 32, wherein the linker is a polynucleotide encoding a cleavable peptide.

34. The T cell of claim 33, wherein the cleavable peptide is 2A peptide.

35. The T cell of claim 34, wherein the cleavable peptide is P2A, T2A, E2A or F2A.

36. The T cell of any one of claims 31-35, wherein the multispecific protein and the CAR are expressed as a polyprotein.50#14564947v137. The T cell of claim 36, wherein the polyprotein comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 55-60.

38. The T cell of claim 36, wherein the polyprotein comprises an amino acid sequence of any one of SEQ ID NOs: 55-60.

39. The T cell of claim 31, wherein the linker is an internal ribosome entry site (IRES).

40. The T cell of any one of claims 1-32, wherein the first polynucleotide is operably linked to a first promoter.

41. The T cell of claim 40, wherein the first promoter is an inducible promoter.

42. The T cell of claim 41, wherein the inducible promoter is activated by T cell signaling.

43. The T cell of claim 41 or claim 42, wherein the inducible promoter is an NFAT promoter.

44. The T cell of claim 40, wherein the first promoter is a constitutive promoter.

45. The T cell of any one of claims 1-44, wherein the second polynucleotide is operably linked to a second promoter.

46. The T cell of claim 45, wherein the second promoter is an inducible promoter.

47. The T cell of claim 46, wherein the inducible promoter is activated by T cell signaling.

48. The T cell of claim 46 or claim 47, wherein the inducible promoter is an NFAT promoter.

49. The T cell of claim 45, wherein the second promoter is a constitutive promoter.51#14564947v150. The T cell of any one of claims 40-49, wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter.

51. The T cell of any one of claim 1-32, wherein the first polynucleotide is operably linked to a NFAT promoter and the second polynucleotide is operably linked to a constitutively active promoter.

52. A multispecific antibody comprising: an anti-CD79b binding antibody or antibody fragment thereof, or an anti-CD20 binding antibody or antibody fragment thereof; and an anti-CD2 binding antibody or antibody fragment thereof.

53. The multispecific antibody of claim 52, comprising an anti-CD3 binding antibody or antibody fragment thereof.

54. The multispecific antibody of claim 53, wherein the anti-CD2 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 1; a VH CDR2 of SEQ ID NO: 2; a VH CDR3 of SEQ ID NO: 3; a variable light chain (VL) CDR1 of SEQ ID NO: 4; a VL CDR2 of SEQ ID NO: 5; and a VL CDR3 of SEQ ID NO: 6.

55. The multispecific antibody of claim 54, wherein the anti-CD3 antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 10; a VH CDR2 of SEQ ID NO: 11; a VH CDR3 of SEQ ID NO: 12; a variable light chain (VL) CDR1 of SEQ ID NO: 13; a VL CDR2 of SEQ ID NO: 14; and a VL CDR3 of SEQ ID NO: 15.52#14564947v156. The multispecific antibody of any one of claims 53-55, wherein the anti-CD20 binding antibody or antibody fragment thereof comprises: a variable heavy chain (VH) complementary determining region (CDR) 1 of SEQ ID NO: 19; a VH CDR2 of SEQ ID NO: 20; and a VH CDR3 of SEQ ID NO: 21;57. The multispecific antibody any one of claims 52-56, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the anti-CD79b binding antibody or antibody fragment thereof or the anti-CD20 binding antibody or antibody fragment thereof, the anti-CD2 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

58. The multispecific antibody any one of claims 52-56, wherein the multispecific antibody comprises, from N-terminal to C-Terminal, the anti-CD2 binding antibody or antibody fragment thereof, the anti-CD79b binding antibody or antibody fragment thereof or the anti-CD20 binding antibody or antibody fragment thereof, and the anti-CD3 binding antibody or antibody fragment thereof.

59. The multispecific antibody of any one of claims 52-58, comprising a leader sequence, optionally an IgK leader sequence.

60. The multispecific antibody of claim 59, wherein the IgK leader sequence in on the N- terminal of the multispecific antibody.

61. The multispecific antibody of claim 52, comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 61-68.

62. The multispecific antibody of claim 52, comprising an amino acid of any one of SEQ ID NOs: 61-68.

63. A polyprotein encoding the multispecific antibody of any one of claims 52-62, a cleavable linker, and a CAR.53#14564947v164. The polyprotein of claim 63, wherein the CAR comprises: an antigen-binding domain comprising an anti-CD19 antibody or antibody fragment thereof; a CD8 hinge / transmembrane domain; and a 41BB intracellular signaling domain and a CD3-zeta intracellular signaling domain.

65. The polyprotein of claim 64, wherein the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 43.

66. A polynucleotide encoding the multispecific antibody of any one of claims 52-62 or the polyprotein of any one of claims 63-65.

67. A cell comprising the polynucleotide of claim 66.

68. The cell of claim 67, wherein the cell is an immune cell.

69. The cell of claim 68, wherein the immune cell is a T cell.

70. A method of treating a B-cell malignancy in a subject, the method comprising administering the T cell of any one of claims 1-51 or the cell of claim 55 to the subject.

71. The method of claim 70, wherein the B-cell malignancy is lymphoma or leukemia.

72. The method of claim 71, wherein the lymphoma is Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphoma, Burkitt lymphoma, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma or Lymphoplasmacytic lymphoma.

73. The method of claim 71, wherein the cell is Hairy cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), Chronic lymphocytic leukemia (CLL), or Prolymphocytic leukemia (PLL).54#14564947v174. The method of any one of claims 70-73, wherein the B-cell malignancy expresses CD19.

75. The method of any one of claims 70-73, wherein the B-cell malignancy expresses CD20 and / or CD79b.

76. A method of delivering a multispecific antibody to a cell expressing a target antigen in a subject, the method comprising administering to the subject a T cell of any one of claims 1- 51, wherein the CAR of the T cell comprises an antigen-binding domain that binds to the target antigen of the cell of the subject.

77. A polynucleic acid comprising: a first polynucleotide encoding a multispecific antibody comprising an anti-CD2 binding antibody or antibody fragment thereof; and a second polynucleotide encoding a chimeric antigen receptor (CAR).

78. A polynucleic acid comprising, the first polynucleotide of any one of claims 1-51; and the second polynucleotide of any one of claims 1-51.

79. A method of treating a cancer in a subject, the method comprising administering to the subject: a CAR-T cell; and a trispecific immune effector cell engager (TRIEC).

80. The method of claim 79, wherein the CAR-T cell does not comprise a TRIEC or a polynucleotide encoding a TRIEC.

81. The method of claim 79 or claim 80, wherein the TRIEC comprises: a CD3 binding domain, a CD2 binding domain, and a CD20 binding domain; a CD3 binding domain, a CD2 binding domain, and a CD79b binding domain; and a CD3 binding domain, an EGFR binding domain, and a CD20 binding domain.55#14564947v182. The method of any one of claims 79-81, wherein the CAR-T cell comprises a CD19 binding CAR and / or a polynucleotide encoding a CD 19 binding CAR.56#14564947v1

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