Compositions and methods involving immune cells engineered for binding bispecific antibodies for use in cell therapy

Abstract

Provided herein are antibody binding constructs and immune cells engineered to comprise the same, along with related nucleic acid molecules, polypeptides, vectors, compositions, and methods which allow enhanced effectiveness of an immunotherapy in a subject by specifically engaging the engineered immune cells in a subject.

Description

[0001] Attorney Docket No.: 1115814 1040WO (00018)

[0002] COMPOSITIONS AND METHODS INVOLVING IMMUNE CELLS ENGINEERED FOR BINDING BISPECIFIC ANTIBODIES FOR USE IN CELL THERAPY

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 689,423, filed August 30, 2024, the contents of which is incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING THE SEQUENCE LISTING

[0006] The instant application contains an electronic Sequence Listing which has been submitted in xml (ST.26) format via USPTO Patent Center, herein incorporated by reference in its entirety. Said xml copy named “1115814 1040WO.xml” is 73,746 bytes in size, and was created on August 28, 2025.

[0007] FIELD OF THE DISCLOSURE

[0008] The present disclosure generally relates to tumor cell targeting in particular through engineering immune cells, such as monocytes and macrophages, to enhance antibody therapies directed against tumor cells. More specifically, the present disclosure relates to immune cells genetically modified to express an antibody -binding construct and related compositions, vectors, and methods useful for enhancing the effectiveness of an immunotherapy.

[0009] BACKGROUND OF THE DISCLOSURE

[0010] Cancer immunotherapy has demonstrated exciting clinical results in the setting of numerous solid tumors and hematologic malignancies. The endogenous immune system is typically non-reactive to malignant cells or can be actively immunosuppressive with respect to the body's reaction to the presence of malignant cells. One way to enhance treatment of tumors is to force tumor recognition by the immune system through genetic engineering of leukocytes (e.g., CAR-T cells). Such cells have been successfully developed for adoptive tumor immunotherapy, a method of adoptively reintroducing lymphocytes cultured in vitro into cancer patients to treat tumors. However, the efficacy of CAR-T cell therapy can be reduced by inherent heterogeneous and fluidity of expression of tumor-associated antigens.

[0011] An alternative therapeutic approach, antibody-based therapy, is frequently used for treating cancers and other diseases. Responses to antibody therapy have typically focused on the direct

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[0013] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) inhibitory effects of these antibodies on the tumor cells (e.g., inhibition of growth factor receptors and the subsequent induction of apoptosis), but the in vivo effects of these antibodies may be more complex and may involve the host immune system. For instance, it has been observed that tumors, especially solid tumors, have a complex microenvironment. Tumors include not only tumor cells and T cells, but also macrophages, fibroblasts, and other types of cells. The complex, and immunosuppressive, tumor microenvironment can limit contact between immune cells and tumor cells, hamper the cytotoxic activity of immune cells, and diminish the effectiveness of tumor killing associated with antibody-based therapies. Tumor antigen heterogeneity further limits the durability of responses and compromises the effectiveness of antibody-based therapies. Despite the initial success that has been seen with antibody therapies, including bispecifics, many significant challenges remain.

[0014] Thus, a need exists in the art for more effective treatments for cancers that improve specificity for and activity against tumor cells. The compositions and methods disclosed herein provide novel anti-tumor therapies and novel methods for enhancing the activity of immune cells, such as antigen-presenting cells and phagocytes, including monocytes and macrophages, to specifically target tumor cells and / or enhance the immune response that is triggered by antibody therapy.

[0015] SUMMARY OF THE DISCLOSURE

[0016] Provided herein are antibody binding constructs comprising: an extracellular domain; and a transmembrane domain. In some embodiments of the above antibody binding constructs, said construct further comprises an at least first intracellular signaling domain. In some embodiments of the above antibody binding constructs, said construct further comprises an at least first intracellular co-stimulatory signaling domain.

[0017] Provided herein are antibody-binding constructs, comprising: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto; and b) at least a first transmembrane domain, wherein said antibody-binding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of: i) a CD3d protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto; or ii) a CD3g protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto. In some embodiments of the

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[0019] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) antibody -binding constructs, said second ectodomain has an amino acid sequence of a CD3d protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto. In some embodiments of the antibody-binding constructs, said second ectodomain has an amino acid sequence of a CD3g protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto.

[0020] In some embodiments of the above antibody-binding constructs, said first ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2.

[0021] In some embodiments of the above antibody -binding constructs, said at least first extracellular domain further comprises an amino acid sequence encoding said second ectodomain.

[0022] In some embodiments of the above antibody-binding constructs, said antibody binding construct further comprises a second extracellular domain. In some embodiments, said second extracellular domain comprises an amino acid sequence encoding said second ectodomain.

[0023] In some embodiments of the above antibody-binding constructs, said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4.

[0024] In some embodiments of the above antibody-binding constructs, said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

[0025] In some embodiments of the above antibody -binding constructs, said CD3e ectodomain or fragment or variant thereof and said second ectodomain form a heterodimer.

[0026] In some embodiments of the above antibody -binding constructs, said CD3e ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, and said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 4.

[0027] In some embodiments of the above antibody -binding constructs, said CD3e ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2 and said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 6.

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[0029] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0030] In some embodiments of the above antibody-binding constructs, said antibody binding construct comprises a first polypeptide comprising said at least first extracellular domain and said at least first transmembrane domain. In some embodiments, said antibody -binding construct further comprises a second polypeptide comprising said second extracellular domain.

[0031] In some embodiments of the above antibody-binding constructs, said antibody-binding construct further comprises at least a first intracellular signaling domain. In some embodiments, said at least first intracellular signaling domain comprises a signaling domain of a CD3z, HVEM, 4-1BB, CD28, NKG2D, DAP10, PI3K, CD40, MyD88, TRIF, STING, RIG- 1, MDA5, Dectin-1, or a FcsRly protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto.

[0032] In some embodiments of the above antibody-binding constructs, said antibody-binding construct further comprises at least a first intracellular co-stimulatory signaling domain. In some embodiments, said at least first co-stimulatory intracellular signaling domain comprises an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as SEQ ID NO: 8 or 9, or a functional fragment or variant thereof that retains co-stimulatory activity.

[0033] In some embodiments of the above antibody -binding constructs, said at least first transmembrane domain is derived from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto.

[0034] In some embodiments of the above antibody-binding constructs, said antibody-binding construct comprises: i) an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 22, 23, or 24; ii) a first amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 22 and a second amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 23; iii) a first amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NOs: 22 and a second amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 24; iv) the amino acid sequence set forth as any one of SEQ ID NOs: 22, 23, or 24; v) a first amino acid sequence set forth as SEQ ID NO: 22 and a second amino acid sequence set forth as SEQ ID NO: 23; or vi) a first amino

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[0036] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) acid sequence set forth as SEQ ID NO: 22 and a second amino acid sequence set forth as SEQ ID NO: 24.

[0037] Also provided herein are nucleic acid molecules comprising a polynucleotide comprising a nucleotide sequence encoding any of the above antibody binding constructs, any of the above first polypeptides, and / or any of the above second polypeptides.

[0038] In some embodiments of the above nucleic acid molecules, said nucleic acid molecule comprises RNA, such as mRNA.

[0039] Also provided herein are vectors comprising at least a first nucleic acid molecule, wherein said first nucleic acid molecule comprises any of the above nucleic acid molecules.

[0040] In some embodiments of the vectors, said vector further comprises an effector molecule expression cassette comprising a polynucleotide sequence encoding said effector molecule. In some embodiments, said effector molecule comprises an antibody, or binding fragment(s) thereof. In some embodiments, said antibody comprises a multi-specific or multi-valent antibody, such as a bispecific, a trispecific, or tetraspecific antibody.

[0041] In some embodiments of the above vectors, said antibody binding construct and said effector molecule are encoded by a single nucleic acid molecule.

[0042] In some embodiments of the above vectors, said vector further comprises at least a second nucleic acid molecule. In some embodiments, said at least second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence encoding said second polypeptide or said effector molecule.

[0043] In some embodiments of the above vectors, said vector comprises a first nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said first polypeptide and a second nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said second polypeptide.

[0044] In some embodiments of the above vectors, said vectors comprise a second nucleic acid molecule, and further comprise at least a third nucleic acid molecule.

[0045] In some embodiments of the above vectors, said vector comprises a first nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said first polypeptide and a second nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said second polypeptide.

[0046] In some embodiments of the above vectors, said vector comprises: i) a single-stranded mRNA or a single-stranded or double-stranded DNA; ii) a plasmid, a cosmid, or a transposon;

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[0048] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) iii) a viral vector; iv) a nano-particle, such as a virus-like nanoparticle or a lipid nanoparticle; and / or v) a liposome.

[0049] Also provided herein are engineered immune cells comprising any of the above antibody-binding constructs, any of the above nucleic acid molecules, and / or any of the above vectors.

[0050] In some embodiments of the above engineered immune cells, said ectodomain of a CD3e protein or fragment or variant thereof, and / or said second ectodomain is absent on said immune cell in nature, or is present on the surface of said immune cell in lower amounts in the absence of said antibody-binding construct, such as the cell in nature, as compared to said engineered immune cell.

[0051] Also provided herein are engineered immune cells expressing an antibody-binding construct on the surface of the cell, wherein said antibody-binding construct comprises: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein comprising an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) at least a first transmembrane domain, wherein said antibody-binding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of: i) a CD3d protein comprising an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or ii) a CD3g protein comprising an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6, and wherein said immune cell is a monocyte, macrophage, dendritic cell, neutrophil, or NK cell, or precursor thereof.

[0052] In some embodiments of the above engineered immune cells, said antibody-binding construct is bound or associated with a multi-specific antibody capable of binding to said CD3e on the surface of the cell. In some embodiments, said multi-specific antibody is further bound or associated with an antigen on the surface of a tumor cell. In some embodiments, the engineered immune cell further comprises a nucleic acid sequence encoding said multi-specific antibody.

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[0055] Also provided herein are cell populations comprising a plurality of any of the above engineered immune cells.

[0056] Also provided herein are pharmaceutical compositions comprising any of the above antibody-binding constructs, any of the above nucleic acid molecules, any of the above vectors, any of the above engineered immune cells, any of the above cell populations, and a pharmaceutically acceptable carrier.

[0057] Also provided herein are methods of stimulating a biological response in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of any of the above pharmaceutical compositions.

[0058] In some embodiments of the above methods of stimulating a biological response said stimulation of a biological response: i) increases in vivo cytokine production in a tumor microenvironment in said subject; ii) reduces antigen escape of a tumor cell in said subject; iii) increases epitope spreading in said subject; iv) increases pathogenic cell killing, such as tumor cell killing, in said subject; v) stimulates a therapeutic vaccination response in said subject; vi) associates a monocyte or macrophage cell with a tumor cell in said subject; and / or vii) reduces a symptom associated with cancer, inflammation, an inflammatory disease, autoimmunity, an autoimmune disease, a neurodegenerative disease, a vascular disease, an ocular disease, a hematologic disorder and / or an infection.

[0059] In some of the embodiments of the above methods of stimulating a biological response, said method comprises administering at least a second therapy. In some embodiments, said at least second therapy comprises an antibody therapy, a vaccine, a T-cell based therapy, and / or bispecific T cell engager (BiTE®) therapy.

[0060] Also provided herein are methods of expressing an antibody-binding construct on the surface of an immune cell, said method comprising introducing into said cell at least any of the above nucleic acid molecules or vectors, thereby producing an engineered immune cell expressing said antibody -binding construct on the surface of the cell.

[0061] In some embodiments of the above methods, said method includes: i) expression of at least one gene in the engineered immune cell is increased compared to a control cell; ii) an immune response is stimulated in the engineered immune cell compared to a control cell; and / or ii) phagocytosis and / or trogocytosis activity of the engineered immune cell is enhanced compared to a control cell, wherein said control cell is said immune cell lacking said at least first nucleic acid molecule. In some embodiments of the above methods, said gene is a pro-

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[0063] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) inflammatory cytokine gene or anti-inflammatory cytokine gene of said engineered immune cell.

[0064] In some embodiments of the above methods, secretion of an effector molecule from said engineered immune cell is increased compared to a control cell. In some embodiments of the above methods, said effector molecule comprises a small molecule, an enzyme, a cytokine, a cytotoxin, a granzyme, a checkpoint inhibitor, an antibody, a polynucleotide, a peptide, a polypeptide, an antibiotic, an anti-viral, an anti-fungal, an anti-inflammatory, or a derivative thereof, or an agent comprising any combination thereof. In some embodiments of the above methods, said effector molecule promotes increased expression of at least one gene of an at least second immune cell in said subject in vivo. In some embodiments of the above methods, said at least second immune cell is a T cell, DC cell, NK cell, neutrophil, monocyte, or macrophage.

[0065] Also provided herein are methods of making an engineered immune cell comprising an antibody-binding construct expressed on the surface of said cell, said method comprising contacting a target immune cell with any of the above nucleic acid molecules and / or any of the above vectors.

[0066] In some embodiments of the above methods of making an engineered immune cell comprising an antibody-binding construct expressed on the surface of said cell, said target immune cell is an isolated immune cell, or precursor thereof.

[0067] In some embodiments of the methods of making an engineered immune cell comprising an antibody -binding construct expressed on the surface of said cell, said contact occurs in vivo.

[0068] In some embodiments of the methods of making an engineered immune cell comprising an antibody-binding construct expressed on the surface of said cell, said target immune cell is a T cell, a monocyte, a macrophage, a dendritic cell, a neutrophil, a mast cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, an eosinophil, a basophil, a myeloid cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a hematopoietic stem cell (HSC), a HSC-derived cell, a precursor cell, a bone marrow cell, a stem cell, a pluripotent stem cell, a mesenchymal stromal cell (MS SC), and induced pluripotent stem cell (iPSC), or an iPSC-derived cell.

[0069] Also provided herein are methods of making any of the above engineered immune cells, said method comprising contacting said immune cell with a vector comprising at least a first nucleic acid molecule encoding said antibody -binding construct.

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[0071] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIGS. 1A-1B show schematics depicting the orientation of domains of eight exemplary antibody binding constructs comprising an extracellular domain, hinge domain, transmembrane domain, and intracellular signaling domain. FIG. 1A depicts four constructs comprising a CD3e ectodomain with a native CD3e signal peptide (“1” and “7”) or a GMCSF signal peptide (“2” and “8”). FIG. IB depicts four constructs comprising either a CD3d ectodomain with a native CD3d signal peptide (“3”); a CD3g ectodomain with native CD3g signal peptide (“4”); a CD3d ectodomain with a heterologous GMCSF signal peptide (“5”); or a CD3g ectodomain with a heterologous GMCSF signal peptide (“6”).

[0074] FIGS. 2A-2E show that the ectodomain of a CD3e subunit protein and the ectodomain of an at least a second CD3 subunit protein (i.e., CD3d or CD3g) is required for CD3e ectodomain surface expression on cells and antibody binding. Cells having a CD3 e-ectodomain containing antibody binding construct only introduced via transduction (FIG. 2A) or a CD3 -ectodomain containing antibody binding expression construct introduced via transduction, and further comprising a second CD3 subunit protein ectodomain (e.g., CD3d or CD3g) containing antibody binding expression construct introduced via electroporation (FIGS. 2B-2E) were analyzed for fluorescence (“GFP+”) and CD3-antibody binding ("CD3+”) via flow cytometry. “GFP+CD3+” percentage indicates the proportion of the analyzed cells that expressed GFP and a CD3 epsilon domain on the surface in the respective box of each panel. FIG. 2A depicts cells comprising a CD3 e-ectodomain expression construct (i.e., construct 1 of FIG. 1A), which is not detected on the surface. FIG. 2B depicts cells comprising the construct of FIG. 2A and construct 3 of FIG. IB. FIG. 2C depicts cells comprising the construct of FIG. 2A and construct 4 of FIG. IB. FIG. 2D depicts cells comprising the construct of FIG. 2A and construct 5 of FIG. IB. FIG. 2E depicts cells comprising the construct of FIG. 2A and construct 6 of FIG. IB.

[0075] FIG. 3 shows that presence of a CD3 e-ectodomain containing expression construct in cells lacking CD3 (i.e., WT THP-1 cells) was essential for detection of CD3e ectodomain on the surface of the cells electroporated with CD3d-ectodomain expression construct (i.e., construct 3 of FIG. IB). A population of THP-1 cells was analyzed for fluorescence (“GFP+”) and CD3-antibody binding (“CD3+”) via flow cytometry using a UCHT1 antibody. “GFP+CD3+” percentage in the box indicates the proportion of the analyzed cell population that expressed GFP and CD3e on the surface.

[0076] FIGS. 4A-4E show CD3e surface expression on THP-1 cells transduced (FIGS. 4A-4D) or electroporated (FIG. 4E) with CD3 constructs detected using multiple antibodies with binding

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[0078] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) regions with homology to current bispecific antibody therapies. Groups include: CD3 positive control Jurkat cell line (“T cell line”); THP-1 cells transduced with a CD3e construct only (i.e., construct 1 of FIG. 1A) (“CD3e only”); THP-1 cells transduced with constructs containing both CD3e and CD3g ectodomains (i.e., construct 1 of FIG. 1A and construct 4 of FIG. IB) (“CD3e / CD3g”); THP-1 cell electroporation control (“THP-1 control”); and THP-1 cells coelectroporated with mRNA expression constructs containing CD3e and CD3d ectodomains (i.e., construct 7 of FIG. 1A and construct 3 of FIG. IB) (“lx CD3e / CD3d mRNA” and “2x CD3 / CD3d mRNA”). Antibody binding was analyzed by flow cytometry and is depicted as expression intensity (x-axis) and percentage of population normalized for the indicated cell population (y-axis). FIG. 4A is a histogram showing UCHT1 anti-CD3 clone binding to the indicated cell populations. FIG. 4B is a histogram showing 0KT3 clone binding to the indicated cell populations. FIG. 4C is a histogram showing SK7 clone binding to the indicated cell populations. FIG. 4D is a histogram showing HIT3 A clone binding to the indicated cell populations. FIG. 4E is a histogram showing 0KT3 clone binding to the indicated cell populations.

[0079] FIG. 5 shows that CD3e ectodomain surface expression and subsequent antibody binding intensity is dependent on the signal peptide used. THP-1 cells were transduced with: an expression construct comprising a CD3e ectodomain (i.e., construct 1 of FIG. 1A) (“CD3e only”); a CD3e ectodomain-containing expression construct and a CD3g ectodomain-containing expression construct, each containing their cognate signal peptide, respectively (i.e., construct 1 of FIG. 1A and construct 4 of FIG. IB) (“CD3e / g native SP”); or a CD3e ectodomain-containing expression construct and a CD3g ectodomain-containing expression construct comprising a GM-CSF signal peptide (i.e., construct 1 of FIG. 1A and construct 6 of FIG. IB) (“CD3e / g heterologous SP”). Antibody binding was analyzed by flow cytometry and is depicted as expression intensity (x-axis) and percentage of population normalized for the indicated cell population (y-axis).

[0080] FIG. 6 shows cellular activation of nuclear factor NFkB activity in THPl-Dual™ Cells transduced with antibody-binding constructs comprising a CD3e ectodomain (i.e., construct 1 of FIG. 1A). THPl-Dual™ Cells were transduced with: construct 1 (“CD3e only”); construct 1 and a construct comprising a CD3g ectodomain comprising a native CD3g signal peptide (SP) (i.e., construct 4 of FIG. IB) (“CD3e / g native SP”); or construct 1 and a construct comprising a CD3g ectodomain comprising a GM-CSF signal peptide (“CD3e / g heterologous SP”) (i.e., construct 6 of FIG. IB) NfkB reporter activity in each cell line was assessed following activation with various concentrations of plate-bound anti-CD3 antibody, as indicated, compared to an LPS control.

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[0082] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0083] FIG. 7 shows enhanced tumor cell killing and reduced tumor cell expansion by THP1 cells comprising antibody binding constructs as determined using a phagocytosis assay. THP1 cells were transduced with: an expression construct encoding an antibody binding construct comprising a CD3e ectodomain (i.e., construct 1 of FIG. 1A) (“CD3e only”); or construct 1 and an expression construct encoding an antibody binding construct comprising a CD3g ectodomain comprising a GM-CSF signal peptide (i.e. construct 6 of FIG. IB) (“CD3e / CD3g”), and incubated with Raji target cells at a ratio of 3 : 1 (3 THP1 cells to 1 Raji) in the presence of anti-BCMA / anti-CD3 bispecific antibody. Target cell fold expansion was analyzed over the indicated time period and compared to tumor cells incubated with only bispecific antibody control.

[0084] FIG. 8 is a schematic depicting exemplary antibody binding constructs of the present disclosure. “TM domain”, transmembrane domain; “Costim domain”, intracellular co-stimulatory domain; “signaling domain”, intracellular signaling domain; “d”, CD3d ectodomain; “g”, CD3g ectodomain; “e”, CD3e ectodomain.

[0085] DETAILED DESCRIPTION

[0086] The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

[0087] I. Definitions

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0089] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.

[0090] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0091] The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range. When used in reference to numerical ranges, cutoffs, or specific values means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined,

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[0093] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of an assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0094] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0095] Various embodiments of this disclosure may be presented in a range format. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.

[0096] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number “and” a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. The recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ^0 and ^2 if the variable is inherently continuous.

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[0098] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0099] As used herein, the term “gene” or “coding sequence”, herein used interchangeably, refers to a functional nucleic acid unit encoding a protein, polypeptide, or peptide. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

[0100] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g, chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The term "fragment" will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and / or consisting of a nucleotide sequence of contiguous nucleotides identical to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially or and / or consist of, oligonucleotides having a length of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0101] The terms “bispecific T cell engager (BiTE®) molecule” as used herein refers to any molecule within a class of artificial bispecific monoclonal antibodies created by linking the targeting regions of two antibodies having one arm that is engineered to bind with a protein found on the surface of cytotoxic T cells, and the other arm is designed to bind to a specific protein found primarily on tumor cells (Baeuerle PA, Kufer P, Bargou R. Curr Opin Mol Ther. 2009; 11 :22-30). As would be understood in the art, a BiTE® is a bispecific antibody having one arm of the antibody that engages with a T cell. When both targets of the BiTE® are engaged, the BiTE® molecule forms a bridge between the targets (i.e. cytotoxic T cell and the tumor cell), which enables the T cell to recognize the tumor cell and fight it through an infusion of toxic molecules. A person having skill in the art would understand that the tumor-binding arm of the molecule can be altered to create different BiTE® molecules that target different types of cancer.

[0102] The term “BiTE®-like molecule” refers to a molecule that is a derivative, and / or a variant, of a BiTE® molecule, and / or any molecule that functions to redirect immune effector cells against cancer cells with the aim of triggering an efficient tumor cell killing. A BiTE®-like molecule can have a similar bispecific antibody structure as a BiTE® molecule (i.e., bispecific monoclonal

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[0104] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) antibody created by linking the targeting regions of two antibodies), but having one arm engineered to bind an immune cell other than a T cell. As such, a BiTE®-like molecule is envisaged as engaging a broader range of immune cell types than a traditional BiTE® molecule, which engages T cells. A person having skill in the art would understand that the T cell-binding arm of any BiTE® molecule can be altered to create a BiTE®-like molecule capable of binding to any target immune cell. A BiTE®-like molecule may have one arm (i.e., “tumor binding arm”) that is designed to bind to a specific protein found primarily on tumor cells (i.e., “tumor cell protein), and a second arm (i.e., “immune cell arm”) that is engineered to bind with a protein found on the surface of any immune cell (i.e., “immune cell protein”), such as , a monocyte, a macrophage, a dendritic cell, a neutrophil, a mast cell, a natural killer (NK) cell, a B cell, a tumorinfiltrating lymphocyte (TIL), an innate lymphoid cell, an eosinophil, a basophil, a myeloid cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a hematopoietic stem cell (HSC), a HSC-derived cell, a precursor cell, a bone marrow cell, a stem cell, a pluripotent stem cell, a mesenchymal stromal cell (MS SC), and induced pluripotent stem cell (iPSC), or an iPSC-derived cell. In some embodiments, the BiTE®-like molecule has one arm that binds to a target on a tumor cell, and one arm that binds to the antibody -binding construct disclosed herein. In some embodiments, the BiTE®-like molecule can bind to any of the engineered immune cells disclosed herein. A person having skill in the art would understand that the tumor-binding arm of the molecule can be altered to create BiTE®-like molecules that target different types of cancer. It would be understood that one aspect of a BiTE®-like molecule is to engage and redirect an immune cell to target a tumor cell. As such, a BiTE®-like molecule as described herein refers to any molecule that can engage and / or redirect an immune cell to target a cancer or tumor cell (e.g., a “T cell engager”, or “immune cell engager”). Any immune cell is envisaged as being engaged and / or redirected by the BiTE®-like molecules as described herein. Molecules that function to redirect immune cells (regardless of antigen binding specificity) can share a similar structure. For instance, most immune cell engagers (ICEs) are trans-binding bispecific antibodies (bsAbs) usually consisting of two linked single-chain fragment variables (scFvs) that originate from different monoclonal antibodies: one scFv recognizes a surface TAA, whereas the other is specific for a certain membrane molecule expressed on effector immune cells (see, Fenis, Aurore et al. “New immune cell engagers for cancer immunotherapy.” Nature reviews. Immunology vol. 24,7 (2024): 471-486. doi: 10.1038 / s41577-023-00982-7; Fuca, G et al. “Immune cell engagers in solid tumors: promises and challenges of the next generation immunotherapy.” ESMO open vol. 6,1 (2021): 100046; and Choi, Bryan D et al. “Bispecific antibodies engage T

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[0106] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) cells for antitumor immunotherapy.” Expert opinion on biological therapy vol. 11,7 (2011): 843- 53, the contents of which are incorporated herein in their entirety). A BiTE®-like molecule or ICE may have more than binding moieties that bind to a specific protein found primarily on tumor cells and / or and more than two binding moieties engineered to bind with a protein found on the surface of an immune cell. For instance, when a BiTE®-like molecule or ICE can comprise two, or more than two, “tumor-binding arms”, wherein a first tumor-binding arm comprises a binding moiety that binds to a first tumor cell protein, and wherein an at least second tumor binding arm comprises an at least second binding moiety that binds to an at least second tumor cell protein. In such instances, said first and second tumor cell protein(s) can be the same or different. In some instances, said first and second tumor cell protein(s) are different, thereby enabling the BiTE®- like molecule or ICE to engage two cancer cell markers and / or two cancer cell types. Additionally, a BiTE®-like molecule or ICE can comprise two, or more than two, “immune cell arms”, wherein a first immune cell arm comprises a binding moiety that binds to a first immune cell protein, and wherein an at least second immune cell arm comprises an at least second binding moiety that binds to an at least second immune cell protein. In such instances, said first and second immune cell protein(s) can be the same or different. In some instances, said first and second immune cell protein(s) are different, thereby enabling the BiTE®-like molecule or ICE to engage and / or redirect two different immune cell types (e.g., two different cell types expressing different cell specific markers) against one, two, or more than two cancer cell types. There is no limit the to the number of tumor binding arms, tumor cell proteins, immune cell arms, and / or immune cell proteins envisaged within the scope of the BiTE®-like molecules or ICEs as disclosed herein, so long as said BiTE®-like molecule or ICE retained the capacity to redirect and / or engage at least one immune cell against at least one cancer cell.

[0107] The term “effector molecule” as used herein in relation to an engineered immune cell refers to any molecule that is secreted by an engineered immune cell disclosed herein that contributes to any of the methods disclosed herein. For example, an effector molecule can contribute to increasing in vivo cytokine production in a tumor microenvironment; reducing antigen escape of a tumor cell; increasing epitope spreading; increasing tumor cell killing; and / or stimulating a therapeutic vaccination response. In some instances, the effector molecule acts on a cell that is different from the cell in which it is secreted. A skilled artisan would understand that molecules released by cells can impact gene expression in surrounding cells, and that this cellular “cross-talk” can modulate multiple cell types from an effector molecule released by a single cell type. For example, the effector molecule is released by an engineered monocyte or macrophage

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[0109] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) and increases or decreases gene expression in a T cell. In such instances, the effector molecule can be secreted by a monocyte or macrophage can enhance tumor killing by a T cell. The effector molecule can be any molecule that is expressed, produced, and / or secreted by an effector immune cell. In particular, binding of an antibody to the antibody binding construct on the surface of the engineered immune cell will promote increased secretion of an effector molecule. An effector molecule can be any molecule that can be produced by an engineered immune cell, such as a polynucleotide, or polypeptide, or a molecule comprising a polynucleotide or polypeptide, or a derivative of a polynucleotide or polypeptide. The effector molecule will be encoded by a gene comprising a polynucleotide presented within the engineered immune cell. The gene encoding the effector molecule can be located in the genome of the engineered immune cell. Alternatively, the gene encoding the effector molecule can be present on any nucleic acid molecule present within the engineered immune cell, such as a plasmid or a linear DNA fragment. In some embodiments, the effector molecule is naturally encoded by the immune cell (i.e., the gene is located in the genome of the immune cell type of the engineered immune cell in nature, without human intervention). In other embodiments, the effector molecule gene is comprised within a vector disclosed herein.

[0110] A “monocyte” refers to a precursor to macrophage or dendritic cells. Monocytes circulate in the bloodstream and differentiate into macrophages or dendritic cells when they infiltrate tissues. An “isolated nucleic acid” refers to a nucleic acid segment or fragment that has been separated from sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a is genome that it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, that naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0111] “Macrophage” refers to a type of innate immune cell that phagocytose cellular material and may act as an antigen presenting cell to activate other immune cells. Macrophages further stimulate and regulate inflammation by releasing cytokines.

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[0114] “Dendritic Cell” refers to a professional antigen presenting cell that can most efficiently activate other immune cells against new epitopes. Dendritic cells further stimulate and regulate inflammation by releasing cytokines. “Natural Killer cell”, “NK cell”, or “NK-LGL cell”, used interchangeably herein, refers to a type of immune cell that has granules (small particles) with enzymes that can kill tumor cells or cells infected with a virus. A natural killer cell is a type of white blood cell.

[0115] “Neutrophil” refers to a type of white blood cell that is an important part of the immune system and helps the body fight infection. When microorganisms, such as bacteria or viruses, enter the body, neutrophils are one of the first immune cells to respond. They travel to the site of infection, where they destroy the microorganisms by ingesting them and releasing enzymes that kill them. Neutrophils also boost the response of other immune cells. A neutrophil is a type of granulocyte and a type of phagocyte.

[0116] The term “ectodomain” as used herein refers to the domain of a membrane protein that extends into the extracellular space (i.e., the space outside a cell). The ectodomain would be understood by a person having skill in the art as the part of the protein (e.g., antibody binding construct) that initiates contact on the surface of the cell, subsequently leading to signal transduction. Methods for determining the ectodomain of a membrane protein would be known by those having skill in the art. Activity of ectodomain variant polypeptides can be measured by the ability of the polypeptide to bind to an intended target molecule of a native polypeptide.

[0117] The term “extracellular domain” refers to a region of the antibody binding construct that extends outside of a cell from the cell surface. The extracellular domain is extracellular (i.e., outside of the cell) and is associated with the membrane of the cell (i.e., membrane-bound) through a linkage to a transmembrane domain. Through this linkage with a transmembrane domain the extracellular domain can relay signals between the cell's external and internal environments. The extracellular domain can interact with signaling molecules, antibodies, or any molecule that binds to the extracellular domain to trigger intracellular responses. An extracellular domain can provide an activation signaling event upon binding of a target molecule, such as an antibody. In a preferred embodiment, the extracellular domain comprises a polypeptide comprising an amino acid sequence. In another preferred embodiments, the extracellular domain comprises an amino acid sequence encoding an ectodomain of a surface marker or surface protein of an immune cell, or a fragment thereof. The antibody binding constructs disclosed herein comprise at least a first extracellular domain. The antibody binding constructs disclosed herein can comprise more than one extracellular domain. In such instances, the antibody binding construct

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[0119] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) can comprise two extracellular domains. Also disclosed herein are variant extracellular domain polypeptides. Activity of extracellular domain native and variant polypeptides can, for example, be measured by the ability of the polypeptide to bind to a target molecule, such as an antibody.

[0120] The term “intracellular signaling domain” as used herein refers to all or a portion of one or more domains of a molecule (here the antibody binding construct) that provides for activation of a cell. An intracellular signaling domain provides for activation of an engineered immune cell disclosed herein. Intracellular domains of the antibody binding constructs disclosed herein mediate a signal by interacting with cellular mediators to result in proliferation, differentiation, activation and other effector functions. In some embodiments, the intracellular signaling domain can comprise all or portions of a CD3z, HVEM, 4-1BB, CD28, NKG2D, DAP10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, or fragments, or variants thereof, or combinations thereof. Activity of intracellular signaling domain variant polypeptides can, for example, be measured by the ability of the polypeptide to mediate a signal by interacting with cellular mediators to result in proliferation, differentiation, activation and / or other effector functions upon binding of the extracellular domain to a target molecule.

[0121] “Intracellular co-stimulatory domain” and “co-stimulatory domain”, terms used interchangeably herein, refer to a signaling moiety that provides to a cell a signal which, in addition to the primary signal provided by for instance the intracellular signaling domain mediates a cellular response, including, but not limited to, activation, proliferation, differentiation, and / or cytokine secretion. A co-stimulatory domain can include all or a portion of, but is not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. In embodiments, the co-stimulatory domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and / or cytokine secretion in the engineered immune cells disclosed herein. Activity of co-stimulatory domain variant polypeptides can, for example, be measured by the ability of the polypeptide to mediate a cellular response in the cell, such as, activation, proliferation, differentiation, and / or cytokine secretion upon binding of the extracellular domain to a target molecule.

[0122] The term “transmembrane domain” refers to a domain of a polypeptide that spans a membrane. Transmembrane domains are regions of a polypeptide that are often hydrophobic, so that they prefer to be inserted into the cell membrane such that the parts of the polypeptide on either side of the domain are on opposite sides of the membrane. The transmembrane domain can

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[0124] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) play multiple roles including, but not limited to, anchoring a polypeptide to the membrane, signal transduction, protein trafficking, facility molecular transport. Often, in a membrane protein, a transmembrane domain links (i.e., functionally connects) an extracellular domain to an intracellular domain. Activity of transmembrane domain variant polypeptides can be measured, for example, by the ability of the polypeptide to anchor the antibody binding construct to the membrane and / or facilitate signal transduction in the polypeptide upon binding of the extracellular domain to a target, such as an antibody.

[0125] The term “signal peptide domain” or “signal peptide sequence”, used interchangeably herein, refers to a short peptide typically present at the amino-terminus of a polypeptide that function to prompt a cell to translocate the polypeptide to the cellular membrane. A signal peptide domain typically mediates translocation of the polypeptide to, into, and / or across the cell membrane. Activity of signal peptide domain variant polypeptides can, for example, be measured by the ability of the polypeptide to mediate translocation of a polypeptide to, into, and / or across a cell membrane.

[0126] As used herein, a “mutation” is any change in a nucleic acid sequence. Nonlimiting examples comprise insertions, deletions, duplications, substitutions, inversions, and translocations of any nucleic acid sequence, regardless of how the mutation is brought about and regardless of how or whether the mutation alters the functions or interactions of the nucleic acid. For example, and without limitation, a mutation may produce altered enzymatic activity of a ribozyme, altered base pairing between nucleic acids (e.g., RNA interference interactions, DNA-RNA binding, etc.), altered mRNA folding stability, and / or how a nucleic acid interacts with polypeptides (e.g. DNA- transcription factor interactions, RNA-ribosome interactions, gRNA-endonuclease reactions, etc.). A mutation might result in the production of proteins with altered amino acid sequences (e.g. missense mutations, nonsense mutations, frameshift mutations, etc.) and / or the production of proteins with the same amino acid sequence (e.g. silent mutations). Certain synonymous mutations may create no observed change in a cell while others that encode for an identical protein sequence nevertheless result in an altered cell phenotype (e.g. due to codon usage bias, altered secondary protein structures, etc.). Mutations may occur within coding regions (e.g., open reading frames) or outside of coding regions (e.g., within promoters, terminators, untranslated elements, or enhancers), and may affect, for example and without limitation, gene expression levels, gene expression profiles, protein sequences, and / or sequences encoding RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.

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[0128] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0129] As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. Similarly, the terms “engineered” or “recombinant” in reference to a phagocyte, cell, gene, nucleic acid and / or protein as used herein, refer to a phagocyte, cell, gene, nucleic acid and / or protein that has been altered through human intervention. Accordingly, the term “naturally occurring” as used herein in reference to a phagocyte, cell, gene, nucleic acid and / or protein as used herein, refer to a phagocyte, cell, gene, nucleic acid and / or protein existing in nature and without any human intervention. Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and / or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a phagocyte, cell, gene, nucleic acid and / or protein herein described.

[0130] As used herein, the term “expression” or “expressing” refers to the transcription and / or translation of a particular nucleic acid sequence driven by a promoter. An expression construct or expression vector can permit transcription of a particular nucleic acid sequence in a cell (e.g., a phagocytic cell). An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. An expression cassette typically comprises at least three components: a promoter sequence, an open reading frame encoding gene(s) of interest, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. An expression cassette can be formed by manipulable fragment of DNA carrying and capable of expressing, one or more genes of interest optionally located between one or more sets of restriction sites. Expression cassettes typically comprise further regulatory sequences additional to the promoter to regulate the expression of the gene or genes within the open reading frame herein also indicated as a coding region of the expression cassette. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter of the present invention and a heterologous nucleotide is a functional link that allows for expression of the heterologous nucleic acid molecule. Operably linked elements may be contiguous or non-contiguous. When

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[0132] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. Two protein coding regions can be operably linked through a spacer. Any spacer disclosed herein can link two protein coding regions of the antibody binding construct. For instance, wherein the antibody binding construct comprises a first extracellular domain and a second extracellular domain, said first extracellular domain and said second extracellular domain can be linked by at least one spacer. At least one spacer can link any of the domains of the antibody binding constructs disclosed herein. In some embodiments, one spacer links at least two domains of the antibody binding constructs disclosed herein. The cassette may additionally contain at least one additional gene to be co-transformed into a cell. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or DNA constructs. The expression cassette may additionally contain selectable marker genes. Other elements that may be present in an expression cassette include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as those that confer certain binding affinity or antigenicity to the recombinant protein produced from the expression cassette.

[0133] The term “polypeptide” as used herein indicates an organic linear, circular, or branched polymer composed of two or more amino acid monomers and / or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full-length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer, peptide, or oligopeptide. In particular, the terms “peptide” and “oligopeptide” usually indicate a polypeptide with less than 100 amino acid monomers. In particular, in a protein, the polypeptide provides the primary structure of the protein, wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds, and additional bonds identifiable by a skilled person. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of alpha-amino acid residues covalently linked by peptide bond or a synthetic covalent linkage. The two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C -terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N- terminal end (NFF-group), which is the end where the amino group is not involved in a peptide bond to the C-terminal end (-COOH group) which is the end where a COOH group is not involved

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[0135] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) in a peptide bond. Proteins and polypeptides can be identified by x-ray crystallography, direct sequencing, immunoprecipitation, and a variety of other methods as understood by a person skilled in the art. Proteins can be provided in vitro or in vivo by several methods identifiable by a skilled person. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and / or analogs thereof are joined through chemically-mediated condensation of an organic acid (-COOH) and an amine (-NH2) to form an amide bond or a “peptide” bond.

[0136] "Amino acid" as used herein refers to a compound having a free carboxyl group and a free unsubstituted amino group on the a carbon, which may be joined by peptide bonds to form a peptide active agent as described herein. Amino acids may be standard or non-standard, natural or synthetic, with examples (and their abbreviations) including but not limited to: Asp=D= Aspartic Acid; Ala=A=Alanine; Arg=R=Arginine; Asn=N=Asparagine; Cys=C=Cysteine;

[0137] Gly=G=Glycine; Glu=E=Glutamic Acid; Gln=Q=Glutamine; His=H=Histidine; Ile=I=Isoleucine; Leu=L=Leucine; Lys=K=Lysine; Met=M=Methionine; Phe=F=Phenylalanine; Pro=P=Proline; Ser=S=Serine Thr=T=Threonine; Trp=W=Tryptophan; Tyr=Y=Tyrosine; Val=V= Valine; Om=Ornithine; Nal=2-napthylalanine; Nva=Norvaline; Nle=Norleucine; Thi=2 -thienylalanine; Pcp=4-chlorophenylalanine; Bth=3-benzothienyalanine; Bip=4,4'-biphenylalanine;

[0138] Tic=tetrahydroisoquinoline-3-carboxylic acid; Aib=aminoisobutyric acid; Anb=a- aminonormalbutyric acid; Dip=2, 2-diphenylalanine; Thz=4-Thiazolylalanine. A "basic amino acid" refers to any amino acid that is positively charged at a pH of 6.0, including but not limited to R, K, and H. An "aromatic amino acid" refers to any amino acid that has an aromatic group in the side-chain coupled to the alpha carbon, including but not limited to F, Y, W, and H.

[0139] The term “fragment” is intended to refer to a portion of a polynucleotide or a portion of an amino acid sequence. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and / or 3' end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Generally, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.

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[0142] "Variant" amino acid or protein is intended to mean an amino acid or protein derived from the native amino acid or protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and / or C-terminal end of the native protein; deletion and / or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. "Variants" is intended to mean substantially similar sequences. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein. Biologically active variants of a native polypeptide will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native sequence as determined by sequence alignment programs and parameters described herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. Biologically active variants of a native polypeptide will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity of the native sequence.

[0143] The terms “surface protein” or “surface marker” are used interchangeably herein and refer to a molecule that is expressed or displayed on the surface of immune cells. Cell surface proteins traverse or are anchored / embedded in the plasma membrane, playing a crucial role in various essential and distinct cellular functions along the plasma membrane, which mediates intercellular communication and regulating cellular interactions with the extracellular milieu, including nutrient and ion transportation, intercellular interactions, receptor-mediated signaling transduction, enzymatic reactions, and immune recognition. In some instances, a surface protein can be a transmembrane protein, extending through the lipid bilayer, with part of their mass on either side. Cell surface proteins include extracellular domains that are relatively independent from the membrane; a feature that allows for the isolation of soluble extracellular fragments that can be useful for structural studies. Due to their critical biological function and unique subcellular location, CSPs have been proposed as valuable resource for the identification of targets in immune and targeted therapy. Additionally, they have been served as informative biomarkers for assays related to early disease detection, diagnosis, and prediction. Cell surface markers are proteins or carbohydrates attached to the plasma membrane of the cell and expressed uniquely by each cell type. As such, cell surface markers are thought to conveniently serve as markers of specific cell types. For example, T cell and B cell surface markers are used to identify their lineage and stage in the differentiation process. One common cell surface markers are CD antigen, also known as CD

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[0145] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) molecules and clusters of differentiation. In terms of physiology, CD molecules can act in numerous ways, often acting as receptors or ligands important to the cell. A signal cascade is usually initiated, altering the behavior of the cell. Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. CD for humans is numbered up to 371. In addition to being useful in defining leukocytes, CD markers have been associated with important cellular functions. For example, CD4 & CD8 are critical in antigen recognition.

[0146] As used herein, “function” of a gene, a peptide, a protein, or a molecule refers to activity of a gene, a peptide, a protein, or a molecule.

[0147] “Introducing,” “introduce,” and “introduced” (and grammatical variations thereof) in the context of a polynucleotide and / or polypeptide of interest means presenting a nucleotide sequence of interest (e.g., polynucleotide, a nucleic acid construct, and / or a guide nucleic acid) and / or polypeptide of interest to a host organism or cell of said organism (e.g., a mammalian cell) in such a manner that the nucleotide sequence and / or polypeptide gains access to the interior of a cell. In some embodiments, “introducing” includes inserting a nucleic acid molecule (e.g., a recombinant DNA construct) into a cell, by means of transformation, transfection, or transduction. The nucleic acid molecule may be incorporated into the genome of the cell (e.g., nuclear chromosome or mitochondrial chromosome), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0148] As used therein, a “subject” that may be treated by methods of the present disclosure include both human subjects for medical and / or therapeutic purposes and animal subjects for veterinary and drug screening and development purposes. Other suitable animal subjects are, in general, mammalian subjects such as primates, bovines, ovines, caprines, porcines, equines, felines, canines, lagomorphs, rodents ( e.g ., rats and mice), etc. Human subjects are the most preferred. Human subjects include fetal, neonatal, infant, juvenile, adult and geriatric subjects.

[0149] The term "anti-tumor effect" as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the proliferation rate, a decrease in the number of metastases, an increase in life expectancy, and / or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to delay the occurrence of tumor in the first place.

[0150] As used herein, the term "autologous" is meant to refer to any material derived from the same individual to whom it is later to be re-introduced.

[0151] "Allogeneic" refers to a graft derived from a different animal of the same species.

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[0153] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0154] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0155] The term "antibody" as used herein refers to any molecule comprising a binding domain that binds to the extracellular domain of the antibody-binding construct disclosed herein. An antibody can refer to full-length immunoglobulins as well as to fragments thereof. An antibody can also refer to a receptor, or fragment or variant thereof, that retains a binding domain. Full- length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies. An antibody can include immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. An antibody can also be comprised in part by aptamers or tagbinding moieties such as biotin / streptavidin / avidin. The antibody can be generated or derived using any method available in the art. For example, recombinant techniques for generating antibodies are well known in the art. In some embodiments, the antibody can be generated using genetic engineering techniques. In some embodiments, the antibody can be derived from click chemistry. Antibody structure and / or production method are not envisaged as limiting the antibodies disclosed herein. Accordingly, any antibody capable of binding to the antibody binding constructs disclosed herein can be used. In some instances, the antibody is capable of binding to a CD3e subunit ectodomain. In some instances, the CD3e subunit is in a heterodimer (e.g., in a heterodimer with a CD3d or CD3g heterodimer). Residues of a CD3e ectodomain that are targeted by antibodies, such as multi-specific or bispecific antibodies, are known in the art. The CD3e ectodomain of the antibody-binding construct comprises residues that are necessary for binding and / or associating with an antibody. That is, the CD3e ectodomain of the antibody-binding construct comprises residues that are necessary for binding and / or associating with a binding domain that targets CD3e. In some instances, an antibody, or binding domain thereof, is capable of binding to and / or associating with an amino acid sequence present within the CD3e ectodomain. Without wishing to be bound by theory, it has been suggested that amino acid residues in the N- terminus of the CD3e ectodomain are important for binding to many known multi-specific antibodies. A specific domain that has been suggested as involved in antibody binding within the CD3e ectodomain comprises the first six amino acid residues set forth in the CD3e ectodomain sequence set forth as SEQ ID NO: 2. In such instances, the antibody, or binding domain thereof, is capable of binding to and / or associating with a CD3e ectodomain comprising an amino acid

[0156] 25

[0157] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) sequence comprising the first six amino acid residues set forth in the CD3e ectodomain sequence set forth as SEQ ID NO: 2. Thus, in some instances, the CD3e ectodomain of the antibody binding construct comprises an amino acid sequence having the first six amino acid residues set forth in the CD3e ectodomain sequence set forth as SEQ ID NO: 2. In a particular instance, the CD3e ectodomain of the antibody binding construct comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said CD3e ectodomain the first six amino acid residues set forth in the CD3e ectodomain sequence set forth as SEQ ID NO: 2. In another particular instance, the CD3e ectodomain of the antibody binding construct comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said CD3e ectodomain comprises an amino acid sequence having the: i) D at residue 1; ii) G at residue 2; iii) N at residue 3; iv) E at residue 4; v) E at residue 5; and / or vi) M at residue 6, of the amino acid sequence set forth as SEQ ID NO: 2. In another particular instance, the CD3e ectodomain of the antibody binding construct comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said CD3e ectodomain comprises an amino acid sequence having the residue: i) E34; ii) G46; iii) E48; iv) R79; v) G80; vi) S81; vii) K82; viii) P83; and / or ix) D85, of the amino acid sequence set forth in SEQ ID NO: 2. In another particular instance, the CD3e ectodomain of the antibody binding construct comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said CD3e ectodomain comprises an amino acid sequence having the residue: i) E34; ii) N43; iii) 144; iv) G46; v) D47; vi) E48; vii) S55; viii) Y77; ix) R79; x) G80; xi) S81; xii) K82; xiii) P83; xiv) E84; and / or xv) D85, of the amino acid sequence set forth in SEQ ID NO: 2. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody

[0158] 26

[0159] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (1), based on the amino acid sequences of their constant domains.

[0160] A “multi-specific antibody” refers to an antibody that specifically binds to at least two distinct antigens or at least two distinct epitopes within the same antigen. In some embodiments, the multi-specific antibody binds to two antigens. In some embodiments, the multi-specific antibody binds to more than two antigens, such as three (tri-specific) or four (tetra-specific) antigens). The multi-specific antibodies disclosed herein are capable of binding to at least a first antigen comprising a surface marker of an immune cell and an at least second antigen comprising an antigen on the surface of a cancer cell.

[0161] “Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody can have crossreactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or can bind an epitope that is shared between two or more distinct antigens.

[0162] “Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibody can be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent. “Mutation” refers to an engineered or naturally occurring alteration in a polypeptide or polynucleotide sequence when compared to a reference sequence. The alteration can be a substitution, insertion or deletion of one or more amino acids or polynucleotides.

[0163] “Multi-specific” or “multispecific”, used interchangeably herein, refers to an antibody that specifically binds at least two distinct antigens or at least two distinct epitopes within the same antigen. Multispecific antibody can bind for example two, three, four or five distinct antigens or distinct epitopes within the same antigen.

[0164] “CD3” refers to a human antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) complex and which consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon (a) (“CD3a” or “CD3e”,

[0165] 27

[0166] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) used interchangeably herein), CD3 delta (5) (“CD35” or “CD3d”, used interchangeably herein), CD3 zeta (Q (“CD3 ” or “CD3z”, used interchangeably herein) and CD3 gamma (y) (“CD3y” or “CD3g”, used interchangeably herein).

[0167] Human CD3 epsilon comprises the amino acid sequence of:

[0168] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGS EILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLR ARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQR GQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 21).

[0169] The extracellular domain (ectodomain) of CD3 epsilon is:

[0170] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 2).

[0171] The term "antibody fragments" portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. Holliger, P. and Hudson, J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p. 1126-1136). Examples of antibody fragments include, without being limited to, include scFv, Fab, Fv, Fab', F(ab')2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units. “Single chain variable fragments” or “single chain antibodies” or “scFv” are one type of antibody fragment. scFv are fusion proteins comprising the variable heavy (VH) and variable light (VL) of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full- length immunoglobulins, but retain the specificity of the original immunoglobulin.

[0172] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab1, F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

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[0174] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0175] Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321 :522-525; Reichmann et al. (1988) Nature 332:323-329; Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0176] "Fully human" refers to an immunoglobulin, such as an antibody, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.

[0177] The numbering system to identify amino acid residue positions in the VH and VL of an antibody can follow a system known to one of skill in the art, including Kabat (Wu and Kabat (1970) J Exp Med. 132(2):211-50; Borden and Kabat (1987) PNAS, 84:2440-2443; Kabat et al. U.S. Department of Health and Human Services, 1991), Chothia (Chothia and Lesk (1987) J Mol. Biol., 196(4): 901-917; Chothia et al. (1989) Nature 342:877-883), and the "AHo" system described by Honegger & Pluckthun (2001) Journal of Molecular Biology 309:657-670.

[0178] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CHI domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241 : 544-546), which consists of a VH domain; and (vi) an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to

[0179] 29

[0180] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883). Such single chain antibodies are also intended to be encompassed within the term "antigen -binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448).

[0181] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s). A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain embodiments, an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0182] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0183] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, K and 1 light chains refer to the two major antibody light chain isotypes.

[0184] The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

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[0186] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0187] By the term "specifically binds," as used herein with respect to an antibody or antigen binding domain, is meant an antibody or antigen binding domain which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody or antigen binding domain that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody or antigen binding domain as specific. In another example, an antibody or antigen binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody or antigen binding domain as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, antigen binding domain, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody or antigen binding domain recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0188] An "immune response" refers to the reaction of a subject to the presence of an antigen, which may include at least one of the following: making antibodies, developing immunity, developing hypersensitivity to the antigen, and developing tolerance. The term “enhance an immune response” as used herein implies that the reaction of a subject to the presence of an antigen is increased and / or amplified in the presence of a CAR-modified phagocytic cell of the disclosure as compared to the reaction of a subject to the presence of an antigen in the absence of a CAR-modified phagocytic cell of the disclosure.

[0189] The terms "treat", "treating", or "treatment of' indicates that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.

[0190] The term "therapeutic" as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0191] An "effective" amount as used herein is an amount that provides a desired effect.

[0192] A "therapeutically effective" amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a "therapeutically effective" amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical

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[0194] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0195] “Cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor. Examples of various cancers include but are not limited to, brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and uterine cancer.

[0196] “Tumor cell” or a “cancer cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid or it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo. The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated. Antibody constant chain numbering can be found for example at ImMunoGeneTics website, at IMGT Web resources at IMGT Scientific charts.

[0197] “Relapsed” refers to a cancer that responded to treatment but then returns.

[0198] “Immune cell redirecting therapeutic” refers to a molecule containing two or more binding regions, wherein one of the binding regions specifically binds a cell surface antigen on a target cell or tissue and wherein a second binding region of the molecule specifically binds an antigen on the surface of an immune cell. The immune cell can be a naturally-occurring immune cell, such as a an immune cell isolated from a subject. The immune call can be an engineered immune cell, which comprises, for example, a heterologous, or recombinant, nucleotide sequence. In some instances, the immune cell can be a monocyte, a macrophage, a T cell, a dendritic cell, a myeloid cell, or a

[0199] 32

[0200] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) precursor any of an immune cell. Examples of cell surface antigen include a tumor associated antigen. Examples of an immune cell antigen include, e.g., CD3, or any receptor chain, or functional fragment thereof. This dual / multi -target binding ability recruits immune cells, such as engineered monocytes or T cells, to the target cell or tissue leading to the eradication of the target cell or tissue.

[0201] By the term "modulating," as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0202] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0203] By "target" is meant a cell, organ, or site within the body that is in need of treatment.

[0204] The term "detectable moiety" as used herein includes any suitable detectable group, such as radiolabels (e.g.35S,125I,131I, etc.), enzyme labels (e.g, horseradish peroxidase, alkaline phosphatase, etc.), fluorescence labels (e.g., fluorescein, green fluorescent protein, etc.), etc., as are well known in the art and used in accordance with known techniques.

[0205] The term "agent," or "biological agent" or "therapeutic agent" as used herein, refers to a molecule that may be expressed, released, secreted or delivered to a target by the modified cell described herein. The agent includes, but is not limited to, a nucleic acid, an antibiotic, an antiinflammatory agent, an antibody or antibody fragments thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate or the like, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combination thereof. The agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. The agent may diffuse or be transported into the cell, where it may act intracellularly.

[0206] The term "expand" as used herein refers to increasing in number, as in an increase in the number of phagocytic cells. In some embodiments, the phagocytic cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In some embodiments, the phagocytic cells that are expanded ex vivo increase in number relative to other cell types in the

[0207] 33

[0208] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) culture. The term "ex vivo" refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0209] By the term "modified" as used herein, is meant a changed state or structure of a molecule or cell of the disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0210] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

[0211] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “increased”, “increase”, and the like encompass both a partial reduction and a significant increase compared to a control.

[0212] The wording “fold change” as used herein indicates a measure describing how much a quantity changes between an original and a subsequent measurement. In particular, fold change is defined as the ratio between two quantities. For example, for quantities A and B, the fold change of B with respect to A is B / A. For example, a change from 30 to 60 is defined as a fold-change of 2.

[0213] “Enhance” or “enhanced” refers to enhancement in one or more functions of a test molecule when compared to a control molecule or a combination of test molecules when compared to one or more control molecules. Exemplary functions that can be measured are tumor cell killing, T cell activation, relative or absolute T cell number, Fc- mediated effector function (e.g. ADCC, CDC and / or ADCP) or binding to an Fey receptor (FcyR) or FcRn. “Enhanced” can be an enhancement of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant enhancement.

[0214] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as

[0215] 34

[0216] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0217] The term “isolated” refers to at least partially separated from the natural environment e.g., from a cell. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0218] “Isolated antibody” refers to an antibody that is substantially free of other cellular material and / or chemicals and encompasses antibodies that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0219] As used herein, the terms “exogenous” or “heterologous” in reference to a nucleic acid sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. Thus, a heterologous nucleic acid sequence may not be naturally expressed within a cell or may have altered expression when compared to the corresponding wild type cell. For example, a heterologous polynucleotide encoding a CAR described herein can be a nucleic acid sequence that is not naturally present in a phagocytic cell in which it is present. An exogenous polynucleotide may be introduced into the cell in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and / or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the cell.

[0220] As used herein, by “endogenous” in reference to a gene or nucleic acid sequence or protein is intended a gene or nucleic acid sequence or protein that is naturally comprised within or expressed by a cell. Endogenous genes can include genes that naturally occur in a cell (e.g., phagocytic cell), but that have been modified in the genome of the cell without insertion or

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[0222] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) replacement of a heterologous gene that is from another species or another location within the genome of the modified cell.

[0223] “Homolog” or “homologous sequence” may refer to both orthologous and paralogous sequences. Paralogous sequence relates to gene-duplications within the genome of a species. Orthologous sequence relates to homologous genes in different organisms due to ancestral relationship. Thus, orthologs are evolutionary counterparts derived from a single ancestral gene in the last common ancestor of given two species and therefore have great likelihood of having the same function.

[0224] In some embodiments, the term “homolog” as used herein, refers to functional homologs of genes. A functional homolog is a gene encoding a polypeptide that has sequence similarity to a polypeptide encoded by a reference gene, and the polypeptide encoded by the homolog carries out one or more of the biochemical or physiological function(s) of the polypeptide encoded by the reference gene. In general, it is preferred that functional homologs and / or polypeptides encoded by functional homologs share at least some degree of sequence identity with the reference gene or polypeptide encoded by the reference gene.

[0225] Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment. "Homologous" can refer to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. According to some embodiments, the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence. According to some embodiments, the homology is a global homology, e.g., a homology over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof. The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools which are described in WO2014 / 102774. As applied to the nucleic acid or protein, "homologous" as used herein refers

[0226] 36

[0227] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) to a sequence that has about 50% sequence identity. More preferably, the homologous sequence has about 75% sequence identity, even more preferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0228] As used herein, “sequence identity,” “identity,” “percent identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266: 131-141; Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(l-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-l l; gap extension penalty=-l; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=l 1; gap opening penalty=-5; gap extension penalty=-2; match reward=l; and mismatch penalty=-3. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according

[0229] 37

[0230] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) to the algorithm of Henikoff S and Henikoff J G. (Proc Natl Acad Sci 89: 10915-9 (1992)). Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0231] As used herein, the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0232] “Combination” means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

[0233] “Pharmaceutical composition” refers to composition that comprises an active ingredient and a pharmaceutically acceptable carrier.

[0234] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject.

[0235] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0236] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0237] II. Overview of the Disclosure

[0238] The compositions and methods disclosed herein provide antibody binding constructs useful for generating engineered immune cells to bind antibodies. Any immune cell can be engineered with the antibody binding constructs disclosed herein. The disclosed antibody binding constructs are particularly useful for engineering immune cells to bind antibodies in order to enhance an immune response in a subject that has been administered an antibody immunotherapy. The antibody binding constructs disclosed herein comprise an extracellular domain and a transmembrane domain. When introduced into an immune cell, the extracellular domain, or a fragment thereof, will be displayed on the surface of the immune cell, while the transmembrane domain will be integrated and / or anchored into the cell membrane, generating a stable construct. The extracellular domain of the antibody binding construct can comprise an amino acid sequence

[0239] 38

[0240] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) encoding an ectodomain of any surface protein or surface marker. As such, the immune cells disclosed herein can be engineered to display the ectodomain of any surface protein and / or surface marker on the cell surface. This aspect of the antibody binding construct is particularly useful for generating engineered immune cells that are capable of binding to antibodies. For instance, an immune cell, such as a neutrophil, monocyte, and / or macrophage, can be engineered to express a CD3 subunit protein ectodomain on the cell surface, whereby said CD3 ectodomain can bind to an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is a bispecific antibody that binds to an at least second antigen, such as an antigen on a tumor cell. In addition to engineered immune cells to bind antibodies, the antibody binding constructs also promote cellular activation of engineered immune cells following antibody binding. In particular, the antibody binding constructs can comprise intracellular signaling domains that contribute to activation of at least one gene in the engineered immune cell following binding of the antibody binding construct to an antibody. For instance, following binding to an antibody, the engineered immune cell can activate at least one gene in the engineered immune cell. In some embodiments, the at least one gene can be a pro-inflammatory gene. In alternative embodiments, the at least one gene can be an antiinflammatory gene. As such, one aspect of the present disclosure is the provision of engineered immune cells that can improve the effectiveness and / or efficiency of an immunotherapy, in particular, an immunotherapy comprising at least one multi-specific antibody, or the immune response initiated by an immunotherapy. The immunotherapy can be an antibody therapy (e.g, a bispecific antibody therapy) that targets specific cells in the body by concomitantly binding and / or engaging effector immune cells, such as T cells. In some instances, the immunotherapy when administered alone may engage an immune cell type (e.g., a T cell) that is different from the engineered immune cells disclosed herein. As such, an additional aspect of the present disclosure is to provide immune cells that are engineered to express heterologous surface proteins that enable the engineered cells to engage with an immunotherapy molecule (e.g., a bispecific antibody) and contribute to an immune response and / or killing of target cells. In some embodiments, the engineered immune cells are intended to complement an immunotherapy. Antibody therapies can include administration of multi-specific antibodies, which can be designed so that one arm of the antibody binds to a target antigen of a target cell, and one arm binds to a target antigen on an immune cell. As such, the bispecific antibody serves as a “bridging” molecule that brings an immune cell into close proximity with the target cell contributing to target cell killing.

[0241] Due to structural limitations (e.g., the number of possible targets) of bispecific antibodies, the number of immune cell surface proteins targeted by any given bispecific antibody is limited.

[0242] 39

[0243] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0244] The present application solves the problem of providing an enhanced immune response associated with an antibody therapy by engineering a specific immune cell type to express a heterologous surface protein that enables the engineered cell type to interact with the antibody of the immunotherapy (e.g., the engineered immune cell is bound by the antibody). Importantly, the engineered cell is a cell type that would not be engaged by the antibody therapy in the absence of the heterologous protein that is expressed on the engineered immune cell surface. By engineering immune cells to express surface proteins that are targeted by an antibody in an antibody therapy, the number and / or type of immune cells that contribute to an immune response can be increased.

[0245] The engineered immune cells will enhance the immune response of an immunotherapy comprising an antibody therapy. In some instances, the antibody therapy comprises an antibody that binds to CD3. Bispecific antibodies that bind to CD3 to engage T cells are well known in the art. As such, a standard bispecific antibody that binds CD3 to engage a T cell with tumor cells could be leveraged to also bind and engage an engineered immune cell(s) that in wild-type form in nature does not express CD3, and is engineered to express CD3 on the surface with the antibodybinding construct disclosed herein. In some embodiments, the immune cell, prior to being engineered by introduction of the antibody-binding constructs disclosed herein, expresses low levels of CD3 on the surface (e.g., lower levels compared to a T cell) in wild-type form found in nature (e.g., the human body). In such instances, the immune cell would not be expected to be bound by sufficient anti-CD3 bispecific antibody immunotherapy. The term “sufficient” as used herein when referring to a wild-type immune cell being bound by anti-CD3 bispecific antibody refers to the ability of the anti-CD3 antibody to engage the wild-type immune cells in the immune response. A skilled artisan would understand that a certain amount of anti-CD3 antibody may bind to an immune cell lacking CD3 due to non-specific binding or interaction between the antibody and immune cell surface. Such non-specific binding to a cell surface would be expected to occur at levels lower than binding observed on an immune cell expressing CD3, and thus not be expected to engage the immune cell or enhance the immune response of an immunotherapy compared to the engineered immune cells disclosed herein. Provided herein are immune cells that are engineered to express CD3 on the surface as part of an antibody binding construct.

[0246] The extracellular domain of the antibody binding constructs disclosed herein can be engineered to comprise an amino acid encoding any surface marker protein, or functional fragment or variant thereof. For example, the extracellular domain can comprise an amino acid sequence of an ectodomain of any surface marker protein, or functional fragment or variant thereof. A functional fragment or variant as used herein to describe a surface protein ectodomain refers to

[0247] 40

[0248] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) any portion of the ectodomain that retains the ability to bind to an antibody that binds to said ectodomain. In general, a functional fragment or variant as used herein can describe any portion or domain of an antibody binding construct, or polypeptide, which retains the functional capacity exhibited by a full length sequence, such that the antibody -binding construct functions as intended. For instance, a functional fragment or variant of any domain of an antibody-binding construct retains the ability to promote the function of the antibody -binding construct to bind an antibody and transmit an intracellular signal in the engineered immune cell. A functional fragment or variant of an intracellular domain retains the ability to transmit an intracellular signal internally within the cell. The antibody binding construct can comprise an extracellular domain comprising a CD3 protein ectodomain. For instance, human monocytes, dendritic cells, macrophages, neutrophils, mast cells, granulocytes, and / or NK cells can be engineered with the antibody -binding constructs disclosed herein to express CD3 (e.g., a CD3 protein ectodomain) on the surface of the cell. In such instances, the bispecific antibody that binds CD3 would bind and engage T cells (which naturally express CD3 on the cell surface) and the CD3+ engineered immune cell disclosed herein (e.g., monocytes, macrophages, dendritic cells, neutrophils, mast cells, granulocytes, and / or NK cells, or precursors thereof). The term “CD3+ engineered immune cell” refers to any immune cell disclosed herein comprising an antibody binding construct that displays a CD3 protein ectodomain on the cell surface. In certain embodiments, the engineered immune cell is a granulocyte or precursor thereof. In particular embodiments, the engineered immune cell is an antigen-presenting cell (APC) or precursor thereof. In some instances, the engineered immune cell is a T cell. In some instances, the cell is a monocyte, a macrophage, a dendritic cell, a neutrophil, a mast cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, an eosinophil, a basophil, a myeloid cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a hematopoietic stem cell (HSC), a HSC-derived cell, a precursor cell, a bone marrow cell, a stem cell, a pluripotent stem cell, a mesenchymal stromal cell (MSSC), and induced pluripotent stem cell (iPSC), or an iPSC-derived cell.

[0249] The antibody binding constructs disclosed herein comprise an extracellular domain and a transmembrane domain. In some embodiments, the antibody binding constructs disclosed herein further comprise an intracellular signaling domain. In some embodiments, the antibody binding constructs further comprise an intracellular co- stimulatory signaling domain. Without wishing to be bound by theory, it is possible that the physical binding of an antibody to an extracellular domain on the cell surface can be enough to instigate a phagocytic response. Accordingly, in some embodiments, wherein said engineered immune cell is a phagocytic cell, it may be desirable or

[0250] 41

[0251] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) beneficial to engineer said cell with an antibody binding construct comprising an extracellular domain and a transmembrane domain. In some instances, the antibody binding construct does not comprise an intracellular signaling domain or an intracellular co-stimulatory domain. In such instances, an engineered phagocytic cell that binds to an antibody via an antibody binding construct on the surface would exhibits increased phagocytosis, engulfment, and / or trogocytosis compared to a control cell. The increased phagocytosis, engulfument, and / or trogocytosis can be any level of increased phagocytosis, engulfument, and / or trogocytosis compared to a control disclosed herein.

[0252] In some embodiments, an antibody binding construct can be introduced into an immune cell that is the target of an immunotherapy (e.g., a CD3+ T cell for a CD3 bispecific antibody therapy). In such instances, the extracellular domain of the antibody-binding construct comprises said target of said bispecific antibody. In some embodiments, wherein the antibody binding construct comprises an intracellular signaling domain, the intracellular domain promotes enhanced effector function of said cell. For instance, an intracellular signaling domain associated with increased cytotoxic effector function or cytokine release can be used. The engineered immune cells are particularly effective at enhancing an antibody therapy comprising a multi-specific antibody, such as a bispecific antibody, a tri-specific antibody, or a tetra-specific antibody because the immune cells are engineered to bind to said antibody, and thus incorporate the effector function of the engineered immune cell into the immune response that is initiated as part of the immunotherapy.

[0253] The heterologous surface proteins expressed on the engineered immune cells of the present disclosure are not intended to be limited to CD3. Any surface protein expressed on an immune cell can be expressed on the surface of the engineered immune cells of the present disclosure. For example, a monocyte or macrophage can be engineered to express a T cell surface protein other than CD3. In some embodiments, a monocyte or macrophage can express any known pan T cell marker. The extracellular domain comprises an amino acid sequence of a surface protein, or fragment thereof to be expressed on an engineered immune cell.

[0254] The present disclosure is based, at least in part, on the ability of the disclosed engineered immune cells comprising the disclosed antibody-binding constructs to leverage the immune response initiated by a multi-specific antibody, such as a bispecific or tri-specific antibody. Such antibodies are known to engage effector immune cells in the body to target, for example, malignancies, autoreactive, or infected cells. As such, the immune cells of the present disclosure, which are engineered to comprise an antibody-binding construct, can, when administered to a

[0255] 42

[0256] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) subject in conjunction with a multi-specific antibody, enhance the immune response of an immunotherapy comprising said antibody for increased efficacy by engaging the engineered immune cell with the antibody to harness the natural, or enhanced, effector response of the engineered immune cell. Any immunotherapy comprising an antibody that can engage with the engineered immune cells disclosed herein is envisaged within the scope of the present application. Similarly, any bispecific antibody known in the art can be used. In particular embodiments, the engineered immune cells of the present disclosure comprise an antibody-binding construct which displays a CD3 surface marker on the cell (e.g., a CD3 protein ectodomain). In such instances, the CD3+ engineered immune cells would be expected to engage with any multi-specific antibody that targets CD3. For instance, the multi-specific antibody can engage T cells, which naturally express CD3, in addition to the engineered immune cells which display CD3 on the surface. Thus, the multi-specific antibody can engage multiple, different cell types. A skilled artisan would understand that it is particularly useful for the extracellular domain of the antibody binding construct to comprise a cell surface protein that is targeted by a bispecific antibody. Exemplary, but non-limiting, surface markers that are targeted by known bispecific antibodies include CD3, and its associated subunit proteins, which include: CD3e, CD3d, and CD3g As such, in some instances, the extracellular domain can be engineered to comprise an amino acid sequence of an ectodomain of a CD3e, CD3d, and / or CD3g protein, or a fragment thereof. Any immune cell surface marker and its associated ectodomain displayed on the surface is envisaged for including in the the extracellular domain of the antibody-binding constructs disclosed herein. For example, the extracellular domain can comprise an amino acid sequence of any immune cell surface marker of a human immune cell known to a skilled artisan. For instance, a skilled artisan would understand that the specific ectodomain of interest for use in the antibody binding construct may depend on antibody and target cell (i.e., cancer or tumor cell) specificity. It may be particularly useful for the extracellular domain to comprise an amino acid sequence encoding a cell surface marker that is targeted by a multi-specific antibody, wherein said cell surface marker is expressed at low levels on the desired cell type of said engineered immune cell. In some instances, the extracellular domain can comprise an amino acid sequence of an ectodomain of a CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4- IBB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or functional fragment thereof. Antibody binding constructs comprising an extracellular domain comprising an amino acid sequence of an ectodomain of a CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc

[0257] 43

[0258] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) receptor (FcR), or functional fragment thereof, can be introduced into any known immune cell to generate the engineered immune cells disclosed herein.

[0259] The immune cells of the present disclosure can be engineered with the antibody binding constructs disclosed herein to engage with any bispecific antibody in order to enhance killing of target tumor cells. Any immune cell is envisaged within the scope of the engineered cells disclosed herein. In particular, the methods disclosed herein are useful for re-purposing an immune cell to bind a bispecific antibody that targets a tumor cell, in order to enhance killing of the tumor cell by, at least in part, the engineered immune cell. Thus, a particular aspect of the disclosure is to provide engineered immune cells that are repurposed to target tumor cells, or to enhance an anti-tumor cell immune response.

[0260] The engineered immune cells disclosed herein are particularly effective when used in combination with a bispecific antibody therapy. Bispecific antibodies (BsAb) can redirect cytotoxic effector cells to tumor cells. From a mechanistic perspective, this type of bispecific antibody can recruit immune cells (such as T cells and NK cells) to the tumor area to exert cytotoxic effects. One antigen-binding site of the BsAb binds to specific antigens expressed on tumor cells, while the other one bridges and activates effector cells such as cytotoxic T lymphocytes (CTL) (Clynes, Raphael A, and John R Desjarlais. “Redirected T Cell Cytotoxicity in Cancer Therapy.” Annual review of medicine vol. 70 (2019): 437-450). CD3 is the most common targeted protein expressed on effector cells, which can activate the anti-tumor activity of T cells (Sun, Yanze et al. “Bispecific antibodies in cancer therapy: Target selection and regulatory requirements.” Acta pharmaceutica Sinica. B vol. 13,9 (2023): 3583-3597). CD3 bispecific antibody approaches circumvent the requirement for TCR / HLA-peptide by creating artificial T cell recognition of more general tumor-associated antigens (TAA). Mimicking the cognate TCR / HLA-peptide interaction, these approaches achieve T cell activation by artificially clustering the TCR complex into the T cell / target cell synapse. TAA x CD3 bispecific antibodies achieve this by simultaneously binding one of the extracellular CD3 subunits (most commonly CD3s) on a T cell while binding a TAA on the surface of a tumor cell.

[0261] The antibody binding constructs disclosed herein comprise an extracellular domain; and a transmembrane domain. In some embodiments, the antibody-binding constructs comprise an intracellular signaling domain. In some embodiments, the antibody binding constructs further comprise an intracellular co-stimulatory signaling domain. When expressed in an immune cell, the extracellular domain of the antibody binding construct will be displayed on the surface of the engineered cell. As such, the extracellular domain can be modified in a manner that enables the

[0262] 44

[0263] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) antibody binding construct, and the immune cell in which it is expressed, to bind to any target antibody of interest. For example, the extracellular domain can be engineered to comprise a target antigen of a bispecific antibody in order to enable binding of the antibody with the antibody binding construct. A skilled artisan would understand any antibody target can be effectively engineered into the extracellular domain to promote binding with an antibody. In some instances, comprises an extracellular domain that comprises a target antigen of a receptor (e.g., a ligand). Any known ligand of a receptor can be engineered into the extracellular domain to enable the receptor to bind to the antibody binding construct via its extracellular domain.

[0264] In some embodiments, the antibody binding construct disclosed herein comprises an extracellular domain and a transmembrane domain. In some embodiments, the antibody binding construct disclosed herein comprises an extracellular domain; a transmembrane domain; and an intracellular signaling domain. In some embodiments, the antibody binding construct disclosed herein comprises an extracellular domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co-stimulatory signaling domain. One aspect of the antibody binding constructs of the present disclosure is to enable the display of a surface protein on the surface of an engineered immune cell. As such, the extracellular domain, which will be displayed on the surface of the immune cell, can be engineered to comprise an amino acid sequence of any surface protein of interest to be displayed on the surface of the cell. The structure of the antibody binding construct should support the display of the extracellular domain on the cell surface. In some embodiments, the presence of an extracellular domain and a transmembrane domain is sufficient to display the extracellular domain on the cell surface. The extracellular domain of the antibody binding construct will typically comprise a target molecule, such as an antigen, ligand, or small molecule. This aspect of the extracellular domain distinguishes the antibody binding constructs of the present disclosure from a cell surface receptor, such as a chimeric antigen receptor (CAR), or any antibody, or fragment of a receptor or antibody, in that the extracellular domain does not comprise complementarity determining regions (CDRs) that are present in a receptor or antibody. In preferred embodiments, the extracellular domain does not comprise a receptor or an antibody. In another preferred embodiment, the extracellular domain comprises a surface marker, or surface protein, or a fragment thereof, of an immune cell. A skilled artisan would understand that specific proteins can be specifically associated with individual immune cells, such that individual proteins are either exclusively expressed by a certain cell type, or predominately expressed by a certain immune cell type. In certain instances, a protein is expressed at higher levels on a specific immune cell compared to other known immune cells. As such, a skilled artisan would understand that a

[0265] 45

[0266] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) cell-specific surface marker or surface protein, when comprised in the extracellular domain, can be expressed on any engineered immune cell using the antibody binding constructs disclosed herein. In this manner, the present disclosure provides compositions and methods for expressing a surface protein from one immune cell on the surface of a second immune cell. In some embodiments, the surface protein is not expressed on the immune cell type in nature (i.e., expression of the surface protein is not found on the immune cell type in nature). As such, in certain embodiments, the extracellular domain comprises a protein that is heterologous to the immune cell comprising the antibody binding construct. Any surface marker or surface protein associated with an immune cell can be comprised within the extracellular domain.

[0267] Also disclosed herein are polypeptides encoding the antibody binding constructs of the present disclosure, and nucleic acid molecules comprising a polynucleotide sequence encoding said polypeptides. The polypeptides of the disclosure comprise an amino acid sequence encoding the antibody-binding constructs. Also disclosed herein are vectors comprising the nucleic acid molecules disclosed herein.

[0268] Provided herein are immune cells that have been engineered to comprise the antibody binding constructs disclosed herein. Also disclosed are methods of making said engineered immune cells by contacting a target immune cell with the polynucleotides encoding the antibody binding constructs, or vectors comprising said polynucleotides, disclosed herein. Also disclosed herein are methods of making said engineered immune cells, and methods of using said cells to improve immunotherapy. The engineered immune cells can be generated ex vivo, by contacting an isolated immune cell with a polynucleotide, or vector comprising the same, disclosed herein. Alternatively, the immune cells can be engineered in vivo, in a subject, by delivering the polynucleotides encoding the antibody binding construct, or vector comprising the same, into a subject, such that the target immune cell is contacted with the polynucleotide, or vector, in a subject, wherein the polynucleotide, or vector, are introduced into said target immune cell in vivo. In such instances, the methods comprise a combination therapy that involves administration of any of the engineered immune cells in combination with an immunotherapy. In these particular embodiments, the immunotherapy can be an antibody therapy.

[0269] In some instances, the antibody binding constructs and engineered immune cells disclosed herein that can be used to augment efficacy of anti-CD3 bispecific antibody therapies. For example, engineered CD3 expression in human monocytes, dendritic cells, macrophages, neutrophils, mast cells, NK cells, and / or T-cells (including gamma delta) will augment efficacy of anti-CD3 bispecific therapies. Thus, a standard bispecific that engages a T-cell (via CD3 binding)

[0270] 46

[0271] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) with tumor cells could be leveraged to also engage another immune cell(s) that is normally CD3lo / negbut was engineered to be CD3+. In the case of T cells that are already CD3+ (in this example), additional intracellular domains could be devised to support function of the bispecific. Any such engineered cell could be utilized with any CD3-binding bispecific that is already approved in the clinic.

[0272] Disclosed herein are engineered immune cells that are genetically engineered to express an antibody binding construct wherein there is an extracellular domain; a transmembrane domain; an intracellular signaling domain; and an intracellular co-stimulatory signaling domain, wherein the intracellular co-stimulatory domain comprises an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein, or a functional fragment or variant thereof that retains co-stimulatory activity. In some embodiments, the immune cell is a phagocytic cell and / or a cytotoxic cell capable of killing a target cell. In some instances, the engineered immune cells have increased phagocytosis and / or trogocytosis, increased cytokine secretion, and / or increased secretion of proinflammatory factors. In some embodiments, the nucleic acid molecule comprising a polynucleotide encoding the antibody binding construct further comprises an effector molecule expression cassette. The effector molecule expression cassette can comprise a polynucleotide sequence encoding an effector molecule gene. In some instances, the polynucleotide sequence encodes more than one effector molecule gene. Exemplary, but non-limiting, effector molecule genes can include cytokines, chemokines, aptamers, peptides, small molecules, scFvs, and / or checkpoint inhibitors. The effector molecule genes can encode any product that may enhance or promote an immune response. Expression, production and / or secretion of any effector molecule gene product from an engineered immune cell can occur independent of binding of the antibody binding construct of said engineered immune cell to an antibody. Alternatively, the expression, production and / or secretion of any effector molecule gene product from an engineered immune cell can be engineered to be dependent upon binding of an antibody binding construct to an antibody. It can be particularly effective for the engineered immune cells to secrete an effector molecule that aids in the immune response. In some instances, the secreted effector molecule can enhance or support T-cells that are engaged by the bispecific therapy.

[0273] In some embodiments, the immune cells are monocytes or macrophages having increased Ml proinflammatory phenotype or increased M2 immunosuppressive phenotype. In such instances, the effector molecule gene product can encode an effector molecule that, either independent or dependent upon binding of the antibody binding construct to the antibody, secretes

[0274] 47

[0275] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) an effector molecule that enhances the Ml proinflammatory phenotype or M2 immunosuppressive phenotype. In such embodiments, the effector molecule can comprise any secreted molecule that is secreted by an Ml or M2 polarized macrophage. In some embodiments, the engineered immune cell exhibits increased proliferation and / or increased antigen presentation to T cells as compared to a relevant control.

[0276] The antibody binding constructs disclosed herein can comprise an intracellular signaling domain or an intracellular co-stimulatory signaling domain that enhances at least one cellular response of the engineered immune cell upon antibody binding compared to a control. In some embodiments, the at least one cellular response comprises increased expression of pro- inflammatory cytokines, increased phagocytosis, increased antigen cross-presentation, and / or increased trogocytosis In some embodiments, the engineered immune cells exhibit increased expression of pro-inflammatory cytokines, increased phagocytosis, increased antigen crosspresentation and / or increased trogocytosis In some embodiments, the antibody binding constructs comprise an intracellular costimulatory signaling domain comprising an HVEM protein, or a functional fragment thereof. For example, the engineered immune cells can have increased expression of a pro-inflammatory cytokine as compared to a relevant control. For instance, the increase in expression of the pro-inflammatory cytokine can be 0.001% to 10,000-fold. In particular, the engineered immune can cells have increased phagocytosis and / or trogocytosis. The increase in phagocytosis and / or trogocytosis can be 0.001% to 10,000%. In some embodiments, the increase in phagocytosis is at least 20%.

[0277] The increased expression of pro-inflammatory cytokines, increased phagocytosis, and / or increased trogocytosis of the engineered immune cells having an intracellular HVEM costimulatory signaling domain are hallmarks of an increased Ml pro-inflammatory phenotype of the Engineered immune cell. In some embodiments, engineered immune cell of the disclosure having an Ml phenotype change the tumor microenvironment such that anti-tumor immune responses are enhanced. In some embodiments, the environment is changed to promote defense mechanisms against infectious cells.

[0278] For example, increased phagocytosis and / or trogocytosis of the Engineered immune cell of the disclosure can lead to increased engulfment or destruction of tumor or infectious cells. The Engineered immune cell of the disclosure are particularly effective at limiting cell expansion of a target cell, or a plurality of target cells, within a cell population. Other functions that may be enhanced in Engineered immune cell having an Ml phenotype include antigen presentation in an adaptive immune response of captured antigens from a tumor or pathogenic agent, recruitment of

[0279] 48

[0280] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) other immune cells (e.g., cytotoxic T cells, natural killer (NK) cells, dendritic cells) to a tumor or infection site, release of reactive oxygen species or reactive nitrogen species toxic to tumor or infectious cells, and / or promotion of CD4+ T helper cell differentiation into a Thl subtype that can further enhance anti-tumor responses, including secretion of interferon-y and subsequent activation of macrophages, dendritic cells, NK cells, and CD8+ cells.

[0281] Polypeptides, nucleic acids, vectors, compositions, and methods related to the antibodybinding constructs and engineered immune cells of the disclosure are also provided.

[0282] III. Antibody Binding Constructs

[0283] Disclosed herein are antibody binding constructs useful for engineering immune cells to engage with an antibody. For instance, the antibody binding constructs are particularly useful for engineering an immune cell to display an extracellular domain on the surface of an immune cell. In some embodiments, the extracellular domain comprises a target that is bound by an antibody. The present disclosure also provides an engineered immune cell genetically modified to express an antibody binding construct. The antibody binding constructs of the present disclosure comprise an extracellular domain that is displayed on an immune effector cell surface and specifically binds a target molecule, such as an antibody. In some embodiments, the antibody binding constructs disclosed herein comprise an extracellular domain and a transmembrane domain. In some embodiments, the antibody binding constructs comprise a signal transduction domain capable of mimicking a T cell receptor-mediated signaling pathway. A "signaling pathway" or "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. In some instances, the extracellular domain of the antibody binding construct can comprise a surface receptor. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting the signal across the plasma membrane of a cell. In some instances, the antibody binding construct can comprise a fragment or portion of a surface receptor or co-receptor. For instance, the extracellular domain can comprise a protein or a protein complex of a T cell co-receptor. In another embodiment, the antibody binding construct can comprise the transmembrane domain and / or intracellular signaling or co-stimulatory domain of a cell surface receptor. Cell-surface receptors are transmembrane proteins that bind signal molecules in the extracellular space and generate different intracellular signals on the opposite side of the plasma membrane.

[0284] The antibody binding constructs disclosed herein can have an extracellular domain fused to a transmembrane domain and / or a signaling pathway domain. Any signaling pathway domain

[0285] 49

[0286] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) known to those skilled in the art in view of the present disclosure can be used with the antibody binding constructs disclosed herein. In some instances, the signaling pathway can be a signaling pathway domain of a chimeric antigen receptor (CAR). T cells expressing the first-generation CAR having CD3z as the signal transduction domain often become anergic and fail to elicit a potent immune response (Kershaw et al, Clin. Cancer Res. 12(20 Pt 1) :6106 (2006)). To solve this problem, second and third generation CARs that have one and two co-stimulatory signaling domains derived from CD28, 4-1BB or ICOS have been developed (Doth et al., Immunol. Rev. 257(1): 107 (2014)). These CARs have been shown to successfully mimic T cell receptor- mediated signal transduction upon antigen stimulation, leading to proliferation and activation of CAR-T cells (Maus et al, Blood 123(17):2625 (2014)). The CAR signaling pathway can be from a first-generation CAR, a second-generation CAR, a third-generation CAR, or a next-generation CAR known to a skilled artisan. The transmembrane domain and / or signaling pathway of any CAR known to a skilled artisan can be used to generate the antibody-binding constructs disclosed herein. The antibody binding constructs of the present disclosure can comprise more than one of any domain disclosed herein. For instances, an antibody binding construct can comprise more than one extracellular domain; more than one transmembrane domain; more than one intracellular signaling domain; and / or more than one intracellular co-stimulatory domain. In some embodiments, the antibody binding construct comprises more than one signal peptide. The antibody binding construct can comprise any extracellular domain, transmembrane domain, intracellular domain, and / or intracellular co-stimulatory domain, or combination thereof, disclosed herein.

[0287] The antibody binding construct, when used in combination with an antibody, such as a bispecific antibody, can help immune cells such as monocytes or macrophages find and kill cancer cells that have the specific protein (e.g., tumor associated antigen) that is recognized by the bispecific antibody. The term “bispecific antibody” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody can have crossreactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes , or can bind an epitope that is shared between two or more distinct antigens. Any bispecific antibody that can bind to the antibody binding construct and an at least second molecule is envisaged within the scope of the present disclosure. In some instances, the at least second molecule is a tumor antigen, such as a tumor-associated antigen that is on the surface of a tumor

[0288] 50

[0289] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) cell or cancer cell. Any tumor-associated antigen known to those skilled in the art is envisaged within the scope of the present disclosure.

[0290] The antibody binding constructs disclosed herein can be introduced into any immune cell using any method available to those skilled in the art for introducing nucleic acid molecules into the cells, as disclosed herein. The antibody binding constructs can be introduced into cells directly as polypeptides. Polynucleotides encoding the antibody binding construct polypeptides are also disclosed herein. Polynucleotides encoding the antibody binding constructs can be introduced into immune cells using any method available in the art. Polypeptides are provided comprising an amino acid sequence encoding the antibody-binding construct disclosed herein. In some instances, the antibody binding construct is encoded by more than one polypeptide. For example, when the antibody binding construct comprises two extracellular domains, a first extracellular domain can be encoded by a first polypeptide and the second extracellular domain can be encoded by a second polypeptide. In some embodiments, the antibody binding construct comprising two extracellular domains is encoded by a single polypeptide. The antibody-binding constructs disclosed herein can comprise at least a first polypeptide sequence. In some instances, the antibody-binding constructs can comprise more than one polypeptide. In a particular instance, the antibody-binding construct comprises two polypeptides. In some instances, the antibody-binding construct comprises more than two polypeptides. In a particular instance, the antibody-binding construct disclosed herein comprises a first polypeptide sequence. In such instances, the first polypeptide comprises at least a first extracellular domain and at least a first transmembrane domain. The at least first extracellular domain of the antibody-binding construct can further comprise an amino acid sequence encoding said second ectodomain. As such, the first polypeptide can comprise both the amino acid sequence encoding the first ectodomain and the amino acid sequence encoding the second ectodomain.

[0291] Phagocytic cells such as macrophages or monocytes can be removed from a blood, tumor, or ascites fluid of a patient and modified so that they express an antibody binding construct that binds to a specific bispecific antibody. In such instances, the bispecific antibody that binds to the extracellular domain of the antibody binding construct also binds an antigen on tumor cells or a cancer cell. When the bispecific antibody is bound to both the antibody binding construct and a tumor cell antigen, the bispecific antibody functions as a “bridge” molecule that co-localizes engineered immune cells with tumor cells. Therefore, an antibody binding construct can target cancers by redirecting an immune cell, such as a phagocyte, such as a monocyte or macrophage, expressing the antibody binding construct specific for a bispecific antibody, wherein said bispecific antibody binds to at least one tumor associated antigen(s). In embodiments of a

[0292] 51

[0293] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) genetically modified phagocytic cell of the present disclosure and related compositions and methods herein described, antibody binding constructs can comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain of the antibody binding constructs can comprise an intracellular signaling domain. In some instances, the intracellular domain can comprise an intracellular co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain and an intracellular co-stimulatory signaling domain. In such instances, the co-stimulatory signaling domain, can comprise a co-stimulatory signaling domain of a herpes virus entry mediator (HVEM) protein as described herein. Phagocytic cells modified to express an antibody binding construct comprising an intracellular co-stimulatory signaling domain of a HVEM protein described herein can have increased phagocytosis, increased cytokine secretion, increased secretion of proinflammatory factors, increased Ml proinflammatory phenotype, increased M2 immunosuppressive phenotype, increased proliferation, and / or increased antigen presentation to T cells, as compared to a relevant control.

[0294] A. Extracellular domain

[0295] An antibody binding construct expressed by a modified immune cell of the disclosure can comprise an extracellular domain that binds to a target molecule located extracellular to the modified immune cell. Exemplary target molecules can include: an antibody, a receptor, a small molecule, or an antigen, or a functional fragment thereof. The extracellular domain comprises a target binding domain which mediates binding to a target molecule. A “target binding domain” when used in connection with the extracellular domain disclosed herein refers to a portion of the extracellular domain that binds to the target molecule. A “functional fragment” when used to refer to a target molecule, including an antibody, receptor, or antigen targeted by the antibody binding construct disclosed herein, refers to any portion or fragment of the target molecule that retains the ability to bind to the extracellular domain. In preferred embodiments, the target molecule will be an antibody, or a binding domain or fragment thereof, wherein the binding domain of the antibody binds to the extracellular domain. The antibody can comprise a multi-specific or multi-valent antibody, such as a bispecific, a trispecific, or tetraspecific antibody. In a preferred embodiment, the extracellular domain comprises an amino acid sequence that is bound by a bispecific antibody, or fragment, or binding domain thereof.

[0296] Any suitable antibody known to those skilled in the art in view of the present disclosure is envisaged as a suitable target molecule for the antibody binding ectodomain disclosed herein. For example, the antibody binding extracellular domain can comprise a target binding domain (e.g., an

[0297] 52

[0298] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) amino acid sequence) that is bound by any antibody known to those skilled in the art. Antibodies approved for immunotherapy in a subject will be particularly useful to bind the extracellular domain.

[0299] As used herein, the term “antibody” refers to any molecule comprising a binding domain that enables the molecule to bind to the extracellular domain of the antibody binding constructs disclosed herein. For instance, the binding domain of an antibody can bind to the target binding domain of the extracellular domain. The binding domain and target binding domain can comprise amino acid sequences capable of mediating binding. A skilled artisan would understand that antibodies (i.e., molecules comprising binding domains that interact with the extracellular domain) can comprise a wide range of molecular structures. Any structure or format of an antibody is envisaged within the present application. Antibodies can, for example, include bispecific, tri- specific, or tetra-specific antibodies; recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy -chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule, or bispecific antibodies generated by arm exchange. Any bispecific antibody format is envisaged as within the scope of the presently disclosed construct. Exemplary bispecific formats include dual targeting molecules including Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech) and mAb2 (F-Star), Dual Variable Domain (DVD)-Ig (Abbott), DuoBody (Genmab), Ts2Ab (Medlmmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee) and TvAb (Roche), ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS) and Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech), Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR- like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain

[0300] 53

[0301] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) antibodies. Various formats of bispecific antibodies have been described, for example in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 and in Nunez-Prado et al, (2015) Drug Discovery Today 20(5):588-594.

[0302] In some embodiments, the antibody comprises a bispecific antibody. Exemplary bispecific antibodies that can be target molecules include: catumaxomab (Removab®), blinatumomab (Blincyto®), amivantamab-vmjw (Rybrevant®), tebentafusp-tebn (Kimmtrak®), faricimab-svoa (Vabysmo®), cadonilimab (fF±S Jt®), mosunetuzumab (Lunsumio®), teclistamab (Tecvayli®), glofitamab (COLUMVI®), epcoritamab (Epkinly®), talquetamab-tgvs (TALVEY™), tarlatamab (Imdelltra™), or elranatamab (ELREXFIO®).

[0303] In certain embodiments, the bispecific antibody comprises a bispecific T cell engager (BiTE®), or a (BiTE®)-like molecule. For instance, when the antibody is a (BiTE®)-like molecule, said antibody can be a derivate of a (BiTE®) wherein one arm has been modified to target an immune cell other than a T cell.

[0304] The bispecific antibody can be of any allotype. It is expected that allotype has no influence on properties of the bispecific antibodies, such as binding or Fc-mediated effector functions. Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert el al, (2003) N Engl J Med 348:602-08). The extent to which therapeutic antibodies induce an immune response in the host can be determined in part by the allotype of the antibody (Stickler et al, (2011) Genes and Immunity 12:213-21). Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody. In some embodiments, the bispecific antibody comprises one or more Fc substitutions that reduces binding of the bispecific antibody to a Fey receptor (FcyR) and / or reduces Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0305] Any suitable epitope comprising a domain capable of binding to a target molecule of interest can be comprised with the extracellular domains disclosed herein. Antibodies are a particular target molecule of interest for the extracellular domains disclosed herein. As such, the antibody binding constructs of the present disclosure comprise extracellular domains that are particularly effective for binding antibodies. In some instances, the antibodies bound by the constructs can be multi-specific or multi-valent antibodies.

[0306] Antibodies that target immune cells by binding surface markers, or proteins, on the surface of immune cells, are known in the art. Engineering the extracellular domain to comprise an

[0307] 54

[0308] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) immune cell marker that is recognized by an antibody will allow binding of the antibody by the constructs disclosed herein. In some instances, the extracellular domain comprises a surface marker, or surface protein, or a fragment thereof, of an immune cell. Exemplary, non-limiting, immune cell surface markers that the extracellular domain can include: CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or functional fragment thereof. In some embodiments, the extracellular domain comprises a pan-T cell marker. The extracellular domain can bind to an antibody at any location of the antibody. The extracellular domain of the constructs disclosed herein may comprise more than one cell surface protein, or fragment(s) thereof. A skilled artisan would understand that the extracellular domains disclosed herein can be engineered to comprise any combination of known cell surface proteins or fragments, based on the intended target molecule(s). For instance, the extracellular domain may comprise a surface marker from multiple distinct immune cells, such that the antibody binding construct binds to multiple immune cells. A skilled artisan would understand that distinct surface markers are associated with specific immune cells and would be capable of engineering a suitable extracellular domain to comprise the desired markers, or fragments thereof.

[0309] The antibody binding constructs of the present disclosure are particularly effective at engaging antibodies, such as multi-specific antibodies, including bispecific antibodies. The antibody binding constructs can bind to any antibody known to those skilled in the art. In particular, antibodies that are capable of engaging an immune cell and a tumor cell (e.g., a bispecific antibody) are particularly of interest in binding the extracellular domain. One aspect of the present disclosure is to provide antibody binding constructs comprising an extracellular domain that is targeted by an antibody, such that when expressed on an immune cell, said antibody binding construct can target said immune cell for binding by said antibody that recognizes. In the broadest embodiment, any immune cell can express an antibody binding construct comprising an extracellular domain comprising a surface protein or fragment thereof from any immune cell. For instances, the extracellular domain can comprise a protein or fragment thereof this is heterologous to said immune cell expressing said antibody binding construct. In another embodiment, the extracellular domain can comprise a protein or fragment thereof this is present at low levels on said immune cell expressing said antibody binding construct compared to levels on another immune cell. In such instances, it may be beneficial for a first immune cell to express a surface protein of an at least second immune cell. As such, the extracellular domain of the antibody binding construct can comprise a surface protein, or fragment of a surface protein of an immune

[0310] 55

[0311] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) cell, such as a T cell. For instance, the antibody binding constructs disclosed herein can be expressed on a monocyte or macrophage. When expressed on a monocyte or macrophage, said extracellular domain of the antibody binding construct can comprise a surface protein found on a T cell. In some instances, When expressed on a monocyte or macrophage, said extracellular domain of the antibody binding construct can comprise a surface protein found on a T cell, a monocyte, a macrophage, a dendritic cell, a neutrophil, a mast cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, an eosinophil, a basophil, a myeloid cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a hematopoietic stem cell (HSC), a HSC-derived cell, a precursor cell, a bone marrow cell, a stem cell, a pluripotent stem cell, a mesenchymal stromal cell (MS SC), and induced pluripotent stem cell (iPSC), or an iPSC-derived cell.

[0312] In order to engage with antibodies, the antibody binding construct comprises an extracellular domain comprising a molecule, or a variant or fragment thereof, that can be bound by said antibody. For example, the extracellular domain can comprise an amino acid sequence of a surface protein that is bound by an antibody. In some instances, the extracellular domain comprises an ectodomain of a surface marker, or surface protein, or a fragment thereof, of an immune cell. Exemplary, non-limiting, surface proteins comprising ectodomains that can be comprised within the extracellular domain of the disclosed antibody binding constructs include: CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4- 1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or fragment thereof, or a combination thereof. For example, the extracellular domain can comprise an amino acid sequence of an ectodomain of any surface protein known in the art. A skilled artisan would readily understand how to identify the ectodomain of a surface protein using methods available in the art.

[0313] In some embodiments, the extracellular domain can comprise a protein or protein subunit, or fragment thereof, of a T cell receptor (TCR) complex. In such instances, the extracellular domain is capable of binding to an antibody that binds to a TCR, or a protein subunit of a TCR (e.g., CD3).

[0314] The T-cell receptor (TCR) is a complex of integral membrane proteins that participate in the activation of T-cells in response to an antigen. Stimulation of TCR is triggered by MHC (major histocompatibility complex) molecules on cells with the antigen. Engagement of the TCR initiates positive and negative cascades that ultimately result in cellular proliferation, differentiation, cytokine production, and / or activation-induced cell death. These signaling

[0315] 56

[0316] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) cascades regulate T-cell development, homeostasis, activation, acquisition of effector’s functions and apoptosis. TCR is composed of six different chains that form the TCR heterodimer responsible for ligand recognition. CD3 molecules are assembled together with the TCR heterodimer. CD3 possess a characteristic sequence motif for tyrosine phosphorylation, known as IT AMs (immunoreceptor tyrosine-based activation motifs). The TCR polypeptides themselves have very short cytoplasmic tails, and all proximal signaling events are mediated through the CD3 molecules. TCR-CD3 complex interaction plays an important role in mediating cell recognition events.

[0317] In some instances, the extracellular domain of the antibody binding construct disclosed herein comprises an amino acid sequence encoding an ectodomain of a CD3 protein, or a fragment thereof. Any CD3 protein is suitable for use within extracellular domain. For instance, the extracellular domain can comprise an amino acid sequence encoding an ectodomain of a CD3epsilon (CD3e), CD3delta (CD3d), and / or CD3gamma (CD3g), or a fragment thereof, or a combination thereof.

[0318] CD3 proteins have an N-terminal extracellular region, a transmembrane domain and a cytoplasmic tail where the immunoreceptor tyrosine activation motifs (IT AMs) are located. The extracellular domains of CD3 epsilon (a) (CD3a or CD3e), CD3 gamma (y) (CD3y or CD3g) and CD3 delta (5) (CD35 or CD3g) contain an immunoglobulin-like domain, so are therefore considered part of the immunoglobulin superfamily. The CD3 protein complex was first identified by immunoprecipitation experiments using human T cells. The determination of glycosylation state of the individual CD3a, CD3y and CD35 polypeptide chains was subsequently determined using endoglycosaminidases and specific anti-CD3 antibodies. CD3a, CD3y and CD35 are highly homologous and are believed to be derived from a single ancestral gene, mapping to human chromosome 11 band q23, through gene duplication (Clevers et al. 1988).

[0319] CD3 epsilon (a) is a non-glycosylated polypeptide chain of 20 kDa. The existence of an epitope on the a polypeptide that is conserved among many species has made it possible to obtain the antibody clone, CD3-12 which has a very broad species cross-reactivity for the CD3 marker (Jones et al. 1993). Both CD3y and CD35 are glycosylated and have a molecular weight of 25-28 kDa and 20 kDa respectively (Norman 1995).

[0320] CD3 zeta (Q (also known as CD247) is a non-glycosylated polypeptide with a molecular weight of 17 kDa that shares no sequence similarity with the other CD3 polypeptide chains. CD3(^ was first discovered by virtue of the fact that it coprecipitates with the TCR complex. CD3(^ maps

[0321] 57

[0322] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) to human chromosome 1 band q22-q25 underscoring the fact that CD3(^ is a distinct genetic component of the CD3 protein complex (Weissman et al. 1988).

[0323] CD3 eta (q) is an alternatively spliced product of the gene that encodes CD3 C, (Clayton et al. 1991) and has an apparent molecular weight of 23 kDa (Orloff et al. 1989). CD3 C, is predominantly found as a homodimer, but a fraction (approximately 10%) of CD3 C, is found complexed as a heterodimer with CD3 q and in this form is thought to confer different signaling properties (Orloff et al. 1989).

[0324] CD3 omega (Q) is a non-glycosylated polypeptide chain of 28 kDa that associates with the TCR complex during TCR assembly in the endoplasmic reticulum, but does not associate with the TCR complex at the cell surface (Pettey et al. 1987, Clevers et al. 1988, Neisig et al. 1993). It is thought that CD3 Q might facilitate complex assembly and / or be involved in the retention of unassembled CD3 protein complexes in the endoplasmic reticulum leading to their subsequent degradation in the lysosomes.

[0325] IT AMs are required for initiation of the signaling cascade as they recruit protein tyrosine kinases, signaling intermediates and adapter molecules. Other immune receptors, such as FcR and BCR, contain far fewer ITAMs than the TCR: CD3 complex, however, T cell development and activation is influenced by the number, location and type of ITAMs present within the complex (Guy and Vignali 2009). This suggests that the TCR is able to vary its signaling leading to diverse T cell responses.

[0326] The extracellular domain can comprise an amino acid sequence encoding an ectodomain of a CD3e, CD3d, and / or CD3g, or a fragment thereof, or a combination thereof. In some instances, the extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3 epsilon protein, or a fragment thereof. In some instances, the extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3 delta protein, or a fragment thereof. In some instances, the extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3 gamma protein, or a fragment thereof. In some instances, it may be suitable for the extracellular domain to comprise an amino acid sequence encoding an ectodomain of more than one CD3 protein, or fragment thereof. For instance, the ectodomain from more than one protein can be comprised within the extracellular domain of the antibody binding construct such that a heterodimeric or heterotrimeric protein is comprised within the extracellular domain.

[0327] The extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3e, CD3d, and / or CD3g protein. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity

[0328] 58

[0329] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) to a sequence set forth as any one of SEQ ID NOs: 2, 4, or 6, or functional fragment thereof. A functional fragment when used to refer to the extracellular domain refers to any fragment or portion that retains the ability to bind to an antibody. Methods for determining the binding of a protein fragment to an antibody, such as binding assays, are known and available in the art. A skilled artisan would understand that the amino acid residues that mediate binding of a sequence to an antibody will be included in the extracellular domain domain.

[0330] In some instances, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3e protein. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to the sequence set forth as SEQ ID NO: 2, or a functional fragment thereof. A “functional fragment” when used in connection with the extracellular domain refers to any fragment or protein that retains the ability to bind to the antibody. In some instances, the extracellular domain comprises an amino acid sequence set forth as SEQ ID NO: 2. In some instances, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3d protein. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to the sequence set forth as SEQ ID NO: 4, or a functional fragment thereof. In some instances, the extracellular domain comprises an amino acid sequence set forth as SEQ ID NO: 4. In some instances, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3g protein. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to the sequence set forth as SEQ ID NO: 6, or a functional fragment thereof. In some instances, the extracellular domain comprises an amino acid sequence set forth as SEQ ID NO: 6.

[0331] The extracellular domain of the antibody binding constructs disclosed herein can comprise an amino acid sequence encoding an ectodomain of more than one protein. In some instances, the extracellular domain can comprise an amino acid sequence encoding an ectodomain of more than one CD3 protein. In such instances, the extracellular domain of the antibody binding construct can comprise an amino acid sequence encoding an ectodomain of two CD3 proteins, or fragments thereof. Without wishing to be bound by theory, when an antibody binding construct comprises an extracellular domain comprising an amino acid sequence encoding an ectodomain of two CD3 proteins, said CD3 ectodomains may interact to form a heterodimer (e.g., a CD3epsilon / CD3delta, or CD3epsilon / CD3gamma heterodimer), which may enhance intracellular signaling in the engineered immune cell and / or bind to an antibody that is specific for said heterodimer. A skilled

[0332] 59

[0333] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) artisan would understand methods available in the art for generating an antibody binding construct wherein the extracellular domain comprises a CD3 heterodimer. When the extracellular domain comprises a CD3 heterodimer, the two CD3 proteins within the heterodimer can be fused to different transmembrane and / or intracellular signaling domains. For instance, each CD3 protein or protein fragment within the heterodimer may be fused to a hinge region or intracellular domain, wherein each CD3 protein is fused to a different hinge region, transmembrane domain, and / or intracellular signaling domain. Alternatively, CD3 subunits in the expressed heterodimer could be expressed inline and thereby fused together.

[0334] When the extracellular domain comprises an amino acid sequence of an ectodomain of more than one protein, said extracellular domain can comprises an amino acid sequence encoding an ectodomain of two CD3 proteins, or fragments thereof.

[0335] In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3e protein, or a functional fragment thereof, and an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3d protein, or a functional fragment thereof. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and an amino acid sequence comprising SEQ ID NO: 4. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 2, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0336] In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3e protein, or a functional fragment thereof, and an amino acid sequence having at least 90% sequence identity to an ectodomain of a CD3g protein, or a functional fragment thereof. In such instances, the extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the extracellular domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and an amino acid sequence comprising SEQ ID NO: 6. In some embodiments, the extracellular domain comprises an amino acid sequence of SEQ ID NO: 2, and an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. The antibody binding constructs of the present disclosure can comprise more than one of any domain disclosed herein. For instance, an antibody binding construct can comprise more than one extracellular

[0337] 60

[0338] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) domain; more than one transmembrane domain; more than one intracellular signaling domain; and / or more than one intracellular co-stimulatory domain. In some embodiments, the antibody binding construct comprises more than one signal peptide. In such instances, the more than one extracellular domain, transmembrane domain, intracellular signaling domain, intracellular costimulatory domains, and / or signal peptides comprised within an antibody binding construct, or a polypeptide encoding the same, can be the same or different. For instance, an antibody binding construct can comprise more than one extracellular domains. In such instances, an antibody binding construct can comprise two extracellular domains. In such instances, an antibody binding construct can comprise a first extracellular domain, and an at least second extracellular domain. In some embodiments, said first extracellular domain and said at least second extracellular domain comprise different amino acid sequences. The extracellular domains can be considered “different” by directly comparing the amino acid sequences comprised within the extracellular domain. For instances, two extracellular domains, when compared, may be considered different when they share no more than 90% sequence identity between them. In some instances, the extracellular domains share no more than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1% sequence identity. A skilled artisan would readily understand that two extracellular domains that are different may be functionally distinguishable. For example, two extracellular domains that share less than 90% sequence identity can differ in antibody binding, which, without wishing to be bound by theory, will likely be dependent upon the amino acid sequence of the extracellular domain. A skilled artisan would understand that when an antibody binding construct comprises more than one extracellular domain (i.e., the target of an antibody binding to the antibody -binding construct) it may be of particular interest that the more than one extracellular domain comprise an amino acid sequence having a different sequence identity between the two sequences. The antibody binding construct can comprise any extracellular domain, transmembrane domain, intracellular domain, and / or intracellular costimulatory domain, or combination thereof, disclosed herein.

[0339] When the antibody binding construct comprises more than one extracellular domain, the binding construct can comprise a first extracellular domain comprising an amino acid sequence encoding an ectodomain of a first surface protein or surface marker, and an at least second extracellular domain comprising an amino acid sequence encoding an ectodomain of an at least second surface protein or surface marker. In some embodiments of the antibody-binding construct, said antibody binding construct comprises two extracellular domains. In such embodiments, the antibody binding construct comprises a first extracellular domain and a second extracellular

[0340] 61

[0341] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) domain, wherein said first extracellular domain comprises an amino acid sequence encoding a first ectodomain of a first surface protein or surface marker, or fragment thereof, or variant having at least 90% sequence identity thereto, and said second extracellular domain comprises an amino acid sequence encoding a first ectodomain of a first surface protein or surface marker, or fragment thereof, or variant having at least 90% sequence identity thereto. When the antibody binding construct comprises two extracellular domains, the first and / or second extracellular domain can comprise an amino acid sequence encoding an ectodomain of a surface marker, or surface protein, or a fragment thereof, of an immune cell. When the antibody binding construct comprises two extracellular domains, the first and / or second extracellular domain can comprise an amino acid sequence encoding an ectodomain of a surface protein, or a fragment thereof, wherein said surface protein comprises CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or functional fragment thereof, or a combination thereof. When the antibody binding construct comprises two extracellular domains, the first extracellular domain can comprise an amino acid sequence encoding an ectodomain of a first surface protein, wherein said first surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or a fragment or variant having at least 90% sequence identity thereto. When the antibody binding construct comprises two extracellular domains, the second extracellular domain can comprise an amino acid sequence encoding an ectodomain of a second surface protein, wherein said second surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or a fragment or variant having at least 90% sequence identity thereto.

[0342] In some embodiments, wherein the antibody binding construct comprises two extracellular domains, the first extracellular domain can comprise an amino acid sequence encoding an ectodomain of a first surface protein, wherein said first surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4- IBB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or a fragment or variant having at least 90% sequence identity thereto, and the second extracellular domain can comprise an amino acid sequence encoding an ectodomain of a second surface protein, wherein said second surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor

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[0344] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0345] (FcR), or a fragment or variant having at least 90% sequence identity thereto. In such instances, said first surface protein or said at least second surface protein can be a CD3 protein, or functional fragment or variant thereof. In some instances, said first surface protein and said at least second surface protein are both CD3 subunit proteins. In some embodiments, the ectodomain of said first surface protein and the ectodomain of said second surface protein form a heterodimer.

[0346] In some embodiments, the antibody binding construct comprises two extracellular domains. In such instances, the first extracellular domain comprises an amino acid sequence encoding an ectodomain of a first CD3 protein, and the second extracellular domain comprises an amino acid sequence encoding an ectodomain of a second CD3 protein. In such instances, said first and said second CD3 proteins can be the same or different CD3 proteins. In some embodiments, said first and said second CD3 protein can be any one of CD3e, CD3g, or CD3g.

[0347] In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3d and / or CD3g protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto. In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3d protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto. In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain comprises an amino acid sequence encoding an ectodomain of a CD3g protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto.

[0348] When the antibody binding construct comprises two extracellular domains, said first extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. When the antibody binding construct comprises two extracellular domains, said second extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. When the antibody binding construct comprises two extracellular domains, said first extracellular domain can comprise an amino acid sequence having at least 90%

[0349] 63

[0350] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) sequence identity to SEQ ID NO: 2, and said second extracellular domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0351] When the antibody binding construct comprises two extracellular domains, said first extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. When the antibody binding construct comprises two extracellular domains, said second extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. When the antibody binding construct comprises two aextracellular domains, said first extracellular domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and said second extracellular domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6.

[0352] In some instances, the extracellular domain can be engineered to bind to a receptor, such as a chimeric antigen receptor (CAR). In such instances, the extracellular domain is engineered to comprise a ligand, or fragment thereof, that is recognized by a receptor. Any ligand known by a skilled artisan can be utilized in the extracellular domain to target and bind a receptor.

[0353] The antibody-binding constructs disclosed herein are particularly useful for expressing surface proteins or surface markers or portions or fragments thereof on the surface of an immune cell. Specifically, the extracellular domain of the antibody-binding construct will be displayed on the surface of the engineered immune cell. As such, the extracellular domain can comprise an amino acid sequence of any known immune cell surface protein or surface marker, or the ectodomain thereof, of interest to be displayed on the surface of the engineered cell. In some embodiments, the extracellular domain comprises an amino acid sequence of an ectodomain of a surface protein or a surface marker, or a fragment thereof. The cell surface protein or marker can be naturally expressed on the immune cell, or, alternatively, can be heterologous to the immune cell type. The expression of a protein or marker on an engineered immune cell enables said engineered immune cell to be targeted (i.e., bound) by an antibody that recognizes and binds to said surface marker, or the surface displayed portion thereof. One particular benefit of the antibody-binding constructs of the present disclosure is their usefulness in generating an engineered immune cell that can be targeted by a multi-specific antibody. The extracellular domain of the antibody binding construct will typically comprise a target molecule, such as an antigen, ligand, or small molecule, or an amino acid encoding the same. The term “antigen” as used herein refers to a target molecule that is recognized and binds to a binding molecule, such as an antibody or receptor, or any other molecule comprising a binding domain. An antigen can be a protein, such as a cell surface protein, or a polypeptide sequence that binds to the binding domain

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[0355] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) of an antibody or receptor. In a preferred embodiment, the extracellular domain comprises a polypeptide comprising an amino acid sequence encoding a surface protein or a fragment thereof. A skilled artisan would understand that when, for example, the extracellular domain comprises a surface protein or surface marker, or fragment or variant thereof, said surface protein or surface marker may not comprise a binding domain of an antibody or receptor. In such instances, said surface protein or surface marker, or fragment or variant thereof, can be targeted by the binding domain of an antibody or receptor, and thus, can bind to said antibody or receptor. The extracellular domain comprises a target of an antibody or receptor. In such embodiments, this aspect of the extracellular domain distinguishes the antibody binding constructs of the present disclosure from other binding molecules, such as a cell surface receptor, including a chimeric antigen receptor (CAR), or any antibody, or fragment of a receptor or antibody. In some embodiments, the extracellular domain of the disclosed antibody binding constructs does not comprise a receptor or an antibody. In some embodiments, the extracellular domain comprises a surface marker, or surface protein, or a fragment or variant thereof, or a combination thereof, of an immune cell. Any immune cell known to a skilled artisan is envisaged within the scope of the immune cell from which a surface protein or surface marker, or fragment or variant thereof, or combination thereof, can be used.

[0356] One purpose of the extracellular domain is to provide a target for a multi-specific antibody, such that said antibody binds to the extracellular domain displayed on the surface of an engineered immune cell. Accordingly, any extracellular domain that binds to an antibody, in particular a multi-specific antibody, is envisaged as within the scope of the present disclosure. For example, an extracellular domain comprising a binding domain may be particularly useful when expressed in the engineered immune cells. For example, an engineered immune cell comprising an antibody binding construct comprising an extracellular domain comprising a binding domain that binds an antibody would allow said engineered immune cell to directly engage with said antibody. In some embodiments, the antibody binding domain of the extracellular domain can bind to any portion or fragment of an antibody. In some instances, the extracellular domain can comprise a binding domain that recognizes the binding domain of an antibody. For instance, the extracellular domain can comprise an amino acid sequence comprising at least one complementarity determining region (CDR) that bind to a receptor or an antibody. The CDRs of the extracellular domain can bind to the binding domain of an antibody. In some embodiments, the extracellular domain can comprise an anti -idiotypic receptor, such as a CAR or antibody. Anti-idiotype receptors or antibodies bind to the variable region of another antibody. The variable region of an antibody comprises a

[0357] 65

[0358] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) paratope (antigen-binding site), which is the region that makes direct contact with an antigenic epitope. Each paratope is made up of six complementarity-determining regions (CDRs). When one antibody binds to an idiotype of another antibody it is referred to as an anti -idiotypic antibody. An idiotype can be defined as the specific combination of idiotopes present within an antibody's complement determining regions (CDRs). A single idiotope, is a specific region within an antibody's Fv region which binds to the paratope (antigen-binding site) of a different antibody. Therefore, an idiotope can be considered almost synonymous with an antigenic determinant of an antibody.

[0359] In some embodiments, the extracellular domain is operably linked to another domain of the antibody binding construct, such as the hinge region or the transmembrane domain or the intracellular domain, for expression in the cell. For example, a polypeptide comprises an amino acid sequence encoding the extracellular domain operably linked at its C-terminus to an amino acid sequence of a hinge region and / or a transmembrane domain.

[0360] In some instances, a polynucleotide sequence encoding the extracellular domain is operably linked at its 3’ end to a polynucleotide sequence encoding a hinge region and a polynucleotide encoding a transmembrane domain and a polynucleotide sequence encoding an intracellular domain.

[0361] In a particular instance, the antibody-binding construct can comprise: a) a first extracellular domain having an amino acid sequence set forth as SEQ ID NO: 2; b) a first transmembrane domain; and a second ectodomain having an amino acid sequence set forth as SEQ ID NO: 4. In another particular instance, the antibody-binding construct can comprise: a) a first extracellular domain having an amino acid sequence set forth as SEQ ID NO: 2; b) a first transmembrane domain; and a second ectodomain having an amino acid sequence set forth as SEQ ID NO: 6.

[0362] When the antibody-binding construct comprises a first polypeptide, said first polypeptide can comprise an amino acid sequence encoding a first ectodomain and an amino acid sequence encoding a second ectodomain. The first ectodomain can comprise an amino acid sequence set forth as SEQ ID NO: 2, and the second ectodomain can comprise an amino acid sequence set forth as SEQ ID NO: 4. The first ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. That is, the first ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. The second ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity

[0363] 66

[0364] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) to the amino acid sequence set forth as SEQ ID NO: 4. That is, the second ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. Alternatively, when the antibodybinding construct comprises a first polypeptide comprising an amino acid sequence encoding a first ectodomain and an amino acid sequence encoding a second ectodomain, the first ectodomain can comprise an amino acid sequence set forth as SEQ ID NO: 2, and the second ectodomain can comprise an amino acid sequence set forth as SEQ ID NO: 6. The first ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. That is, the first ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. The second ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6. That is, the second ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

[0365] Within a first polypeptide, the first ectodomain sequence and the second ectodomain sequence can be separated by a spacer or linker. The spacer can comprise an amino acid sequence. The amino acid sequence of the linker can be at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, or greater than 10 amino acids. In some instances, the spacer between the first ectodomain and second ectodomain is 1-10 amino acids, 5-15 amino acids, 10-20 amino acids, 15-25 amino acids, 20-30 amino acids, 25-35 amino acids, 30-40 amino acids, 35-45 amino acids, 40-50 amino acids, 45-55 amino acids, 50-100 amino acids, or greater than 100 amino acids. Without wishing to be bound by theory, the presence of a spacer can serve to allow the first and the second ectodomains to form a heterodimer and / or interact with a multi-specific antibody.

[0366] In some instances, the antibody-binding construct further comprises a second extracellular domain. The second extracellular domain can comprise an amino acid sequence encoding a second ectodomain. In such instances, the second ectodomain can be a different ectodomain sequence that the first ectodomain sequence. One of skill in the art would readily understand how to determine if

[0367] 67

[0368] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) the first and second ectodomain are different based on, for instance, sequence identity, as described elsewhere herein. When the antibody-binding construct comprises a first extracellular domain sequence and a second extracellular domain sequence, the antibody-binding construct can comprise two polypeptides. For instance, in such a construct, a first polypeptide can comprise a first extracellular domain sequence comprising a first ectodomain sequence, and a second polypeptide can comprise a second extracellular domain sequence comprising a second ectodomain sequence. The first ectodomain sequence can be a CD3e protein ectodomain sequence, or a fragment or variant thereof. The second ectodomain sequence can be a CD3d protein ectodomain sequence, or a fragment or variant thereof. Alternatively, the second ectodomain sequence can be a CD3g protein ectodomain sequence, or a fragment or variant thereof.

[0369] In a particular instance, the antibody-binding construct disclosed herein comprises a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an amino acid sequence encoding a first ectodomain wherein said first ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, and wherein said second polypeptide sequence comprises an amino acid sequence encoding a second ectodomain, wherein said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 4. The first ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. That is, the first ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. The second ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. That is, the second ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4.

[0370] In another particular instance, the antibody-binding construct disclosed herein comprises a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an amino acid sequence encoding a first ectodomain wherein said first ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, and wherein said second polypeptide sequence comprises an amino acid sequence encoding a second ectodomain, wherein said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 6. The first ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. That is, the first ectodomain sequence can comprise an amino acid sequence

[0371] 68

[0372] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. The second ectodomain sequence can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6. That is, the second ectodomain sequence can comprise an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

[0373] The antibody-binding construct disclosed herein can comprise a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an amino acid sequence encoding a first ectodomain comprising an amino acid sequence set forth as SEQ ID NO: 2, and wherein said second polypeptide sequence comprises an amino acid sequence encoding a second ectodomain comprising an amino acid sequence set forth as SEQ ID NO: 4, wherein said first polypeptide further comprises: i) an amino acid sequence encoding a first transmembrane domain; and ii) an amino acid sequence encoding a first intracellular costimulatory domain, and said second polypeptide further comprises: i) an amino acid sequence encoding a first transmembrane domain.

[0374] The antibody-binding construct disclosed herein can comprise a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an amino acid sequence encoding a first ectodomain comprising an amino acid sequence set forth as SEQ ID NO: 2, and wherein said second polypeptide sequence comprises an amino acid sequence encoding a second ectodomain comprising an amino acid sequence set forth as SEQ ID NO: 6, wherein said first polypeptide further comprises: i) an amino acid sequence encoding a first transmembrane domain; and ii) an amino acid sequence encoding a first intracellular costimulatory domain, and said second polypeptide further comprises: i) an amino acid sequence encoding a first transmembrane domain.

[0375] The antibody-binding construct disclosed herein can comprise: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein comprising an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said antibody-binding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of: i) a CD3d protein comprising an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or ii) a CD3g protein

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[0377] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) comprising an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

[0378] Disclosed herein are polynucleotides encoding any of the antibody binding constructs of the present application. The polynucleotide can comprise an amino acid sequence encoding any polypeptide disclosed herein. The polynucleotide can comprise a nucleotide sequence encoding any of the extracellular domains disclosed herein. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an ectodomain of a surface protein or surface marker, or a fragment thereof. The surface protein or surface marker can be a protein or marker of an immune cell. The surface protein can be CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or fragment thereof, or a combination thereof, and the polynucleotide can comprise a nucleotide sequence encoding an ectodomain of the surface protein. The surface protein can be a CD3 subunit protein, such as CD3e, CD3g, and / or CD3g. In some embodiments, the polynucleotide encoding the extracellular domain comprises a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 59, 60, and / or 61.

[0379] In some embodiments, wherein the antibody binding construct comprises two extracellular domains, the first extracellular domain can comprise an amino acid sequence encoding an ectodomain of a first surface protein, wherein said first surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4- IBB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or a fragment or variant having at least 90% sequence identity thereto, and the second extracellular domain can comprise an amino acid sequence encoding an ectodomain of a second surface protein, wherein said second surface protein is CD3, CD14, CD56, CD28, CD4, CD8, CD5, CD7, signal regulatory protein alpha (SIRPalpha), 4-1BB, a pan-T cell marker, or a fragment, or subunit thereof, or an Fc receptor (FcR), or a fragment or variant having at least 90% sequence identity thereto. In such instances, said first surface protein or said at least second surface protein can be a CD3 protein, or functional fragment or variant thereof. In some instances, said first surface protein and said at least second surface protein are both CD3 subunit proteins. In some embodiments, the ectodomain of said first surface protein and the ectodomain of said second surface protein form a heterodimer.

[0380] In some embodiments, the antibody binding construct comprises two extracellular domains. In such instances, the first extracellular domain can be encoded by a polynucleotide

[0381] 70

[0382] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) comprising a nucleotide sequence encoding an ectodomain of a first CD3 protein, and the second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a second CD3 protein. In such instances, said first and said second CD3 proteins can be the same or different CD3 proteins. In some embodiments, said first and said second CD3 protein can be any one of CD3e, CD3g, or CD3g.

[0383] In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3d and / or CD3g protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto. In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3d protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto. In some embodiments of an antibody binding construct comprising two extracellular domains, the first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3e protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto, and the second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence encoding an ectodomain of a CD3g protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto.

[0384] When the antibody binding construct comprises two extracellular domains, said first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 59. When the antibody binding construct comprises two extracellular domains, said second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 60. When the antibody binding construct comprises two extracellular domains, said first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence

[0385] 71

[0386] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) identity to SEQ ID NO: 59, and said second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 60.

[0387] When the antibody binding construct comprises two extracellular domains, said first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 59. When the antibody binding construct comprises two extracellular domains, said second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 61. When the antibody binding construct comprises two extracellular domains, said first extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 59, and said second extracellular domain can be encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 61.

[0388] The choice of extracellular domain domain depends upon the intended target molecule. A preferred target molecule is an antibody, or binding domain(s) thereof. For example, the extracellular domain may be chosen to recognize (i.e., to bind to) an antibody that binds to a particular cell surface marker on a target cells, such as a tumor or cancer cell or otherwise infectious cell to be killed. For example, the extracellular domain may be chosen to recognize a multi-specific antibody that binds to a cell surface marker on a target cell associated with a particular disease state. Exemplary antibodies useful for immunotherapy are well known in the art. As described herein, bispecific antibodies that target CD3 and a tumor-associated antigen are well known in the art. A skilled artisan would understand that the extracellular domain can be engineered to comprise a target protein of a known multi-specific antibody.

[0389] In some embodiments, the extracellular domain binds to an antibody that also binds to a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest. The tumor antigen can comprise one or more antigenic cancer epitopes. Nonlimiting examples of tumor associated antigens include: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer);

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[0391] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018)

[0392] TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca- Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (R0R1); Fms-Like Tyrosine Kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (S SEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF- I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY- ESO-1); Cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1 A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer

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[0394] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos- related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen- 1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2

[0395] (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase- related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T Cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (UPV E6); human papilloma virus E7 (UPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of

[0396] IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). The antibody disclosed herein that binds to the extracellular domain can comprise a multi-specific antibody, such as a bispecific antibody, a tri-specific antibody, a tetra-specific antibody, or any configuration or fragment of a multi-specific antibody that binds to at least the extracellular domain and a target molecule. Any target molecule disclosed herein is envisaged within the scope of being bound by the antibody.

[0397] In some embodiments, the extracellular domain binds to an antibody that can also target an antigen present on the surface of a viral particle. Examples of viral particles include influenza virus, equine infectious anemia virus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), lassa fever virus, herpes simplex virus, varicella zoster virus, cytomegalovirus, epstein-barr virus, variola virus, adeno virus, papilloma virus, parvo virus,

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[0399] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) measles virus, mumps virus, respiratory syncytial virus, para influenza virus, corona virus, rubella virus, rabies virus, human T-cell lymphotropic virus, picoma virus, hepa DNA virus, flavivirus, deltavirus, calicivirus, polio virus, zika virus, west nile virus, SARS, rubella, norovirus, human papillomavirus, malaria, human T-lymphotropic virus, and / or helicobacter pylori.

[0400] Exemplary viral antigens include any surface protein and / or polypeptide present on the surface of the above listed viral particles. Examples of such surface proteins and / or polypeptides include: Zika capsid protein (C), Zika envelope protein (E), Zika precursor membrane protein (PrM), WNV glycoprotein E, WNV small membrane protein M, VZV glycoprotein E (gE), VZV gB, VZV gH, CoV nucleocapsid (N), CoV envelope (E), CoV membrane (M), Rubella El, Rubella E2, Norovirus Group VP1, HPV 11, HPV capsid protein LI, HPV capsid protein L2, Lassa Fever Virus GP1, Lassa Fever Virus GP2, influenza neuraminidase (NA, N1 to N11), influenza hemagglutinin (HA, Hl to H18), HTLV-1 Envelope, HTLV-1 gp21, HTLV-1 mosaic, HIV gpl20, HIV gp41, hepatitis A virus (HAV) capsid protein VP1, HAV capsid protein VP2, HAV capsid protein VP3, hepatitis B surface antigen (HbsAg), hepatitis B core antigen (HbcAg), herpes simplex virus glycoprotein (gB), herpes simplex virus glycoprotein (gC), and / or herpes simplex virus glycoprotein (gD).

[0401] In some embodiments, the extracellular domain binds to an antibody that can also target an antigen present on the surface of a pathogenic bacteria cell. One of skill would understand that the term pathogenic when used in the context of a microorganism, or product therefrom, refers to, for instance, a cell or molecule, that contributes to at least one symptom of a disease or disorder in a subject described herein. It would be understood that there are microorganisms present within a subject that are not considered pathogenic. In some instances, a microorganism, or product thereof, is pathogenic if it contributes to at least one symptom of a disease or disorder in a subject. Non-limiting examples of target bacteria include: Escherichia spp., Salmonella spp., Bacillus spp., Corynebacterium Clostridium spp., Clostridium spp., Pseudomonas spp., Clostridium spp., Lactococcus spp., Acinetobacter spp., Mycobacterium spp., Myxococcus spp., Staphylococcus spp., Streptococcus spp., or cyanobacteria. In some embodiments, non-limiting examples of bacteria include Escherichia coli, Salmonella enterica, Bacillus subtilis, Clostridium acetobutylicum, Clostridium ljungdahlii, Clostridium difficile, Acinetobacter baumannii, Mycobacterium tuberculosis, Myxococcus xanthus, Staphylococcus aureus, Streptococcus pyogenes, or cyanobacteria. In some embodiments, non-limiting examples of bacteria include Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Streptococcus pneumonia, carbapenem-resistant Enteroacteriaceae, Staphylococcus epidermidis, Staphylococcus salivarius,

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[0404] Corynebacterium minutissium, Cory neb acterium pseudodiphtheriae, Corynebacterium stratium, Corynebacterium group GI, Corynebacterium group G2, Streptococcus pneumonia, Streptococcus mitis, Streptococcus sanguis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia, Serratia marcescens, Haemophilus influenzae, Moraxella sp., Neisseria meningitidis, Neisseria gonorrhoeae, Salmonella typhimurium, Actinomyces spp., Porphyromonas spp., Prevotella melaninogenicus, Helicobacter pylori, Helicobacter felis, or Campylobacter jejuni.

[0405] An engineered immune cell of the disclosure can also be modified to express additional targeting ligands on the surface of the phagocytic cell in addition to the antibody binding constructs described herein. Additional targeting ligands comprise molecules configured to associate with any molecule presented on a target cell of interest, such as a target associated with an organ, a tissue, or an extracellular matrix of a target cell of interest. In some embodiments, additional targeting ligands bind to molecules associated with a particular state of a target cell, such as a cancerous condition. An additional targeting ligand can be specific to one target or be configured to bind multiple target molecules. Suitable target molecules recognized by an additional target ligand can include a protein (e.g., a receptor, a tumor marker, a transmembrane protein), a nucleic acid (e.g., DNA, RNA), and / or a carbohydrate (e.g., a monosaccharide, disaccharide, or polysaccharide) that is present on the surface of a target cell. Exemplary targeting ligands include an RGD-containing peptide, a small molecule (e.g., a peptide) mimetic ligand, and an antibody or antibody fragment specific for a particular target.

[0406] B. Transmembrane Domain

[0407] An antibody binding constructs of the disclosure comprise a transmembrane domain that connects the extracellular domain to the intracellular domain. In some embodiments, wherein the antibody binding constructs comprise an intracellular signaling domain, said transmembrane domain can connect the extracellular domain to the intracellular domain. The transmembrane domain can function to stabilize the antibody binding construct as a whole. The transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other molecules.

[0408] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,

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[0411] CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain can be from any type I transmembrane protein such as CD4, CD28 or HVEM or a functional fragment or variant thereof that retains co-stimulatory activity. In some embodiments, the transmembrane domain is a CD8 transmembrane domain or a functional fragment or variant thereof. In some embodiments, the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof. In some embodiments, the transmembrane domain is a CD28 transmembrane domain or a functional fragment or variant thereof. In some embodiments, the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof.

[0412] In some embodiments, the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 20. A functional fragment or variant of a CD8a transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 20. In some embodiments, a functional fragment of a CD8a transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 20.

[0413] In some embodiments, the transmembrane domain is a CD8a transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 33. A functional fragment or variant of a CD8a transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 33. In some embodiments, the CD8a transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 20, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 33. In some embodiments, the functional fragment or variant of a CD8a transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 20, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 33, or a polynucleotide sequence comprising a

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[0415] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 33.

[0416] In some embodiments, the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10. A functional fragment or variant of a HVEM transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10. In some embodiments, a functional fragment of a HVEM transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 10.

[0417] In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 34. A functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 34. In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 34. In some embodiments, the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 34, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 34.

[0418] In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 35. A functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

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[0421] 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 35. In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 35. In some embodiments, the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 35, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 35.

[0422] In some embodiments, the transmembrane domain is a HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 11. A functional fragment or variant of a HVEM transmembrane domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11. In some embodiments, a functional fragment of a HVEM transmembrane domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 11.

[0423] In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 36. A functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 36. In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 11, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 36. In some embodiments, the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO:

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[0426] 36, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 36.

[0427] In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 37. A functional fragment or variant of a HVEM transmembrane domain can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 37. In some embodiments, the HVEM transmembrane domain or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 11, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 37. In some embodiments, the functional fragment or variant of a HVEM transmembrane domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO:

[0428] 37, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 37.

[0429] The antibody binding constructs of the present disclosure can comprise more than one of any domain disclosed herein. For instance, an antibody binding construct can comprise more than one transmembrane domain. In such instances, the more than one transmembrane domain comprised within an antibody binding construct, or a polypeptide encoding the same, can be the same or different. For instance, an antibody binding construct can comprise more than one transmembrane domain. In some instances, an antibody binding construct can comprise two transmembrane domains. In such instances, an antibody binding construct can comprise a first transmembrane domain, and an at least second transmembrane domain. In some embodiments, said first transmembrane domain and said at least second transmembrane domain can comprise the same or can comprise different amino acid sequences. The transmembrane domains can be considered “different” by directly comparing the amino acid sequences comprised within the transmembrane domain. For instance, two transmembrane domains, when compared, may be considered different when they share no more than 90% sequence identity between them. In some instances, the extracellular domains share no more than 85%, 80%, 75%, 70%, 65%, 60%, 55%,

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[0432] 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1% sequence identity. A skilled artisan would understand that when an antibody binding construct comprises more than one transmembrane domain, it may be of particular interest to have two different transmembrane domains. In some embodiments, it may be beneficial for the two transmembrane domains to be the same, or have a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%. The antibody binding construct can comprise any transmembrane domain known in the art that maintains function of the antibody binding construct.

[0433] When the antibody binding construct comprises more than one transmembrane domain, the binding construct can comprise a first transmembrane domain derived from a first protein, and an at least second transmembrane domain derived from a second protein. In some embodiments of the antibody-binding construct, said antibody binding construct comprises two transmembrane domains. In such embodiments, the antibody binding construct comprises a first transmembrane domain and a second transmembrane domain, wherein said first transmembrane domain is derived from a first protein, or fragment thereof, or variant having at least 90% sequence identity thereto, and said second transmembrane domain derived from a second protein, or fragment thereof, or variant having at least 90% sequence identity thereto. When the antibody binding construct comprises two transmembrane domains, the first and / or second transmembrane domains can comprise an amino acid sequence encoding the transmembrane domain of any protein known in the art. When the antibody binding construct comprises two transmembrane domains, the first and / or second transmembrane domain can be derived from a protein, wherein said protein is alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. When the antibody binding construct comprises two transmembrane domains, the first transmembrane domain can be derived from a first protein, wherein said first protein is alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. When the antibody binding construct comprises two transmembrane domains, the second transmembrane domain can be derived from a second protein, wherein said second protein is alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154,

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[0436] HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof.

[0437] In some embodiments, wherein the antibody binding construct comprises two transmembrane domains, the first transmembrane domain can be derived from a first protein, wherein said first protein is alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof, and the second transmembrane domain can be derived from a second protein, wherein said second protein is alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. In such instances, said first surface protein or said at least second protein can be a CD8 protein, or functional fragment or variant thereof. In some instances, said first protein and said at least second protein are both CD8 proteins. In some instances, said first protein is CD8a. In some instances, said second protein is CD8a. In some instances, said first protein is HVEM. In some instances, said second protein is HVEM. In some instances, said first protein is CD8a, and the second protein is CD8a. In some instances, said first protein is CD8a, and the second protein is HVEM. In some instances, said first protein is HVEM, and the second protein is CD8a. In some instances, said first protein is HVEM, and the second protein is HVEM.

[0438] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20.

[0439] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. When the antibody binding construct comprises two

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[0441] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.

[0442] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11.

[0443] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.

[0444] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11.

[0445] When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. When the antibody binding construct comprises two transmembrane domains, said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. When the antibody binding construct comprises two transmembrane domains, said first transmembrane domain can comprise an amino acid sequence

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[0447] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) having at least 90% sequence identity to SEQ ID NO: 11, and said second transmembrane domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 l.In some embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge.

[0448] In some embodiments, the transmembrane domain may be a hydrophobic alpha helix that spans across the membrane of the cell (e.g., macrophage or T cell). The transmembrane domain can be naturally associated with one or more of the domains in the antibody binding construct.

[0449] In some embodiments, the transmembrane domain may be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. A triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain.

[0450] The antibody-binding construct disclosed herein can comprise: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein comprising an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) at least a first transmembrane domain, wherein said antibodybinding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of: i) a CD3d protein comprising an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or ii) a CD3g protein comprising an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

[0451] C. Intracellular Domain

[0452] An antibody binding construct expressed by an engineered immune cell of the disclosure can comprise at least one intracellular domain. In some embodiments, the antibody binding constructs disclosed herein can comprise more than one intracellular domains. The intracellular (z.e., cytoplasmic) domain can be based on, or derived from, the intracellular domain of any known receptor protein, such as a chimeric antigen receptor (CAR). A skilled artisan would understand that any intracellular domain that functions with the extracellular domain can be used in the antibody binding constructs disclosed herein. An intracellular domain, such as that of a CAR, is a signaling domain that transduces the event of receptor antigen binding to an intracellular signal that contributes to activation and / or transduction of signals in a cell in which

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[0454] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) the antibody binding construct is expressed. In some embodiments, absent appropriate costimulatory signals, this event is insufficient for useful cell activation and proliferation. Cell activation can include cytokine production, clonal proliferation, differentiation, and survival. A skilled artisan would understand that cell activation will be specific for each immune cell.

[0455] Examples of an intracellular domain for use in the disclosure include the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in a cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0456] Examples of an intracellular domain include a fragment or domain from one or more molecules or receptors including: T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), FcsRly (Fc Epsilon R1 gamma), Dectin- 1 (CLEC7A), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, CD27, CD28, 4- 1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 1 id, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof. In some embodiments, the intracellular signaling domain comprises a CD3z, 4-1BB, FcsRIy, PI3K, CD40, MyD88, TRIF, STING, RIG-1 , MDA5, Dectin- 1 (CLEC7A), or a FcsRly signaling domain, derivative, variant, or a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. In some embodiments, the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof. In some embodiments, the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof having an amino acid sequence set forth as SEQ ID NO: 15 or 16. A functional fragment or variant of a CDS zeta intracellular

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[0458] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) signaling domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ II) NO: 15 or 16. In some embodiments, a functional fragment or variant of a CD3 zeta intracellular signaling domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 15 or 16. In some embodiments, the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 32. In some embodiments, the intracellular signaling domain includes a CD3 zeta intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 32, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 32. CD3 zeta signaling domains are described in U.S. Patent No. 7,446,190 and U.S. Patent No. 8,911,993.

[0459] In some embodiments, the intracellular signaling domain includes a FceRly signaling domain or a functional variant thereof. In some embodiments, the intracellular signaling domain includes a FceRly signaling domain or a functional variant thereof having an amino acid sequence set forth as SEQ ID NO: 18. A functional fragment or variant of a FceRly signaling domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 18. In some embodiments, a functional fragment or variant of a FceRly signaling domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81 %, 80%, 79%, 78%, 77%, 76%, 75%, or less of the amino acid sequence set forth as SEQ ID NO: 18. In some embodiments, the intracellular signaling domain includes a FceRly signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 38. In some embodiments, the intracellular signaling domain includes a FceRly signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 38, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 38

[0460] In some embodiments, the intracellular signaling domain includes a Dectin- 1 intracellular signaling domain or a functional variant thereof. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain or a functional variant thereof of a Dectin- 1

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[0462] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) protein having an amino acid sequence set forth as SEQ ID NO: 14. A functional fragment or variant of a Dectin- 1 intracellular signaling domain can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an intracellular signaling domain of a Dectin- 1 protein having the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, a functional fragment or variant of a Dectin- 1 intracellular signaling domain can include a fragment that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%>, or less of the amino acid sequence of an intracellular signaling domain of a Dectin- 1 protein set forth as SEQ ID NO: 14. In some embodiments, the intracellular signaling domain includes a Dectin- 1 intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 49. In some embodiments, the intracellular signaling domain includes a Dectin- 1 intracellular signaling domain or a functional variant thereof encoded by the nucleotide sequence set forth as SEQ ID NO: 49, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; sequence identity to the amino acid sequence set forth as SEQ ID NO: 49.

[0463] The antibody binding constructs of the present disclosure can comprise more than one of any domain disclosed herein. For instance, an antibody binding construct can comprise more than one intracellular signaling domain. In such instances, the more than one intracellular signaling domain comprised within an antibody binding construct, or a polypeptide encoding the same, can be the same or different. For instance, an antibody binding construct can comprise more than one intracellular signaling domain. In some instances, an antibody binding construct can comprise two intracellular signaling domains. In such instances, an antibody binding construct can comprise a first intracellular signaling domain, and an at least second intracellular signaling domain. In some embodiments, said first intracellular signaling domain and said at least second intracellular signaling domain can comprise the same or can comprise different amino acid sequences. The intracellular signaling domains can be considered “different” by directly comparing the amino acid sequences comprised within the intracellular signaling domain. For instance, two intracellular signaling domains, when compared, may be considered different when they share no more than 90% sequence identity between them. In some instances, the extracellular domains share no more than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1% sequence identity. A skilled artisan would understand that when an antibody binding construct comprises more than one intracellular signaling domain, it may be of

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[0465] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) particular interest to have two different intracellular signaling domains. In some embodiments, it may be beneficial for the two intracellular signaling domains to be the same, or have a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%. The antibody binding construct can comprise any intracellular signaling domain known in the art that maintains function of the antibody binding construct.

[0466] When the antibody binding construct comprises more than one intracellular signaling domain, the binding construct can comprise a first intracellular signaling domain derived from a first protein, and an at least second intracellular signaling domain derived from a second protein. In some embodiments of the antibody-binding construct, said antibody binding construct comprises two intracellular signaling domains. In such embodiments, the antibody binding construct comprises a first intracellular signaling domain and a second intracellular signaling domain, wherein said first intracellular signaling domain is derived from a first protein, or fragment thereof, or variant having at least 90% sequence identity thereto, and said second intracellular signaling domain derived from a second protein, or fragment thereof, or variant having at least 90% sequence identity thereto. When the antibody binding construct comprises two intracellular signaling domains, the first and / or second intracellular signaling domains can comprise an amino acid sequence encoding the intracellular signaling domain of any protein known in the art. When the antibody binding construct comprises two intracellular signaling domains, the first and / or second intracellular signaling domain can be derived from a protein, wherein said protein is a CD3z, HVEM, 4- IBB, CD28, NKG2D, DAP 10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. When the antibody binding construct comprises two intracellular signaling domains, the first intracellular signaling domain can be derived from a first protein, wherein said first protein is a CD3z, HVEM, 4-1BB, CD28, NKG2D, DAP10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. When the antibody binding construct comprises two intracellular signaling domains, the second intracellular signaling domain can be derived from a second protein, wherein said second protein is a CD3z, HVEM, 4- IBB, CD28, NKG2D, DAP 10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof.

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[0469] In some embodiments, wherein the antibody binding construct comprises two intracellular signaling domains, the first intracellular signaling domain can be derived from a first protein, wherein said first protein is a CD3z, HVEM, 4- IBB, CD28, NKG2D, DAP 10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof, and the second intracellular signaling domain can be derived from a second protein, wherein said second protein is a CD3z, HVEM, 4- IBB, CD28, NKG2D, DAP 10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, a functional fragment or variant thereof having at least 90% sequence identity thereto, or any combination thereof. In such instances, said first protein or said at least second protein can be a CD3z protein, or functional fragment or variant thereof. In some instances, said first protein and said at least second protein are both CD3z proteins, or functional fragment or variant thereof. In some instances, said first protein is CD3z. In some instances, said second protein is CD3z.

[0470] In some instances, said first protein or said at least second protein can be a FcsRly protein, or functional fragment or variant thereof. In some instances, said first protein and said at least second protein are both FcsRly proteins, or functional fragment or variant thereof. In some instances, said first protein is FcsRly. In some instances, said second protein is FcsRly.

[0471] In some instances, said first protein is FcsRly. In some instances, said second protein is CD3z. In some instances, said first protein is CD3z. In some instances, said second protein is Dectin-1. In some instances, said first protein is FcsRly. In some instances, said second protein is Dectin-1.

[0472] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15.

[0473] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. When the antibody binding construct comprises two

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[0475] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

[0476] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0477] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a. Dectin- 1 protein set forth as SEQ ID NO: 14. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a Dectin- 1 protein set forth as SEQ ID NO: 14.

[0478] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID

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[0481] NO: 16, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

[0482] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a Dectin- 1 protein set forth as SEQ ID NO: 14. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a Dectin- 1 protein set forth as SEQ ID NO: 14.

[0483] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0484] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. When the antibody binding construct comprises two intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0485] When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. When the antibody binding construct comprises two

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[0487] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) intracellular signaling domains, said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a Dectin-1 protein set forth as SEQ ID NO: 14. When the antibody binding construct comprises two intracellular signaling domains, said first intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18, and said second intracellular signaling domain can comprise an amino acid sequence having at least 90% sequence identity to an intracellular signaling domain of a Dectin-1 protein set forth as SEQ ID NO: 14.

[0488] In some embodiments of the disclosure, the intracellular signaling domain of the antibody binding construct includes at least 1, at least 2, at least 3, at least 4, or at least 5 immunoreceptor tyrosine-based activation motifs (IT AMs). Generally, any intracellular signaling domain including an ITAM can be suitably used for the construction of antibody binding construct. An “IT AM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YXXL / I separated by 6-8 amino acids, wherein each X is independently any amino acid, producing the conserved motif YXXL / IX(6-8) YXXL / I (YXXL / IXXXXXXXXYXXL / I, SEQ ID NO: 39; YXXL / IXXXXXXXYXXL / I, SEQ ID NO: 62; YXXL / IXXXXXXYXXL / I, SEQ ID NO: 63). ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways. In some embodiments, the intracellular signaling domain includes at least 1, at least 2, at least 3, at least 4, or at least 5 ITAMs derived from CD3z, FcRy, Megfl 0, FcRy, and combinations thereof.

[0489] In some embodiments, the intracellular domain of the antibody binding construct useful in an engineered immune cell of the disclosure includes any portion of one or more co-stimulatory molecules. The intracellular domain used in the antibody binding construct can be selected based on the specific immune cell in which the antibody binding construct is expressed. For example, when the engineered immune cell is a monocyte, it will be useful for the antibody binding construct to comprise an intracellular domain that functions optimally within a monocyte. A skilled artisan would understand that an intracellular domain functions optimally when it contributes to activation and / or transduction of signals in a cell in which the antibody binding construct is expressed.

[0490] The antibody binding constructs disclosed herein can comprise an intracellular costimulatory signaling domain. Co-stimulation is a key event for T cells to exhibit effective effector

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[0492] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) functions, and is mediated by co-stimulatory molecules. In some embodiments, the co-stimulatory domain of a receptor, such as a CAR, is essential for promoting the intracellular signal to initiate CAR cell activation and proliferation. Thus, promotion of such a signal can depend upon the selected co-stimulatory signaling domain and / or combinations thereof. The co-stimulatory signaling domain(s) and / or combinations thereof used in the constructs disclosed herein can be derived from any known co-stimulatory signaling domain used in a CAR, for example, a second- generation or third-generation CAR. Second generation CARs incorporate an intracellular co- stimulatory signaling domain in addition to an intracellular signaling domain (e.g., CD3z, or FcsRly) and can enhance CAR T cell activation. Co-stimulatory molecules are divided into two major families; the CD28 family which includes CD28 and ICOS, and the tumor necrosis factor receptor superfamily (TNFRSF) which includes 4-1BB (TNFRSF9), CD27, CD30, DR3, GITR, 0X40, TNFR2 and herpes virus entry mediator (HVEM, TNFRSF14). So far, co-stimulatory domains derived from CD28 or 4- IBB have commonly been used to construct CARs. A previous study has shown that T cells expressing the second-generation CAR with the 4-lBB-derived co- stimulatory signaling domain persist for more than 6 months in the blood of most patients, whereas CAR-T cells with the CD28-derived co-stimulatory signaling domain become mostly undetectable after 3 months (Zhang et al, Oncotarget 6(32):33961 (2015)). In addition, 4-1BB- mediated co-stimulation selectively induced mitochondrial biogenesis and oxidative metabolism for energy production, resulting in enhanced differentiation and increased in vitro persistence of central memory T cells (Kawalekar et al, Immunity 44(2):380 (2016)). Moreover, 4-lBB-mediated co-stimulation averts T cell exhaustion induced by tonic signaling (Long et al, Nat. Med.

[0493] 21(6):581 (2015)). Therefore, the co-stimulatory signaling domain derived from the TNFRSF appears to function better than the one from the CD28 family in the context of second-generation CAR. Third-generation CARs incorporate two co-stimulatory domains and can promote CAR-T cell proliferation, reduce CAR-T cell apoptosis, and / or increase the the NF- K B pathway (Dai et al. (2020) Frontiers in Immunology 11 :539654).

[0494] HVEM, another member of the TNFRSF, is the main receptor targeted by the Herpes simplex virus to enter host cells (Sedy et al. (2005) Nature Immunology 6(1): 90-98). It was previously described as ATAR (Another TRAF- Associated Receptor) (Hsu et al. (1997) Journal of Biological Chemistry 272(21): 13471-13474) and is also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14). A human HVEM is 283 amino acids in length (UniProt ID Q92956-1; SEQ ID NO: 40), comprising: an extracellular region that includes amino acid residues 39-202; a transmembrane region that includes amino acid residues 203-223; and a cytoplasmic

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[0496] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) region that includes amino acid residues 224-283. HVEM plays a role in effector CD8+T cell effector function and memory T cell development. HVEM deficiency in CD8+T cells is shown to profoundly impair effector CD8+T cell survival and development of protective immune memory (Flynn et al, PLoS One 8(10):e77991 (2013)). B and T lymphocyte attenuator (a ligand of HVEM) interaction with HVEM expressed on CD8+T cells was also reported to promote survival and memory generation in response to a bacterial infection (Steinberg et al., PLoS One 8(10):e77992 (2013)). Additionally, tumor cells which express anti -HVEM single chain antibody induce a potent proliferation and cytokine production of co-cultured T cells (Park et al, Cancer Immunol. Immunother. 61(2):203 (2012)), suggesting HVEM might serve as a potent co-stimulatory signaling entity in T cells.

[0497] HVEM is expressed in peripheral T and B cells, and in resting T and B cells (Ning et al. (2021) Frontiers in Immunology 12:654960). HVEM is constitutively expressed in naive T cells. The HVEM receptor has both stimulatory and inhibitory effects depending on its ligand, and at least five different ligands have been described (Cai & Freeman (2009) Immunological Reviews 229(l):244-258; Del Rio et al. (2010) Journal of Leukocyte Biology 87(2):223-235; Granger & Rickert (2003) Cytokine Growth Factor Rev 14(3-4):289-296; Pasero & Olive (2013) Immunology Letters 151 (1 -2):71 -75; Sorobetea & Brodsky (2018) Cell Host & Microbe 24(2): 187-188). For example, in an inflammatory disease model setting, HVEM binding to the LIGHT ligand of the TNF family and lymphotoxin alpha (LTa) produces a co-stimulatory signal, leading to increased T cell proliferation in vitro and formation of effector and memory T cells (Del Rio et al. (2010) Journal of Leukocyte Biology 87(2):223-235). However, HVEM can generate an inhibitory signal when bound to BTLA or CD 160 (Pasero & Olive (2013) Immunology Letters 151 (1 -2):71 -75). The overexpression of BTLA / HVEM on T cells can contribute to T cell exhaustion (Ning et al. (2021) Frontiers in Immunology 12:654960; Shui et al. (2011) Journal of Leukocyte Biology, 89(4), 517-523).

[0498] The cytoplasmic domain of HVEM is 60 amino acids in length and includes an a-helix, a TRAF domain, and a tail (amino acid residues 224-283 of SEQ ID NO: 4; Hennecke, Derek. 2022. A Comparison of Co-stimulatory HVEM Domains in Second Generation CAR-T Cells. Master's thesis, Harvard University Division of Continuing Education). M83 interacts with adapter proteins, including TRAFs (TRAF5 and TRAF2) (Hsu et al. (1997) Journal of Biological Chemistry 272(21): 13471-13474), which are important signaling molecules downstream of TNF receptors, connecting receptor signaling with kinase (e.g., IKB kinase) activation, which leads to transcription factor (e.g., AP-1, NF AT, NF-KB) activation (Zamegar et al. (2008) Nature

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[0501] Immunology 9(12): 1371-1378). Trancription factor activation is important, for example, in T cell priming and control of tumors in vivo (Barnes et al. (2015) J for Immunotherapy of Cancer 3(1): 1- H).

[0502] In the present disclosure, the term M83 refers to a co-stimulatory signaling domain from an HVEM protein. The co-stimulatory signaling domain from an HVEM protein can include all or part of the transmembrane domain along with all or part of the intracellular region, or can include only all or part of the intracellular region of the HVEM protein. An antibody binding construct comprising an intracellular co-stimulatory signaling domain from an HVEM protein, or a functional fragment or variant thereof that retains co-stimulatory activity, can promote greater phagocytosis in a phagocytic cell as compared to a antibody binding construct comprising a different co-stimulatory signaling domain (e.g., 4-1BB) or as compared to an antibody binding construct lacking a co-stimulatory signaling domain.

[0503] The ‘VEET’ domain of the HVEM protein (corresponding with amino acid positions 269- 272 of SEQ ID NO: 40) is thought to be important for co-stimulatory activity of the HVEM protein (see Ye et al., “The Structural Basis for the Recognition of Diverse Receptor Sequences by TRAF2, Molecular Cell, Vol.4, p. 321-330; and Hsu et al, “ATAR, A Novel Tumor Necrosis Factor Receptor Family Member, Signals through TRAF2 and TRAF5, The Journal of Biological Chemistry, Vol. 272, No. 21, 1997, p. 13471-13474, the contents of which are herein incorporated by reference in their entirety). Without wishing to be bound by theory, it is possible that the VEET domain within the HVEM co-stimulatory signaling domains disclosed herein is important, if not required, to promote co-stimulatory activity. An HVEM protein intracellular co-stimulatory signaling domain, or a functional fragment or variant thereof is thought to retain its co-stimulatory activity so long as it functions to enhance or heighten the response of a engineered immune cell having an antibody binding construct comprising said co-stimulatory domain, or functional fragment or variant thereof, compared to an antibody binding construct lacking an intracellular co- stimulatory signaling domain from an HVEM protein.

[0504] The antibody binding constructs disclosed herein can comprise a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, wherein the encoded domain retains co-stimulatory activity. In some embodiments, wherein the antibody binding construct comprises an HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

[0505] 95

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[0507] 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least one residue corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272).

[0508] In some embodiments, wherein the antibody binding construct comprises an HVEM costimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least one residue corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least one residue selected from the following residues corresponding with SEQ ID NO: 40: V269, E270, E271, or T272.

[0509] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least two residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least two residues selected from the following residues corresponding with SEQ ID NO: 40: V269, E270, E271, or T272.

[0510] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least three residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, can comprise at least three residues selected from the following residues corresponding with SEQ ID NO: 40: V269, E270, E271, or T272.

[0511] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%,

[0512] 96

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[0514] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises the following residues corresponding to SEQ ID NO: 40: V269, E270, E271, or T272.

[0515] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises a V269, corresponding with residue 269 of SEQ ID NO: 40.

[0516] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises a E270, corresponding with residue 270 of SEQ ID NO: 40.

[0517] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or

[0518] 97

[0519] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) functional fragment or variant thereof, comprises a E271, corresponding with residue 271 of SEQ ID NO: 40.

[0520] In some embodiments, wherein the antibody binding construct comprises an HVEM costimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises a E272, corresponding with residue 272 of SEQ ID NO: 40.

[0521] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least one, at least two, at least three, or at least four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272).

[0522] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises a V269, corresponding with residue 269 of SEQ ID NO: 4; a E270, corresponding with residue 270 of SEQ ID NO: 40; a E271, corresponding with residue 271 of SEQ ID NO: 4; and a E272, corresponding with residue 272 of SEQ ID NO: 40.

[0523] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four

[0524] 98

[0525] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least of one of the following: a V269, corresponding with residue 269 of SEQ ID NO: 40; a E270, corresponding with residue 270 of SEQ ID NO: 40; a E271, corresponding with residue 271 of SEQ ID NO: 40; and a E272, corresponding with residue 272 of SEQ ID NO: 40.

[0526] In some embodiments, wherein the antibody binding construct comprises an HVEM costimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8 or 9, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises at least of one of the following: a V269, corresponding with residue 269 of SEQ ID NO: 40; a E270, corresponding with residue 270 of SEQ ID NO: 40; a E271, corresponding with residue 271 of SEQ ID NO: 40; and a E272, corresponding with residue 272 of SEQ ID NO: 40.

[0527] In some embodiments, wherein the antibody binding construct comprises an HVEM co- stimulatory signaling domain, or functional fragment or variant thereof, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoding SEQ ID NO: 8 or 9, said HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises four residues corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40 (i.e., V269, E270, E271, T272). In such embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, comprises a V269, corresponding with residue 269 of SEQ ID NO: 40; a E270, corresponding with residue 270 of SEQ ID NO: 40; a E271, corresponding with residue 271 of SEQ ID NO: 40; and a E272, corresponding with residue 272 of SEQ ID NO: 40.

[0528] In some embodiments, the antibody binding construct comprising a co-stimulatory domain having at least 90% sequence identity to an HVEM co-stimulatory protein set forth as any one of SEQ ID NOs: 8 or 9, or a functional fragment or variant thereof that retains co-stimulatory activity will have a domain comprising amino acid residues VEET corresponding with amino acids at positions 269 to 272 of SEQ ID NO: 40. In some embodiments, the HVEM co-stimulatory protein

[0529] 99

[0530] 4898-2128-9826vl Attorney Docket No.: 1115814 1040WO (00018) of the present disclosure comprises a valine, or functional variant thereof, at an amino acid residue corresponding with position 269 of SEQ ID NO: 40. In some embodiments, the HVEM costimulatory protein of the present disclosure comprises a glutamic acid, or functional variant thereof, at an amino acid residue corresponding with position 270 of SEQ ID NO: 40. In some embodiments, the HVEM co-stimulatory protein of the present disclosure comprises a glutamic acid, or functional variant thereof, at an amino acid residue corresponding with position 271 of SEQ ID NO: 40. In some embodiments, the HVEM co-stimulatory protein of the present disclosure comprises a threonine, or functional variant thereof, at an amino acid residue corresponding with position 272 of SEQ ID NO: 40.

[0531] A nucleic acid sequence disclosed herein can encode a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 8, wherein the encoded domain retains co-stimulatory activity. In some embodiments, a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, has at least 90% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 0, wherein the encoded domain retains co-stimulatory activity. In some embodiments, a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, of the disclosure, has at least 95% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 0, wherein the encoded domain retains co-stimulatory activity. In some embodiments, a nucleic acid sequence encoding a HVEM co-stimulatory signaling domain of the disclosure has 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO: 0. In some embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide sequence set forth as SEQ ID NO: 41. In some embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide sequence set forth as SEQ ID NO: 41, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 41. In some embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide sequence set forth as SEQ ID NO: 42. In some embodiments, the HVEM co-stimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide sequence set forth as SEQ ID NO: 42, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

[0532] 100

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[0534] 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 42. In some embodiments, the HVEM costimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide sequence set forth as SEQ ID NO: 43. In some embodiments, the HVEM costimulatory signaling domain, or functional fragment or variant thereof, is encoded by the nucleotide s...

Claims

Attorney Docket No.: 1115814 1040WO (00018)THAT WHICH IS CLAIMED:

1. An antibody -binding construct, comprising: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto; and b) at least a first transmembrane domain, wherein said antibody-binding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of: i) a CD3d protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto; or ii) a CD3g protein, or fragment, or variant thereof having an amino acid sequence having at least 90% amino acid sequence identity thereto.

2. The antibody-binding construct of claim 1, wherein said CD3e ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2.

3. The antibody -binding construct of claim 1 or 2, wherein said at least first extracellular domain further comprises said amino acid sequence encoding a second ectodomain.

4. The antibody-binding construct of claim 1 or 2, wherein said antibody binding construct further comprises a second extracellular domain.

5. The antibody -binding construct of claim 4, wherein said second extracellular domain comprises said amino acid sequence encoding a second ectodomain.

6. The antibody -binding construct of any one of claims 1-5, wherein said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4.2714898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)7. The antibody -binding construct of any one of claims 1-5, wherein said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6.

8. The antibody -binding construct of any one of claims 1-7, wherein said CD3e ectodomain or fragment or variant thereof and said second ectodomain form a heterodimer.

9. The antibody-binding construct of any one of claims 1-8, wherein said CD3e ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, and said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 4.

10. The antibody-binding construct of any one of claims 1-8, wherein said CD3e ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 2, and said second ectodomain comprises an amino acid sequence set forth as SEQ ID NO: 6.

11. The antibody -binding construct of any one of claims 1-10, wherein said antibody binding construct comprises a first polypeptide comprising said at least first extracellular domain and said at least first transmembrane domain.

12. The antibody-binding construct of claim 11, wherein said antibody-binding construct further comprises a second polypeptide comprising said second extracellular domain.

13. The antibody -binding construct of any one of claims 1-12, wherein said antibodybinding construct further comprises at least a first intracellular signaling domain.

14. The antibody-binding construct of claim 13, wherein said at least first intracellular signaling domain comprises a signaling domain of a CD3z, HVEM, 4- IBB, CD28, NKG2D, DAP10, PI3K, CD40, MyD88, TRIF, STING, RIG-1, MDA5, Dectin-1, or a FcsRly protein, or a functional fragment or variant thereof having at least 90% sequence identity thereto.2724898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)15. The antibody -binding construct of any one of claims 1-14, wherein said antibodybinding construct further comprises at least a first intracellular co-stimulatory signaling domain.

16. The antibody -binding construct of claim 15, wherein said at least first co-stimulatory intracellular signaling domain comprises an amino acid sequence having at least 90% sequence identity to a cytoplasmic domain of a herpes virus entry mediator (HVEM) protein set forth as SEQ ID NO: 8 or 9, or a functional fragment or variant thereof that retains co-stimulatory activity.

17. The antibody -binding construct of any one of claims 1-16, wherein said at least first transmembrane domain is derived from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or NKG2D, or a functional fragment or variant thereof having at least 90% sequence identity thereto.

18. The antibody -binding construct of any one of claims 1-17, wherein said antibodybinding construct comprises: i) an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 22, 23, or 24; ii) a first amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 22, and a second amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 23; iii) a first amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NOs: 22, and a second amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 24; iv) the amino acid sequence set forth as any one of SEQ ID NOs: 22, 23, or 24; v) a first amino acid sequence set forth as SEQ ID NO: 22, and a second amino acid sequence set forth as SEQ ID NO: 23; or vi) a first amino acid sequence set forth as SEQ ID NO: 22, and a second amino acid sequence set forth as SEQ ID NO: 24.2734898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)19. A nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said antibody binding construct of any one of claims 1-18, said first polypeptide of any one of claims 11-18, or said second polypeptide of any one of claims 12-18.

20. The nucleic acid molecule of claim 19, wherein said nucleic acid molecule comprises RNA, such as mRNA.

21. A vector comprising at least a first nucleic acid molecule, wherein said first nucleic acid molecule comprises the nucleic acid molecule of claim 19 or 20.

22. The vector of claim 21, wherein said vector further comprises an effector molecule expression cassette comprising a polynucleotide sequence encoding said effector molecule.

23. The vector of claim 22, wherein said effector molecule comprises an antibody, or binding fragment(s) thereof.

24. The vector of claim 23, wherein said antibody comprises a multi-specific or multivalent antibody, such as a bispecific, a trispecific, or tetraspecific antibody.

25. The vector of any one of claims 22-24, wherein said antibody binding construct and said effector molecule are encoded by a single nucleic acid molecule.

26. The vector of any one of claims 21-25, further comprising at least a second nucleic acid molecule.

27. The vector of claim 26, wherein said at least second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence encoding said second polypeptide or said effector molecule.

28. The vector of claim 27, wherein said vector comprises a first nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said first polypeptide and a second nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said second polypeptide.2744898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)29. The vector of any one of claims 26-28, further comprising at least a third nucleic acid molecule.

30. The vector of claim 29, wherein said vector comprises a first nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said first polypeptide and a second nucleic acid molecule comprising a polynucleotide comprising a nucleotide sequence encoding said second polypeptide.

31. The vector of any one of claims 21-30, wherein said vector comprises: i) a single-stranded mRNA or a single-stranded or double-stranded DNA; ii) a plasmid, a cosmid, or a transposon; iii) a viral vector; iv) a nano-particle, such as a virus-like nanoparticle or a lipid nanoparticle; and / or v) a liposome.

32. An engineered immune cell comprising the antibody-binding construct of any one of claims 1-18, the nucleic acid molecule of claim 19 or 20, or of the vector of any one of claims 21-31.

33. The engineered immune cell of claim 32, wherein said ectodomain of a CD3e protein or fragment or variant thereof, and / or said second ectodomain is absent on said immune cell in nature, or is present on the surface of said immune cell in lower amounts in the absence of said antibody -binding construct, such as the cell in nature, as compared to said engineered immune cell.

34. An engineered immune cell expressing an antibody-binding construct on the surface of the cell, wherein said antibody-binding construct comprises: a) at least a first extracellular domain having an amino acid sequence encoding an ectodomain of a CD3e protein comprising an amino acid sequence set forth as SEQ ID NO: 2, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and2754898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018) b) at least a first transmembrane domain, wherein said antibody-binding construct further comprises an amino acid sequence encoding a second ectodomain having an amino acid sequence of i) a CD3d protein comprising an amino acid sequence set forth as SEQ ID NO: 4, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or ii) a CD3g protein comprising an amino acid sequence set forth as SEQ ID NO: 6, or a fragment or variant thereof having an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6, and wherein said immune cell is a monocyte, macrophage, dendritic cell, neutrophil, or NK cell, or precursor thereof.

35. The engineered immune cell of claim 34, wherein said antibody -binding construct is bound or associated with a multi-specific antibody capable of binding to said CD3e on the surface of the cell.

36. The engineered immune cell of claim 35, wherein said multi-specific antibody is further bound or associated with an antigen on the surface of a tumor cell.

37. The engineered immune cell of claim 35, further comprising a nucleic acid sequence encoding said multi-specific antibody.

38. A cell population comprising a plurality of the engineered immune cell of any one of claims 32-37.

39. A pharmaceutical composition comprising the antibody-binding construct of any one of claims 1-18, the nucleic acid molecule of claim 19 or 20, the vector of any one of claims 21- 31, the engineered immune cell of any one of claims 32-37, or the cell population of claim 38, and a pharmaceutically acceptable carrier.

40. A method of stimulating a biological response in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 39.2764898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)41. The method of claim 40, wherein said stimulation of a biological response: i) increases in vivo cytokine production in a tumor microenvironment in said subject; ii) reduces antigen escape of a tumor cell in said subject; iii) increases epitope spreading in said subject; iv) increases pathogenic cell killing, such as tumor cell killing, in said subject; v) stimulates a therapeutic vaccination response in said subject; vi) associates a monocyte or macrophage cell with a tumor cell in said subject; and / or vii) reduces a symptom associated with cancer, inflammation, an inflammatory disease, autoimmunity, an autoimmune disease, a neurodegenerative disease, a vascular disease, an ocular disease, a hematologic disorder and / or an infection.

42. The method of claim 40 or 41, wherein said method comprises administering at least a second therapy.

43. The method of claim 42, wherein said at least second therapy comprises an antibody therapy, a vaccine, a T-cell based therapy, and / or bispecific T cell engager (BiTE®) therapy.

44. A method of expressing an antibody-binding construct on the surface of an immune cell, said method comprising introducing into said cell at least said nucleic acid molecule of claim 19 or 20 or said vector of any one of claims 21-31, thereby producing an engineered immune cell expressing said antibody-binding construct on the surface of the cell.

45. The method of any one of claims 40-44, whereby: i) expression of at least one gene in the engineered immune cell is increased compared to a control cell; ii) an immune response is stimulated in the engineered immune cell compared to a control cell; and / or iii) phagocytosis and / or trogocytosis activity of the engineered immune cell is enhanced compared to a control cell, wherein said control cell is said immune cell lacking said at least first nucleic acid molecule.2774898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018)46. The method of claim 45, wherein said gene is a pro-inflammatory cytokine gene or anti-inflammatory cytokine gene of said engineered immune cell.

47. The method of claim 46, whereby secretion of an effector molecule from said engineered immune cell is increased compared to a control cell.

48. The method of claim 47, wherein said effector molecule comprises a small molecule, an enzyme, a cytokine, a cytotoxin, a granzyme, a checkpoint inhibitor, an antibody, a polynucleotide, a peptide, a polypeptide, an antibiotic, an anti-viral, an anti-fungal, an antiinflammatory, or a derivative thereof, or an agent comprising any combination thereof.

49. The method of claim 47 or 48, wherein said effector molecule promotes increased expression of at least one gene of an at least second immune cell in said subject in vivo.

50. The method of claim 49, wherein said at least second immune cell is a T cell, DC cell, NK cell, neutrophil, monocyte, or macrophage.

51. A method of making an engineered immune cell comprising an antibody -binding construct expressed on the surface of said cell, said method comprising contacting a target immune cell with the nucleic acid molecule of claim 19 or 20, or the vector of any one of claims 21-31.

52. The method of claim 51, wherein said target immune cell is an isolated immune cell, or precursor thereof.

53. The method of claim 51, wherein said contact occurs in vivo.

54. The method of any one of claims 51-53, wherein said target immune cell is a T cell, a monocyte, a macrophage, a dendritic cell, a neutrophil, a mast cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, an eosinophil, a basophil, a myeloid cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a hematopoietic stem cell (HSC), a HSC-derived cell, a precursor cell, a bone marrow cell, a2784898-2128-9826vlAttorney Docket No.: 1115814 1040WO (00018) stem cell, a pluripotent stem cell, a mesenchymal stromal cell (MS SC), and induced pluripotent stem cell (iPSC), or an iPSC-derived cell.

55. A method of making the engineered immune cell of claim 34, said method comprising contacting said immune cell with a vector comprising at least a first nucleic acid molecule encoding said antibody-binding construct.2794898-2128-9826vl

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