Anti-CD28 antibodies

Abstract

Provided are anti-CD28 binders, their inclusion in multispecific constructs, and methods for use and manufacture thereof.

Description

ANTLCD28 ANTIBODIESSEQUENCE LISTING

[0001] The present application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 4, 2025, is named CROS-006-01WO-Sequence-Listing.xml and is 209 KB in size.BACKGROUND

[0002] Cluster of Differentiation 28 (CD28) is a protein expressed on the surface of T-cells. It is a member of the B7 receptors, that provides co-stimulatory signals that promote T-cell activation and survival. T-cell stimulation involving CD28 leads to increased production of certain interleukins and secretion of other co-factors promoting a T-cell immune response, which for example, may provide positive signals to overcome immune checkpoints. The CD28 molecule is expressed on most resting T cells, where it naturally binds to ligands on the surface of other cells of the immune system to promote the co-stimulatory reaction. Recently, CD28 stimulation has been investigated in conjunction with immunotherapies.

[0003] Evidence has emerged that certain anti-CD28 antibodies, when provided in conjunction with a T-cell receptor (TCR) antigen, may simulate and thereby cause the CD28 co-stimulatory response. Studies of agonistic CD28 antibodies demonstrate that promoting the CD28 co-stimulatory response results in, for example, anti-tumor responses caused by the activation of T-cells that target tumor cells.

[0004] Given the critical role of CD28 in emerging immunotherapies, developing anti-CD28 binders with various CD28 affinities provides a promising path to regulating T-cell activation, whether in the context of an immunotherapy or in producing cellular products ex vivo.BRIEF SUMMARY

[0005] The Summary is provided to introduce a selection of concepts that are further described in the Detailed Description below. This Summary is not intended to identify key or essential featuresof the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0006] Provided herein are novel anti-CD28 binders, including as part of larger bi specific or multispecific constructs. Various embodiments of CD28 binders, antibodies, and fragments thereof are provided, which have diverse anti-CD28 affinities. As the presently disclosed CD28 binders each have a different anti-CD28 affinity, they can be selectively employed and / or incorporated into different therapies and uses (e.g., as part of a multispecific antibodies such as a T-cell engagers, used or included as part of co-therapeutic, used for in vivo or ex vivo immune cell activation, and other uses as described herein), as the different binders are able to provide a desired level of anti-CD28 affinity depending on a particular use. As such, different CD28 binders of the disclosure each possess the optimum anti-CD28 affinity for different uses. Although a strong anti-CD28 affinity is often desired in a therapy incorporating a CD28 binder, some therapies benefit from anti-CD28 binders with intermediate or low anti-CD28 affinities. For example, reducing the relative anti-CD28 affinity can widen the therapeutic window of a potential therapy. Similarly, especially when incorporated into a multi-specific antibody, the ideal anti-CD28 may be dependent upon an interaction with other binders and / or immune cell components, e.g., a pMHC protein complex, CD3, and / or other tumor antigens. Similarly, higher CD28 affinities may result in high maximum cytokine release, with high potency, but may cause a potentially severe toxicity. Accordingly, various anti-CD28 binders of the disclosure each represent a different optimum CD28 affinity.

[0007] The presently disclosed CD28 binders may be themselves or incorporated within antibodies, such as those exemplified herein. Such antibodies that are capable of specifically binding to CD28, each with a different affinity. Such antibodies can, for example, be monoclonal, synthetic / recombinant, or other type of antibody. In certain aspects, the CD28 antibodies are used in a multispecific and / or multivalent antibody, such as T-cell engagers and various bi-, tri- and higher multi-specific antibodies.

[0008] In the immune system, T cells recognize antigens via the T cell receptor (TCR). The TCR recognizes antigen surface epitopes indirectly through antigen-peptide-MHC molecular complexes (pMHC) found on the surface of antigen-presenting cells or target cells (e.g., tumor cells and autoreactive B-cells). A cytotoxic T cell response is initiated by a primary signal, also referred to as Signal 1, which is induced by the TCR binding to the pMHC by molecular complexes of theTCR and CD3 Without wishing to be bound by any particular theory, it is presumed that the Signal 1 activation signal is generated by antigen recognition by the TCR, which is transduced into the T cell by the CD3 peptide chain.

[0009] However, generally, Signal 1 alone is not sufficient to promote a full T cell activation and corresponding immune response. Without co-stimulatory signals, immune responses promoted solely by Signal 1 induce T cell exhaustion, anergy, senescence, and may impair T cell activation, proliferation, differentiation and clonal expansion. In order to induce full T cell activation, an additional co-stimulatory signal is required, which is referred to as “Signal 2”. Co-stimulatory signals, such as those produced by B7 proteins that interact with CD28 costimulatory receptors binding on an immune cell, induce the Signal 2 response, which leads to T cell activation. The presently disclosed anti-CD28 binders of the disclosure may be used in methods and compositions, as disclosed herein, to provide a targeted Signal 2 costimulatory response to induce full immune cell activation. For example, the anti-CD28 binders of the disclosure find utility when incorporated into a bispecific constructs, that are able to bind to both CD28 on an immune cell (e.g., a T cell) and an additional target antigen. Such target antigens may, for example, be expressed by a target cell. In certain aspects, the target cell is an immune cell (e.g., a T cell) and the additional target antigen is expressed by the immune cell. Such bispecific constructs may find utility, for example, in identifying or activating immune cells (e.g., using CD3 as the additional target antigen). As an alternative, for example, the target cell may be a cell associated with a disease or condition, e.g., a tumor cell or autoreactive immune cell, which expresses the target antigen.

[0010] The present disclosure further includes the use of the anti-CD28 binders in other multispecific formats, e.g., trispecific and tetraspecific formats. For example, a multispecific construct of the disclosure may include an anti-CD28 binder and one or more binder for a first target antigen and one or more binder for a second target antigen. In certain aspects, the first and / or second target antigen is expressed on immune cells (e.g., T cells, B cells, NK cells, myeloid cells, and other leukocyte subsets). In such aspects, by binding to CD28 and one or more other targets on an immune cell (e.g., CD3), multispecific constructs of the disclosure may serve as cis-acting antibodies, which target multiple targets on the same cell, thereby providing a means to modulate binding, selectivity, and / or function of the cell. In certain aspects, the multispecific construct is a cis-targeting antibody, and the first and second target antigens are expressed on an immune cell. In certain aspects, the first and / or the second target antigen is expressed by a target cell (e.g., atumor cell). Tn certain aspects, the multispecific construct is a cis targeting antibody, and the first and second target antigens are expressed on a target (e.g., disease) cell. In certain aspects, the first and / or second target antigen is a soluble target (e.g., disease biomarkers, cytokines, growth factors, and other circulating mediators associated with a pathological process).

[0011] In certain aspects, a multispecific construct of the disclosure may include an anti-CD28 binder and one or more binder for a first target antigen and one or more binder for a second target antigen, and one or more binder for a third target antigen. In certain aspects, the first, second, and / or third target antigen is expressed on immune cells. In certain aspects, the first, second, and / or third target antigen expressed by a target cell (e.g., a tumor cell). In certain aspects, the first, second, and / or third target antigens are expressed on a diseased cell (e.g., tumor cells, virally infected cells, autoimmune effector cells, or pathogenic stromal cells). In certain aspects, the first, second, and / or third target antigen is a soluble target (e.g., disease biomarkers, cytokines, growth factors, and other circulating mediators associated with a pathological process).

[0012] In some embodiments, the anti-CD28 antibody or antigen-binding fragment of the disclosure is a fully humanized antibody or antigen-binding fragment. In some embodiments, anti-CD28 antibody or antigen-binding fragment of the disclosure is a chimeric or synthetic antibody or antigen-binding fragment.

[0013] In certain aspects, the present disclosure provides immunoconjugates that comprise an anti-CD28 binder of the disclosure and an effector molecule, e.g., a drug moiety. Further provided herein are antibody-drug conjugates (ADCs) that include a drug conjugated to an anti-CD28 antibody or antigen-binding fragment disclosed herein. In some embodiments, the drug is a small molecule, for example an anti -microtubule agent, an anti-mitotic agent and / or a cytotoxic agent.

[0014] The present disclosure provides, anti-CD28 binders incorporated into multispecific antibodies, such as T cell engagers (TCE) as described herein, and methods of using such antibodies, e.g., for the treatment of cancers. In certain aspects, the subject anti-CD28 binders and antibodies agonistically bind to CD28 costimulatory molecules on T cells, thereby promoting a co-stimulatory response. Thus, the presently disclosed antibodies may be used to selectively enhance, for example, anti-tumor activity of a redirected T-cell. The subject antibodies provided herein are particularly useful when included in TCEs, where their differing affinities for CD28 can be used to fine-tune the activity of the larger construct.

[0015] In certain aspects, the disclosure provides an antibody or antigen binding portion thereof that specifically binds to CD28, (e.g., on an immune cell), wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NO: 158-162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 153-157, or a fragment thereof. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 158-162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 153-157, or a fragment thereof. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is identical to any of SEQ ID NOS: 158-162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to any of SEQ ID NOS: 153-157, or a fragment thereof.

[0016] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region consisting of an amino acid sequence that is at least 90% identical to any of SEQ ID NOS: 158-162, or a fragment thereof, and a light chain variable region consisting of an amino acid sequence that is at least 90% identical to any of SEQ ID NOS: 153-157, or a fragment thereof. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region consisting of an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 158-162, or a fragment thereof, and a light chain variable region consisting of an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 153-157, or a fragment thereof. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises a heavy chain variable region consisting of an amino acid sequence that is identical to any of SEQ ID NOS: 158-162, or a fragment thereof, and a light chain variable region consisting of an amino acid sequence identical to any of SEQ ID NOS: 153-157, or a fragment thereof.

[0017] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 153, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 154, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 160 or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 155, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 156, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 157, or a fragment thereof.

[0018] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 153, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 154, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 160, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 155, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 156, or a fragment thereof; or a heavy chain variable regioncomprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 157, or a fragment thereof.

[0019] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 153, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 154, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 160, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 155, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 156, or a fragment thereof; or a heavy chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is identical to SEQ ID NO: 157, or a fragment thereof.

[0020] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence, wherein: the VH CDR1 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 178-182; the VH CDR2 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 183-187; and / or the VH CDR3 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 188-192. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28 on an immune cell, wherein the antibody or antigen binding portion thereof comprises: a light chain variable region comprising a CDR1, CDR2, and CDR3 sequence, wherein: the VL CDR1 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 163-167; the VL CDR2 sequence comprises an amino acid sequence that is at least 85% identical toany one of SEQ ID NOS: 168-172; and / or the VL CDR3 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 173-177. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28 on an immune cell, wherein the antibody or antigen binding portion thereof comprises: heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence and a light chain variable region comprising a CDR1, CDR2, and CDR3 sequence wherein: the VH CDR1 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 178-182; the VH CDR2 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 183-187; and / or the VH CDR3 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 188-192; and, the VL CDR1 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 163-167; the VL CDR2 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 168-172; and / or the VL CDR3 sequence comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 173-177.

[0021] The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence, wherein: the VH CDR1 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 178-182; the VH CDR2 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 183-187; and / or the VH CDR3 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 188-192. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: a light chain variable region comprising a CDR1, CDR2, and CDR3 sequence, wherein: the VL CDR1 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 163-167; the VL CDR2 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 168-172; and / or the VL CDR3 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 173-177. The disclosure also provides an antibody or antigen binding portion thereof that specifically binds to CD28, wherein the antibody or antigen binding portion thereof comprises: heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence and a light chain variable region comprising a CDR1, CDR2, and CDR3 sequence wherein: the VH CDR1 sequence comprises anamino acid sequence that is identical to any one of SEQ ID NOS: 178-182; the VH CDR2 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 183-187; and / or the VH CDR3 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 188-192; and, the VL CDR1 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 163-167; the VL CDR2 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 168-172; and / or the VL CDR3 sequence comprises an amino acid sequence that is identical to any one of SEQ ID NOS: 173-177.

[0022] In certain aspects, the disclosure provides isolated antibodies or antibody fragments that are multispecific antibodies comprising an anti-CD28 antibody or an antigen-binding portion thereof. In some aspects, the multispecific antibody further comprises an additional antibody or antibody fragment that binds to a target antigen. In particular aspects, the multispecific antibody is a bispecific antibody.

[0023] In some aspects, the target antigen bound by the multispecific antibody is expressed on an immune cell. In other aspects, the target antigen is expressed on the surface of a target cell, or the target antigen is a soluble target.

[0024] In further aspects, the multispecific antibody may include at least one additional antibody or antibody fragment that binds to at least one additional target antigen. In certain aspects, the multispecific antibody comprises at least one antibody or antibody fragment that binds to a second additional target antigen. The target antigen, additional target antigen, and / or second additional target antigen may be expressed on immune cells or on the surface of target cells, or may be soluble targets.

[0025] In some aspects, the soluble target comprises one or more soluble proteins, peptides, carbohydrates, lipids, lipoproteins, steroids, other small molecules, cytokines, soluble receptors, antibodies, antigens, prostaglandins, vitamins, metabolites such as sugars or amino acids, drugs or drug metabolites, DNA or RNA strands in various forms, polysaccharides, chromosomes, genes, cells, cellular membranes or organelles (e.g., nuclei, mitochondria), bacteria, viruses, or other microorganisms, natural metabolites, hormones, pollutants, pesticides, and complexes or components of any of the foregoing.

[0026] In additional aspects, the antibody fragment may comprise a monovalent single-chain variable fragment (scFv), a divalent scFv, a Fab fragment, an F(ab')2 fragment, an F(ab')s fragment, an Fv fragment, or a single-chain antibody.

[0027] In yet other aspects, the multispecific antibody may be selected from a T-cell engager, a TCR-mimetic T-cell engager, a bispecific T-cell engager (BiTE), a tandem antibody (tandAb), a Dual-Affinity Re-Targeting Antibody (DART), a bispecific killer engager (BiKE), a trispecific killer engager (TriKE), or other engineered multispecific formats including Fab-Fc-scFv, “bottle-opener,” Mab-scFv, Mab-Fv, Dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, common light-chain IgG, Cross-Mab, SEED, BEAT, TrioMab, or DuetMab.

[0028] In certain aspects, the additional target antigen and / or second additional target antigen is selected from one or more of 17-IA, 4-1BB, 4Dc, 6-keto-PGF 1 a, 8-iso-PGF2a, 8-oxo-dG, Al Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, AD AMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha- 1 -antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL1 7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137,CD 138, CD 140a, CD 146, CD 147, CD 148, CD 152, CD 164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, ED AR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, Enkephalinase, eNOS, Eot, eotaxinl, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcRI, FEN- 1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP- 14, CDMP-1), GDF-6 (BMP- 13, CDMP-2), GDF -7 (BMP- 12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF- 15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alphal, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp!20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGF A, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF -beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / betal, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / betal, integrin alpha5 / beta3, integrin alpha6, integrin betal, integrin beta2, interferon gamma, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15,Kallikrein LT, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin Beta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1 -alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP- 12, MMP- 13, MMP- 14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Muellerian-inhibitin substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGGI, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PC AD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PEC AM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76, RPA2, RSK, SI 00, SCF / KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP -like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-betaPan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRI, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF1 IB (OPG OCIF, TRI), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM AT AR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (0X40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11 (TRANCE / RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSFIA (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (0X40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-Rl, TRAIL-R2, TRANCE, transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VC AM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrands factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0029] In certain aspects, the target cell is a cancer cell. In certain aspects, the cancer is selected from a carcinoma, a lymphoma, a blastoma, a sarcoma, a leukemia and lymphoid malignancies. In certain aspects, the cancer is selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell / histocyte-rich large B-cell lymphoma, multiple myeloma, myeloid leukemia-protein 1 (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular tumor, breast cancer, colon cancer, hepatocellular cancer (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngolaryngeal cancer, hepatobiliary cancer, central nervous system cancer, esophagus cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell cancel, testicular cancer, and skin cancer.

[0030] In certain aspects, the target cell is associated with a disease or condition. In certain aspects, the condition is an autoimmune or inflammatory disease. In some embodiments, the autoimmune disease or inflammatory disease described above is selected from the group consisting of rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren syndrome, autoimmune hemolytic anemia after transplant rejection, vesicular pemphigoid, Graves disease, Hashimoto thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus and pernicious anemia.

[0031] Also described herein are trispecific TCEs that are capable of specifically binding to pHLA-CGl peptide complex on a target cell (e.g., a tumor cell) and CD3 and CD28 on an immune cell. In certain aspects of the disclosure, the anti-CD28 binders provided herein may be included as part of T cell receptor (TCR) mimetic (TCRm) T cell engager antibodies or fragments thereof that specifically bind to: (i) complexes comprising an HLA class I molecule and a peptide, such as a Cathepsin G (CG) peptide, or fragment thereof (e.g., CGI) and / or derivatives of such a peptide or fragment; (ii) an immune cell associated protein (e.g., an immune cell engager such as CD3); and (iii) CD28 via an anti-CD28 binder of the disclosure. In certain aspects, the complex comprises an HLA class I molecule-CGl complex.

[0032] In certain aspects, a TCE of the disclosure comprises at least three binding domains, comprising: a first binding domain that specifically binds to a Cathepsin G (CG) peptide orfragment thereof (e.g., CGI) and / or derivatives of a CG peptide or fragment; and (ii) a second binding fragment that binds to an immune cell associated CD3; and (iii) a CD28 binder as disclosed herein. In certain aspects, the CG peptide is a CGI peptide.

[0033] In some aspects, TCEs incorporating the anti-CD28 binders of the disclosure target CG and / or CGI, the expression of which is associated with certain diseases, such as cancers (e.g., AML). Cathepsin G (CG) is an intracellular serine protease whose endogenous expression is primarily restricted to cells of myeloid lineage, where it is normally stored in azurophilic granules. Compared to normal hematopoietic progenitors, CG is highly expressed and ubiquitinated in AML blasts and leukemic stem cells, where it is aberrantly localized and processed for antigen presentation. CGI is an HLA-A*02:01 restricted peptide (FLLPTGAEA) derived from the CG protein leader sequence and is abundantly presented by leukemic compared to normal myeloid cells. Thus, CGI represents a highly specific target for treating diseases associated with CG expression, such as AML. Moreover, the detection of CGI -specific cytotoxic T lymphocytes (CTLs) in AML patients following allogeneic stem cell transplantation underscores its attractive potential as a novel immunotherapeutic target in AML.

[0034] In certain aspects, the anti-CD28 binders of the disclosure are incorporated into an antibody or antibody fragment. In certain aspects, the antibody fragment can be a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody. In certain aspects, the antibody can be a monoclonal, chimeric antibody, bispecific antibody, trispecific or other multi-specific antibody, or BiTE. In certain aspects, the antibody can be an IgG antibody or a recombinant IgG antibody or antibody fragment.

[0035] In certain aspects, the anti-CD28 antibodies as described herein are human antibodies or humanized antibodies. In embodiments of the present disclosure, the provided antibodies and antibody fragments thereof specifically bind to CD28 on an immune cell. In further embodiments of the present disclosure, novel anti-CD28 antibodies (or antibody fragments) are incorporated into a multispecific, such as a bispecifc antibody that specifically binds to a target antigen, such as a tumor or other antigen that is a disease marker, and can specifically bind to a second antigen that can engage or otherwise stimulate T-cells, e.g., via the novel anti-CD28 antibody. In some aspects, a multispecific antibody may include an additional antigen binding region that binds to another immune cell protein, e.g., CD3, to activate the immune cell. In some certain aspects, the targetantigen is a tumor antigen. In certain aspects, the tumor antigen is a pHLA-peptide complex, e g., pHLA-CG. In some certain aspects, the tumor antigen is a pHLA-CGl peptide complex.

[0036] In certain aspects, the CD28 antibody or antigen binding portion thereof is incorporated into a TCE as described herein. In certain aspects, the TCE binds to CD28, a target antigen, such as a tumor or other antigen that is a disease marker, second antigen that can engage or otherwise stimulate T-cells.

[0037] In certain aspects, the present disclosure includes methods, compositions, formulations, and various techniques for producing TCE antibodies that bind to CD28 on an immune cell and the CGl / HLA-A*02:01 pMHC complex on a target cell with high affinity. In certain aspects, TCEs of the disclosure, including specific TCEs (TA-6, TA-7, and TA-8) disclosed herein, are CGI (“CTSG”) x CD3 x CD28 trispecific TCEs, and include the novel anti-CD28 binders of the disclosure.

[0038] In some aspects, relative to similar TCEs that bind only to CGI and CD3, trispecific CGI x CD3 x CD28 TCEs, which incorporate the novel anti-CD28 binders, are shown herein to induce a more potent T cell mediated killing in vitro of leukemia cell lines. Surprisingly, at low effector to target ratios, the presently disclosed trispecific CGI x CD3 x CD28 TCEs induce clearly higher cytotoxicity than similar CGI x CD3 TCEs. This finding led to the discovery of a novel property of the immune response induced by the presently disclosed CGI x CD3 x CD28 TCEs; even in TCEs with lower-affinity anti-CD28 domains, the increased cytotoxicity and cytokine secretion remained. Thus, while including the CD28 binding domain increases cytotoxicity, a strong affinity for CD28 is not necessary to induce a strong cytotoxic response.

[0039] In certain aspects, TCEs of the disclosure comprise at least: (i) a first binding domain binds to an HLA class I molecule-CG peptide complex; (ii) a second binding domain comprising a second immune cell protein (e.g. CD3), which may act as an immune cell activator or stimulator, and a third binding domain for an anti-CD28 antibody of the disclosure or a fragment thereof. In certain aspects, the second binding domain binds to CD3 on an immune cell, and the third binding domain binds to CD28 on an immune cell.

[0040] In certain aspects, the HLA class I molecule-CG peptide complex is an HLA class I molecule-CGl complex. In certain aspects, the HLA class I molecule-CG complex includes a CG and / or CGI peptide that has one or more amino acid modifications relative to the wildtype CG or CGI peptide.

[0041] ITn certain aspects, CGI binding TCEs, isolated antibodies or antigen binding portions thereof according to the present disclosure may comprise a heavy chain (HC) variable region sequence, wherein the HC variable region comprises a CDR1 sequence comprising one of SEQ ID NOs: 67-78; and a light chain (LC) variable region sequence, wherein the LC variable region comprises a CDR1 sequence comprising one of SEQ ID NOs: 96-105. In certain aspects, the CGI binding TCEs, isolated antibodies or antigen binding portions thereof further comprise, in the HC variable region, a CDR2 sequence comprising one of SEQ ID NOs: 79-87, and 129-131, and / or a CDR3 sequence comprising one of SEQ ID NOs: 88-95. TCEs, antibodies, or antigen binding portions thereof according to the present disclosure can further comprise, in the LC variable region, a CDR2 sequence comprising one of SEQ ID NOs: 106-117 and / or a CDR3 sequence comprising one of SEQ ID NOs: 118-123.

[0042] In certain aspects, the novel anti-CD28 antibodies, or antigen binding fragments thereof, are incorporated into TCEs comprising a heavy chain variable region (VH) having at least 90% identity to any one of SEQ ID NOs: 158-162; and / or (b) a light chain variable region (VL) having at least 90% identity to any one of SEQ ID NOs: 153-157.

[0043] In certain aspects, the present disclosure includes methods, compositions, formulations, and various techniques for producing the anti-CD28 binders and antibodies incorporating such binders, such as trispecific TCE antibodies that bind to CD28, CD3, and the CGl / HLA-A-*02:01 pMHC complex with high affinity.

[0044] The disclosure provides a TCE or other multispecific antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOS: 133 and 144-146, or a fragment of any thereof. The disclosure also provides a TCE or other multispecific antibody, or antigen binding portions thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, multispecific antibody, or antigen binding portions thereof comprise a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 133, 144-146, or a fragment thereof. The disclosure also provides a TCE or other multispecific antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloidmalignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 133, 144-146, or a fragment thereof. The disclosure also provides a TCE or other antibody, or antigen binding portions thereof that specifically bind to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CG1 binding domain heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 133, 144-146, or a fragment thereof.

[0045] The disclosure also provides a TCE or other antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CG1 binding domain light chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOS: 132, 147-149, or a fragment thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOS: 132, 147-149, or a fragment thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 95% identical to any of SEQ ID NOS: 132, 147-149, or a fragment thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 132, 147-149, or a fragment thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 132, 147-149, or a fragment thereof.

[0046] The disclosure also provides a TCE or other antibody, antigen binding portion thereof that comprises a CD3 binding domain and a CD28 binding domain. In certain aspects, the CD3 binding domain and CD28 binding domain are on the same peptide chain. In certain aspects, the peptide chain is a different peptide chain from one or more peptide chains comprising the pHLA-CGl binding domain. In certain aspects, the CD3 and CD28 binding domains are scFvs. In certain aspects, the CD3 and CD28 scFvs are joined by a linker. In certain aspects, the TCEs of the disclosure comprise a silent Fc IgGl.

[0047] In some certain aspects, TCEs and other antibodies of the disclosure comprise a peptide chain comprising the CD3 and CD28 binding domains. In certain aspects, the peptide chaincomprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 95% identical (for example, at least 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 150-152, or one or more fragments thereof.

[0048] In some embodiments, a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 1-27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 1-27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 1-27, 133, or 134.

[0049] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable regioncomprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 28-54 or 132.

[0050] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 28-54 or 132.

[0051] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specificallybinds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 28-54 or 132.

[0052] In certain aspects, the antigen is CGI, in particular, a 9-mer peptide derived from cathepsin G protein leader sequence, presented by HLA class I, specifically HLA-A*02:01 (SEQ ID NO:61, for example).

[0053] In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 57, 128, and 139, or a fragment of any thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 57, 128 and 139, or a fragment of any thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 57, 128 and 139, or a fragment of any thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NO: 57, 128, and 139, or a fragment of any thereof.

[0054] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162.

[0055] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in any one ofSEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157.

[0056] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 153.

[0057] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavychain region comprising an amino acid sequence as set forth in SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 154.

[0058] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 155.

[0059] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 156.

[0060] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 157.

[0061] In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 57, 128, and 139, or a fragment thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 57, 128, and 139, or a fragment thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that as set forth in any one of SEQ ID NOs: 57, 128, and 139, or a fragment thereof.

[0062] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the thirdpeptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0063] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0064] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with anamino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0065] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0066] Isolated antibodies or antibody fragments as described herein can be a human antibody or human antibody fragment or humanized antibody or humanized antibody fragment.

[0067] In certain aspects, described herein are TCEs, isolated antibodies, or antigen binding portions thereof comprise one or more of a monovalent scFv (single chain fragment variable)antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.

[0068] In certain aspects, the TCE comprises an Fc domain. In certain aspects, the Fc domain comprises one or more amino acid substitutions. In certain aspects, the Fc is silent.

[0069] In certain aspects, the TCE can be or comprise a chimeric antibody, trispecific or other multi-specific antibody, or BiTE. In certain aspects, the TCE can be an IgG antibody or a recombinant IgG antibody or antibody fragment.

[0070] In certain aspects, the TCE antibodies or antibody fragments thereof can exhibit increased binding affinity for CGI presented by HLA-A*02:01 compared to sample from a wild-type subject or subject not having a cancer.

[0071] In certain aspects, anti-CD28 antibodies, including TCEs, as described herein can be conjugated or fused to an imaging agent, a cytotoxic agent, a metal, or a radioactive moiety. In certain aspects, the imaging agent can be a fluorophore. In certain aspects, the radioactive moiety can comprise at least one of Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212. In certain aspects, the antibody can be an immune conjugate or a radio-immune conjugate. In certain aspects, the antibody is an antibody-drug conjugate.

[0072] Also described herein are methods of making a TCE antibodies incorporating the anti-CD28 antibodies of the disclosure, or antibody fragments thereof. Methods as described herein can comprise, for example, culturing a hybridoma or engineered cell as described herein under conditions that allow expression of the TCE or fragment thereof and, optionally, isolating the TCE from the culture.

[0073] In certain aspects, the TCE antibody incorporating the anti-CD28 antibodies of the disclosure comprises a first and second antigen binding domain is a trispecific antibody in heterodimer format, wherein the first and second antigen binding domains independently bind to an HLA class I molecule CG / CG1 peptide complex. In certain aspects, the TCE comprises a third binding domain that comprises two single-chain variable fragments (scFv) that bind CD3 and CD28. In certain aspects, the first and / or second binding domains comprise an scFv that binds to the plurality of HLA class I molecule CG / CG1 peptide antigens. In certain aspects, the first and second binding domains comprise an scFv that binds to the plurality of HLA class I CG / CG1 peptide antigens. In certain aspects, the TCE antibody comprises a first, second and third antigen binding domain is a trispecific antibody in heterodimer format, wherein the first antigen bindingdomain independently binds to an HLA class I molecule CG / CG1 peptide complex. In certain aspects, the TCE comprises a second binding domain that binds to CD3. In certain aspects, the TCE comprises a third binding domain that binds to CD28. In certain aspects, the first binding domain comprises a Fab that binds to the plurality of HLA class I molecule CG / CG1 peptide antigens. In certain aspects, the second binding domain comprises a scFV that binds to CD3. In certain aspects, the third binding domain comprises a scFv that binds to CD28.

[0074] In certain TCE-based antibodies incorporating the anti-CD28 antibodies of the disclosure comprise: a first and third polypeptide comprising the first binding domain; and a second polypeptide comprising the second and third binding domains. In certain aspects, the second polypeptide includes the scFvs of the second and third binding domains, which are in a tandem format. In certain aspects, the first and second polypeptide each comprise a hinge-CH2-CH3 and form a heterodimer. In certain aspects, the Fc is silent.

[0075] In some embodiments, the TCE specifically binds two HLA class I molecules: HLA-A*02:01, HLA-A*02:03, HLA-A*02:06, and HLA-A*02:07. In some embodiments, the TCE is or comprises a single domain antibody, a human single domain antibody, or a humanized single domain antibody. In some embodiments, the TCE is or comprises a murine antibody, a chimeric antibody, a camelid antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is part of a multispecific antibody or a multifunctional antibody.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0077] FIG. 1 provides a schematic of an exemplary TCE of the disclosure, incorporating an anti-CD28 antibody of the disclosure.

[0078] FIG. 2 provides a schematic of an exemplary TCE of the disclosure, incorporating an anti-CD28 antibody of the disclosure.

[0079] FIG. 3 provides a schematic of an exemplary TCE, incorporating an anti-CD28 antibody of the disclosure.

[0080] FIG. 4 summarizes the attributes and biophysical characterization of newly humanized anti-CD28 binders compared to reference molecules. The Humanness Z-scores (VL and VH) represent the degree of sequence humanization of the variable light and heavy chains, where higher scores indicate greater similarity to human germline sequences. BVP binding and CHO binding assess specificity, with greater number representing non-specific background binding. The melting temperature (Tm, °C) provides a measure of thermal stability for each molecule. All binders were tested in a symmetric scFv-based monoclonal antibody format used for initial clone evaluation.

[0081] FIG. 5 provides the results of a CHO and BVP binding assays for the CD28 binders. As shown in FIG. 5, CHO and BVP have low binding, indicating there is no nonspecific polyreactivity binding from the anti-CD28 binding domain of the molecule.

[0082] FIG. 6A shows a table of dissociation constant (KD) values for monovalent humanized anti-CD28 binders in scFv and Fab formats, measured for binding to CD28, measured by biolayer interferometry (BLI). The table groups them into high, medium, and low affinity categories relative to benchmark humanized reference molecule, demonstrating a range of binder strengths to CD28 for incorporation into trispecific antibody structures. FIGS. 6B-6C show BLI sensorgrams binding kinetics. In this experiment, biotinylated CD28 was immobilized on the sensor and associated into 100 nM-0.41 nM antibody in 3-fold dilution series, except CD28 Binder 15 which was run at 500 nM, 300 nM, 200 nM, 100 nM, and 50 nM antibody concentration TCE.

[0083] FIG. 7 illustrates the ‘superagonist cytokine release test’ of the anti-CD28-binder only using a dry coating assay. (Left panel) Schematic representation of the dry coating assay principle: 1 μg / well antibody is immobilized on the surface of a well by air drying, followed by addition of PBMCs. After a 24 h incubation, cytokine levels in the culture supernatant are quantified using a multiplex readout. (Right panel) Cytokine release profile for IFNy, IL-2, and TNFa for various CD28 binding test articles, including CD28 superagonist control TGN1412 (purple). Each data point represents the average mean of a technical duplicate for one donor, with four independent donors tested. As shown, the all new humanized monovalent CD28 binders do not elicit cytokine release. Also shown is the clinically validated CD28 binder, CD28 Binder 13.

[0084] FIGS. 8A-8B show analytical size exclusion chromatography (ASEC) chromatograms of monovalent CD28 binders showing good protein quality and lack of aggregation.

[0085] FIG. 9 provides a summary of characteristics of CD28 humanization of CGI x CD3 x CD28 trispecific TCEs of the disclosure. FIG. 10 provides a summary of characteristics of octet binding affinity of CGI x CD3 x CD28 trispecific TCEs and CD28 bivalents of the disclosure.

[0086] FIGS. 11A-11C provide cytotoxicity assay results for TCE of the disclosure.

[0087] FIGS. 12A-12B provide cytotoxicity assay results for TCE of the disclosure.

[0088] FIG. 13 provides cytokine secretion data for TCE of the disclosure.

[0089] FIG. 14 provides CHO and BVP assay results for TCE of the disclosure.

[0090] FIG. 15 provides quality control protein analytics data for TCE of the disclosure.

[0091] FIG. 16 details the binding affinities of a TCE of the disclosure (TA-8) measured by biolayer interferometry (BLI).

[0092] FIG. 17 displays results of an assay assessing on-cell binding by flow cytometry comparing two TCEs of the disclosure (TA-8 versus TA-2) which further helps determine the binding affinity of the TCE TA-8 to T cells.

[0093] FIG. 18 shows the results of testing a TCEs of the disclosure using U937-A2 cells as a target in a 72-hour killing assay. CD3 cells used were as effector at an effector-to-target (E: T) ratio of 1: 1. The cells were incubated with TA-2 or TA-8 from 1 nM- 0.1 pM at a 10-fold dilution.

[0094] FIG. 19 provides results for a cytotoxicity assessment of a TCE of the disclosure against U937 cells transduced with different HLA-A*02 subtypes via a Luciferase readout.

[0095] FIGS. 20A-20B provide results from an ex vivo study evaluating autologous T cell activation and cytotoxicity mediated by TCEs of the disclosure (TA-8 or TA-2) towards primary AML cells.

[0096] FIG. 21 provides data for an assessment of the pharmacokinetics of a TCE (TA-8).

[0097] FIG. 22 provides data confirming the efficacy of TCEs of the disclosure in vivo as measured by tumor growth inhibition.

[0098] FIG. 23 shows results of a bone marrow colony forming unit (CFU) assay for TCEs TA-2 and TA-8.

[0099] FIG. 24 displays the results of the full alloreactivity panel using TCEs of the disclosure.

[0100] FIG. 25 shows an A / G Scan comparison of TA-8 to TA-2.

[0101] FIG. 26 provides test results from a test of protentional X-reactive peptides to TA-8.

[0102] FIG. 27 shows the IFNy release results from a cytokine bead array (CBA) assay after 24 hours using the TCE TA-8 with a normal cell panel from vital tissues. As shown, no T-cellactivation was observed with TA-8, further reinforcing the specificity of this TCE of the disclosure.

[0103] FIG. 28 summarizes the results of the developability assessment, which show the TCE to have favorable developability characteristics. TA-8 showed minimal change in critical quality attributes after various stated stress (i.e., long-term storage, thermal stress, etc.) showing TA-8 passes specifications.

[0104] FIG. 29 shows an analytical characterization of TA-8. The top panel provides an analytical size exclusion chromatography (SEC) profile demonstrating monomeric purity of TA-8 and minimal aggregate or fragment content. The bottom panel provides a capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) profile of TA-8, illustrating the integrity and absence of degradation products. Together these analyses confirm the structural quality and purity of >95% of TA-8.

[0105] FIG. 30 shows cytotoxicity data in the presence of immunosuppressive cytokines.DETAILED DESCRIPTION

[0106] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

[0107] Provided herein are novel anti-CD28 binding molecules, including antibodies and antigenbinding fragments thereof, as well as multispecific constructs incorporating such anti-CD28 binders. The disclosure encompasses a range of CD28-binding molecules with distinct affinities and functional profiles, enabling selective tuning of CD28-mediated costimulatoiy signaling across diverse therapeutic applications. The presently disclosed anti-CD28 binders may be used as stand-alone antibodies or incorporated into multispecific, multivalent, or modular antibody constructs, including but not limited to bispecific, trispecific, and tetraspecific formats.

[0108] In certain aspects, the CD28 binders of the present disclosure possess variable anti-CD28 affinities, enabling each to be employed in different therapeutic contexts to achieve an optimal degree of costimulatory engagement.

[0109] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0110] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of those certain elements.” As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0111] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel character! stic(s)” of the present disclosure or features of the claims. See, for example, In re Herz, 537 F.2d 549, 551-52, 190 U. S. P. Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0112] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0113] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X”specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0114] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particularpolynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0115] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0116] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (z.e., two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

[0117] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoi somers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581-87 (2013); Xie et al. “Adding amino acids to the genetic repertoire, "Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0118] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0119] The term “identity”, “substantial identity,” “similarity” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0120] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0121] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman Proc. Natl. Acad. Set. (U. S. A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0122] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-10 and Altschul etal. (1977) Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0123] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10‘5, and most preferably less than about IO'20.

[0124] The term “antigen binding protein” or “ABP” is used herein in its broadest sense and includes certain types of molecules such as antibodies, including TCE antibodies, comprising one or more antigen-binding domains (e.g., for CD28 or a tumor antigen) that specifically bind to an antigen or epitope.

[0125] The term “peptide-Human Leukocyte Antigen (HLA)-class I molecule complex”, “peptide-HLA class I molecule”, “HLA class I molecule peptide complex”, and “peptide and HLA class I molecule complex”, and similarly described terms as used herein, refer to the complex of both the HLA class I molecule bound to the presented peptide antigen.

[0126] The term “HLA-CG1 peptide antigen” or “HLA class I molecule-CGl complex” and such similar terms as used herein refers to the peptide-HLA class I molecule complex comprising both the HLA class I molecule bound to a CGI peptide described herein. The term “HLA-CG1 peptide antigen” or “HLA class I molecule-CG complex” and such similar terms as used herein refers to the peptide-HLA class I molecule complex comprising both the HLA class I molecule bound to a CG peptide, which may be CGI, described herein.

[0127] The term “T cell engager” or “TCE”, as used herein, refers to an antibody that binds an immune cell antigen (e.g., CD28 and CD3) and at least one antigen expressed on a target cell, e.g., a tumor cell. In certain aspects, the target antigen is a peptide MHC molecule complex on a different cell. A TCE as disclosed herein is an ABP that includes an immune cell engager (CD3 binding domain), immune cell co-stimulatory engager (CD28 binding domain), and a target binding domain (e.g., a pHLA-CGl binding domain).

[0128] A “HLA-CG1 T cell engager”, “Anti-HLA-CGl TCE antibody”, “HLA-CG1 peptide ABP,” “anti-HLA-CGl peptide ABP,” or “HLA-CG1 peptide-specific ABP” is an ABP, as provided herein, which specifically binds to the peptide-HLA class I molecule complex bound to a CG peptide, which includes variant, truncated, or otherwise engineered CG peptides, such as the CGI peptide. An “HLA-CG1 T cell engager”, “Anti-HLA-CGl T cell engager”, “HLA-CG1 peptide ABP,” “anti-HLA-CGl peptide ABP,” or “HLA-CG1 peptide-specific ABP” is an ABP, as provided herein, which specifically binds to the peptide-HLA class I molecule complex bound to a CGI peptide.

[0129] As used herein, “variable region” refers to a variable sequence that arises from a recombination event, for example, it can include a V, J, and / or D segment of a T cell receptor (TCR) sequence from a T cell, such as an activated T cell.

[0130] The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope. An antigen-binding domain can include antibody CDRs, e g. VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3; as well as TCRCDRs, e.g., aCDRl, aCDR2, aCDR3, CDR1, CDR2, and £CDR3. TCR CDRs are described herein.

[0131] The amino acid sequence boundaries of a TCR or antibody CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including but not limited to the IMGT unique numbering, as described by LeFranc, M.-P, Immunol Today. 1997 Nov;18(l l):509; Lefranc, M.-P., "IMGT Locus on Focus: A new section of Experimental and Clinical Immunogenetics", Exp. Clin. Immunogenet., 15, 1-7 (1998); Lefranc and Lefranc, The T Cell Receptor FactsBook; and M.-P. Lefranc / Developmental and Comparative Immunology 27 (2003) 55-77, all of which are incorporated by reference.

[0132] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a T cell engager protein) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., T cell engager protein and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0133] With regard to the binding of a binder / ABP, such as a a TCE, or other antibody or fragment thereof to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule.

[0134] The term “kd” (sec-1), as used herein, refers to the dissociation rate constant of a particular ABP - antigen interaction. This value is also referred to as the koir value.

[0135] The term “ka” (M'^sec'1), as used herein, refers to the association rate constant of a particular ABP -antigen interaction. This value is also referred to as the konvalue.

[0136] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular ABP -antigen interaction. KD = kd / ka. In some embodiments, the affinity of an ABP is described in terms of the KD for an interaction between such ABP and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.

[0137] The term “KA” (M-1), as used herein, refers to the association equilibrium constant of a particular ABP-antigen interaction. KA = ka / kd.

[0138] An “immunoconjugate” is an ABP conjugated to one or more heterologous molecule(s), such as a therapeutic (cytokine, for example) or diagnostic agent.

[0139] When used herein in the context of two or more ABPs, the term “competes with” or “crosscompetes with” indicates that the two or more ABPs compete for binding to an antigen (e g., HLA-CG1 peptide). In one exemplary assay, HLA-CG1 peptide is coated on a surface and contacted with a first HLA-CG1 peptide ABP, after which a second HLA-CG1 peptide ABP is added. In another exemplary assay, a first HLA-CG1 peptide ABP is coated on a surface and contacted with HLA-CG1 peptide, and then a second HLA-PEPTIDE ABP is added. If the presence of the first HLA-CG1 peptide ABP reduces binding of the second HLA-CG1 peptide ABP, in either assay, then the ABPs compete with each other. The term “competes with” also includes combinations of ABPs where one ABP reduces binding of another ABP, but where no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one ABP reduces binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. A skilled artisan can select the concentrations of the ABPs used in the competition assays based on the affinities of the ABPs for HLA-CG1 peptide and the valency of the ABPs. The assays described in this definition are illustrative, and a skilled artisan can utilize any suitable assay to determine if ABPs compete with each other. Suitable assays are described, for example, in Cox et al., “Immunoassay Methods,” in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih. ov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated by reference in its entirety.

[0140] The term “epitope” means a portion of an antigen that specifically binds to an ABP. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination such as, for example, testing for ABP binding to HLA-CG1 peptides in the context of an HLA Class I molecule.

[0141] As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0142] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alternatively, sequence similarity or dissimilarity can be established by the combined presence or absence of particular nucleotides, or, for translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).

[0143] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0144] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0145] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0146] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), which each include the primary transformed or transfected cell and progeny derived therefrom. Such progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations.

[0147] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of a clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0148] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[0149] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease orcondition that can be treated with an ABP provided herein. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[0150] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0151] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematologic malignancy.

[0152] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0153] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0154] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0155] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[0156] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.

[0157] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.

[0158] The terms “nucleic acids” and “polynucleotides” may be used interchangeably herein to refer to polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can include, but are not limited to coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA, isolated RNA, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Exemplary modified nucleotides include, e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-( carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthioN6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

[0159] As used herein the term “antigen” is a substance that induces an immune response. An antigen can be a neoantigen. An antigen can be a “shared antigen” that is an antigen found among a specific population, e.g., a specific population of cancer patients. Antigens can include HLA-CG1 peptide antigens.

[0160] As used herein the term “neoantigen” is an antigen that has at least one alteration that makes it distinct from the corresponding wild-type antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. In some embodiments, the alteration occurs in tumor or cancer cells. In some embodiments, the alteration does not occur in a non-tumor or a non-cancer cell. In some embodiments, the alteration is absent from normal tissue. A neoantigen can include a polypeptide sequence or a nucleotide sequence. Neoantigens can include HLA- CGI peptide neoantigens.

[0161] As used herein the term “tumor antigen” is an antigen present in a subject’s tumor cell or tissue but not in the subject’s corresponding normal cell or tissue, or derived from a polypeptideknown to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.

[0162] As used herein the term “candidate antigen” is a mutation or other aberration giving rise to a sequence that may represent an antigen.

[0163] As used herein the term “coding region” is the portion(s) of a gene that encode protein.

[0164] As used herein the term “coding mutation” is a mutation occurring in a coding region.

[0165] As used herein the term “ORF” means open reading frame.

[0166] As used herein the term “NEO-ORF” is a tumor-specific ORF arising from a mutation or other aberration such as splicing.

[0167] As used herein the term “missense mutation” is a mutation causing a substitution from one amino acid to another.

[0168] As used herein the term “nonsense mutation” is a mutation causing a substitution from an amino acid to a stop codon or causing removal of a canonical start codon.

[0169] As used herein the term “frameshift mutation” is a mutation causing a change in the frame of the protein.

[0170] As used herein the term “indel” is an insertion or deletion of one or more nucleic acids.

[0171] As used herein the term “non-stop or read-through” is a mutation causing the removal of the natural stop codon.

[0172] Provided herein are novel anti-CD28 binding molecules, including antibodies and antigenbinding fragments thereof, as well as multispecific constructs incorporating such anti-CD28 binders. The disclosure encompasses a range of CD28-binding molecules with distinct affinities and functional profiles, enabling selective tuning of CD28-mediated costimulatory signaling across diverse therapeutic applications. The presently disclosed anti-CD28 binders may be used as stand-alone antibodies or incorporated into multi specific, multivalent, or modular antibody constructs, including but not limited to bispecific, trispecific, and tetraspecific formats.

[0173] In certain aspects, the CD28 binders of the present disclosure possess variable anti-CD28 affinities, enabling each to be employed in different therapeutic contexts to achieve an optimal degree of costimulatory engagement. By way of example, high-affinity CD28 binders may strongly activate T cells and induce robust cytokine responses, while intermediate- or low-affinity binders may promote T-cell costimulation in a controlled manner, widening the therapeutic window and minimizing the risk of cytokine release syndrome (CRS) or other systemic immunetoxicities. Such controlled costimulation is particularly advantageous in combination with T-cell-redirecting antibodies (e.g., CD3- or TCR-based engagers), where excessive activation of CD28 has been associated with hyper-cytokinemia and systemic inflammatory responses, as evidenced in early-generation CD28 superagonists (e.g., TGN1412). Accordingly, the present disclosure provides families of anti-CD28 binders spanning a continuum of affinities and epitope specificities, allowing precise modulation of CD28 signaling strength depending on the therapeutic construct and indication.

[0174] The presently disclosed CD28-binding molecules may be full-length antibodies or antigenbinding fragments (e.g., Fab, scFv, F(ab')2, Fv, single-chain antibodies) and may be monoclonal, recombinant, synthetic, human, humanized, chimeric, or derived from display libraries or immunized transgenic animal systems. In certain aspects, the CD28 antibodies are incorporated into multi specific antibodies such as T-cell engagers (TCEs), TCR-mimetic TCEs (TCRm-TCEs), or other multispecific architectures designed for controlled T-cell activation.

[0175] In the adaptive immune system, T-cell activation requires at least two coordinated signals. A primary activation signal (“Signal 1”) is generated when the T-cell receptor (TCR) complex, associated with CD3 subunits, recognizes an antigenic peptide presented on a major histocompatibility complex (pMHC) on antigen-presenting or target cells. However, Signal 1 alone is insufficient to sustain full T-cell activation. Without a secondary costimulatory signal (“Signal 2”), T cells become anergic, exhausted, or undergo apoptosis.

[0176] Signal 2 is provided through costimulatory receptor-ligand interactions, most prominently via engagement of CD28 on the T-cell surface with B7 family ligands (e.g., CD80 and CD86) expressed on antigen-presenting cells. Engagement of CD28 leads to recruitment of PI3K, AKT, and NF-KB signaling cascades, promoting IL-2 production, clonal expansion, survival, and memory formation. The presently disclosed anti-CD28 binders mimic or modulate this natural costimulatory interaction, providing a controlled Signal 2 in therapeutic settings.

[0177] In certain aspects, the anti-CD28 binders of the disclosure are incorporated into multispecific (e.g., bispecific) constructs that bind both CD28 on immune cells and at least a second target antigen. The second antigen may be expressed on immune effector cells (e.g., CD3, CD2, or CD137 on T cells; CD16 or NKG2D on NK cells) or on diseased target cells (e.g., tumor-associated antigens such as HER2, EGFR, CD 19, BCMA, PSMA, or mesothelin). Such bispecific constructs can provide localized costimulation by engaging CD28 only when the immune cell isbrought into proximity with a target cell expressing the cognate antigen — thus confining activation to the relevant cells (e.g., those located within a tumor microenvironment) and reducing off-target systemic activation.

[0178] In some aspects, the target antigen bound by the multispecific construct is a soluble factor (e.g., cytokine, chemokine, or growth factor) or a disease biomarker, enabling sequestration or modulation of extracellular mediators while concurrently engaging CD28 on immune cells.

[0179] In additional aspects, the anti-CD28 binders are incorporated into larger multispecific constructs (e.g., trispecific, tetraspecific and higher-order constructs). For instance, a trispecific antibody may include an anti-CD28 binder, an immune cell binding domain (e.g., a CD3-binding domain, which may induce Signal 1), and a target antigen (e.g., a tumor / disease antigen) binding domain, thereby enabling co-engagement of both costimulatory and cytotoxic T-cell activation pathways in a single molecule. In yet other aspects, a tetraspecific antibody may include an anti-CD28 binder, an immune cell binding domain, and target antigen-binding domains, or alternatively an immune cell antigen (e.g., CD3 and CD137) and a tumor / disease antigen. Such multispecific constructs may act in cis (binding multiple targets on the same immune cell) or in trans (binding an immune cell and a target cell), enabling fine-tuned modulation of T-cell activation, proliferation, and cytolytic function.

[0180] The design of such constructs may include IgG-based architectures (e.g., DuetMab, SEEDbody, CrossMab, DVD-Ig), scFv-linked architectures (e.g., BiTE, DART, TandAb), or hybrid Fab-Fc-scFv arrangements that balance stability and manufacturability.

[0181] In certain aspects, the disclosure provides immunoconjugates comprising an anti-CD28 binder linked to an effector molecule such as a small-molecule drug, toxin, cytokine, or radionuclide. In certain aspects, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the payload may include cytotoxic agents (e.g., auristatins, maytansinoids, tubulysins, calicheamicins), immune-modulatory small molecules, or signaling modulators that enhance local immune activation.

[0182] The CD28 binders of the disclosure may be engineered by rational mutagenesis, display selection, or structure-guided affinity maturation to achieve optimized binding kinetics and safety profiles. Structural studies (e.g., PDB entries 1YJD, 2J8H) identify the MYPPPY motif on CD28 as the canonical B7 binding interface; thus, non-blocking anti-CD28 antibodies targeting alternative epitopes (distal to this interface) can function as partial agonists with reducedsuperagonistic potential. The presently disclosed binders may target such epitopes or employ Fc silencing substitutions (e.g., L234A / L235A, N297A, L234A / L235A / K322A) to mitigate FcyR and / or Cql -mediated crosslinking and cytokine storm risk.

[0183] In certain aspects, the anti-CD28 binders and multispecific constructs are used in methods for treating cancer, infectious diseases, autoimmune disorders, or conditions characterized by impaired T-cell costimulation. In oncology, they may enhance antitumor immunity by providing controlled CD28 engagement in conjunction with TCR-mediated targeting. In certain aspects, an anti-CD28 binder of the disclosure is an agonist or partial-agonist of CD28.

[0184] In some aspects, the anti-CD28 binders are fully human antibodies or fragments thereof to minimize immunogenicity. In other aspects, the binders are chimeric, humanized, or synthetic antibodies designed for manufacturability, stability, and defined valency.

[0185] In summary, the present disclosure provides a spectrum of anti-CD28 binding molecules and multispecific constructs that enable rational modulation of T-cell costimulation. By varying CD28 affinity, epitope, valency, and construct architecture, these molecules allow precise control of immune activation, thereby improving efficacy and safety in targeted immunotherapies.

[0186] Provided herein are novel anti-CD28 binders, including as part of larger multi-specific constructs such as T-cell engagers (TCEs), as described herein. The subject anti-CD28 antibodies agonistically bind to CD28 costimulatory molecules on T cells, thereby promoting a co-stimulatory response. Thus, the presently disclosed antibodies selectively enhance, for example, anti-tumor activity of a redirected T-cell. The subject antibodies provided herein are particularly useful when included in TCEs, where their differing affinities for CD28 may be used to fine-tune the activity of the larger construct.

[0187] Also described herein are trispecific TCEs that are capable of specifically binding to pHLA-CGl peptide complex on a target cell (e.g., a tumor cell) and CD3 and CD28 on an immune cell. Aspects of the disclosure include, in certain embodiments, TCE antibodies or fragments thereof that specifically bind to: (i) complexes comprising an HLA class I molecule and a Cathepsin G (CG) peptide or fragment thereof (e.g., CGI) and / or derivatives of a CG peptide or fragment; (ii) an immune cell associated protein (e.g., an immune cell engager such as CD3); and (iii) CD28 via an anti-CD28 binder of the disclosure. In certain aspects, the complex comprises an HLA class I molecule-CGl complex.

[0188] In certain aspects, a TCE comprises at least three binding domains, comprising: a first binding domain that specifically binds to a Cathepsin G (CG) peptide or fragment thereof (e.g., CGI ) and / or derivatives of a CG peptide or fragment; and (ii) a second binding fragment that binds to an immune cell associated CD3; and (iii) a CD28 binder as disclosed herein. In certain aspects, the CG peptide is a CGI peptide.

[0189] In some aspects, TCEs target CG and / or CGI, the expression of which is associated with certain diseases, such as cancers (e.g., AML). Cathepsin G (CG) is an intracellular serine protease whose endogenous expression is primarily restricted to cells of myeloid lineage, where it is normally stored in azurophilic granules. Compared to normal hematopoietic progenitors, CG is highly expressed and ubiquitinated in AML blasts and leukemic stem cells, where it is aberrantly localized and processed for antigen presentation. CGI is an HLA-A*02:01 restricted peptide (FLLPTGAEA) derived from the CG protein leader sequence and is abundantly presented by leukemic compared to normal myeloid cells. Thus, CGI represents a highly-specific target for treating diseases associated with CG expression, such as AML. Moreover, the detection of CG1-specific cytotoxic T lymphocytes (CTLs) in AML patients following allogeneic stem cell transplantation underscores its attractive potential as a novel immunotherapeutic target in AML.

[0190] Tumor cells can express intracellular antigens, such as CGI, and may display such antigens on the surface of the tumor cell via Major Histocompatibility Complex (MHC) presentation. MHCs display intracellularly processed protein fragments on the cell surface. In humans, MHC is referred to as Human Leukocyte Antigen (HLA). MHC class I molecules / HLA class I molecules are expressed on the surface of nearly all nucleated cells of humans. MHC class I molecules / HLA class I molecules are dimeric and comprise a transmembrane heavy chain, comprising the peptide antigen binding cleft, and a smaller extracellular chain, beta2 -microglobulin.

[0191] In the canonical pathway, MHC class I molecules / HLA class I molecules present peptides derived from the proteolytic degradation of cellular proteins. Proteins may originate from endogenous de novo biosynthesis or from extra-cellularly sourced proteins through the process of cross-presentation. Canonically, the cleaved peptides are transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) where they are bound to the groove of the assembled class I molecule, and the resultant peptide-MHC class I complex is transported to the cell membrane to enable antigen presentation to T lymphocytes. Alternatively, some proteins contain leader or signal sequences that insert directlyinto the ER, where they are cleaved by signal peptidases resident in the ER and can then bind to MHC class I molecules in a TAP -independent manner for presentation on the cell surface.

[0192] MHC class I / HLA class I genes are highly polymorphic across humans, comprising multiple common alleles for each individual gene. Each MHC allele-peptide complex comprising a specific HLA subtype and a specific peptide fragment presents a protein structure on the cell surface that can be targeted by an immunotherapeutic antigen-binding protein. However, the polymorphic nature of MHC allele / peptide complexes limits the number of possible recipients of therapeutics designed to bind specific MHC allele-peptide complexes. The presently disclosed TCEs may be designed to overcome this MHC-restriction, which allows certain disclosed TCEs to bind to more than one MHC allele-peptide complex for a given peptide derived from a protein of interest, such as CG / CG1. This, in turn, allows such TCEs to provide increased therapeutic activity for diverse groups expressing various MHC alleles. By identifying and targeting CG / CG1 as targets for AML using TCEs, the present disclosure is also able to overcome a standing issue in developing immunotherapeutic approaches for the treatment of AML-the identification of a specific target.

[0193] The TCE antibodies, and their analogues discussed in the examples, which include those referred to herein as ““TA-#” (e.g., “TA-8”), and the like, are characterized in that, in addition to targeting tumor associated peptides / antigens (TAAs) presented by the MHC, they also have at least two binding domains that target an immune cell, CD28 expressed on the surface, along with another immune cell protein (e g., CD3), which serve as immune cell activators and engagers.

[0194] Furthermore, the presently disclosed TCEs are, while avoiding off-target toxicity, able to provide “bystander killing”, which overcomes potential resistance mechanisms that may arise due to heterogeneous tumor antigen expression. In response to certain stimuli, e.g., a viral infection, large numbers of neighboring T cells may become activated in a T cell receptor-independent and cytokine-dependent manner, which is known as “bystander activation”. Bystander T cells lack specificity for any particular antigen target, but nonetheless can play an important immunological role. This is significant as target-specific T-cells can become exhausted over time or encounter target antigen escape / avoidance / downregulation / switching and the like in which the immune cell target is modified or no longer expressed on diseased cells, such as tumor cells, but nonetheless continue to grow. However, these T-cells can be activated by IL-15 release initiated by the activity of target-specific T-cells contacted with TCEs of the disclosure. As a result, bystander-activatedCD8+ T cells are able to enhance the therapeutic effect of the immunotherapy. It has been reported that there is a strong correlation between the cytolytic function of bystander-activated CD8+ T cells and higher levels of tumor clearance in response to immunotherapy. The bystander-activated T cells are able, for example, to provide an immune response (e.g., by secreting cytokines), and exert cytotoxicity facilitated by natural killer cell-activating receptors and cytolytic molecules.

[0195] In certain aspects, the TCEs of the disclosure comprise at least: (i) a first binding domain binds to an HLA class I molecule-CG peptide complex; (ii) a second binding domain that binds to CD28 and a second immune cell protein (e.g., CD3), which may act as an immune cell activator or stimulator. In certain aspects, the second binding domain binds to CD3 on an immune cell and a third binding domain binds to CD28 on an immune cell.

[0196] In certain aspects, the HLA class I molecule-CG peptide complex is an HLA class I molecule-CGl complex. In certain aspects, the HLA class I molecule-CG complex includes a CG and / or CGI peptide that has one or more amino acid modifications relative to the wildtype CG or CGI peptide.

[0197] Described herein are also antibodies, antigen binding regions, and fragments thereof, that may be incorporated into a TCEs as described herein, that are capable of specifically binding to CGI. Such antibodies can be monoclonal, synthetic / recombinant, or other type of antibody. Also described herein are trispecific recombinant antibodies, antigen binding regions, and fragments thereof that are capable of specifically binding both CGI, CD3, and CD28 and incorporated into a TCE as described herein.

[0198] The present disclosure provides compositions comprising novel anti-CD28 antibodies and / or antigen binding portions thereof, which may be or included as part of an anti-CD28 antibody and / or multi-specific antibody molecule. For example, in certain aspects, the novel anti-CD28 antibodies or antigen binding portions thereof, are incorporated into a TCE. Such antibody molecules find utility, for example, in treating certain pathologies, such as cancers, including hematological malignancies. The anti-CD28 antibodies or antigen binding portions thereof may be included in a larger TCE construct that specifically or selectively binds antigen targets related toa disease or condition (e.g., cancer), while the anti-CD28 binding portion binds to CD28 on an immune cell.

[0199] In certain aspects, the anti-CD28 antibodies as described herein are human antibodies or humanized antibodies. In embodiments of the present disclosure, the provided antibodies andantibody fragments thereof specifically bind to CD28 on an immune cell. In further embodiments of the present disclosure, novel anti-CD28 antibodies (or antibody fragments) are incorporated into a TCE that specifically binds to a target antigen, such as a tumor or other antigen that is a disease marker, and can specifically bind to a second antigen that can engage or otherwise stimulate T-cells, e.g., via the novel anti-CD28 antibody. In some certain aspects, such a TCE may include an additional antigen binding region that binds to another immune cell protein, e.g., CD3, to activate the immune cell. In some certain aspects, the target antigen is a tumor antigen. In some certain aspects, the tumor antigen is a pHLA-peptide complex, e.g., pHLA-CG. In some certain aspects, the tumor antigen is a pHLA-CGl peptide complex.

[0200] Provided herein are antibodies or antigen binding portions thereof that specifically bind to CD28. Such antibodies may be, for example, monoclonal antibodies (mAbs) or recombinant / chimeric antibodies (i.e., synthetic antibodies derived from synthetic nucleic acid constructs, such as viral vectors, that may also contain monoclonal Ab sequences as disclosed herein). Such antibodies or antigen binding regions thereof may be incorporated into a larger construct, such as the CGI x CD3 x CD28 TCEs, as described herein.

[0201] In an aspect, provided herein are antigen binding proteins (ABPs), such as the TCE antibodies disclosed herein, or fragments thereof, that bind HLA-CG, preferably HLA-CG1, peptide antigens disclosed herein. In certain aspects, the TCEs bind to a plurality of HLA-CG1 peptide antigen complexes, wherein each of the plurality of HLA-CG1 peptide antigen complexes comprises a distinct HLA subtype. In certain aspects, a TCE antibody or other ABP disclosed herein specifically binds to an HLA-CG1 peptide comprising CGI peptide complexed with an HLA class I molecule.

[0202] In certain embodiments, the HLA-CG (e.g., HLA-CG1) peptide is located in the peptide binding groove of an al / a2 domain of the HLA class I molecule heavy chain.

[0203] In certain aspects, described herein are TCE antibodies or fragments thereof that specifically bind a plurality of complexes comprising an HLA class I molecule and a CGI peptide.

[0204] In certain embodiments, an ABP, such as a TCE antibody, incorporating the anti-CD28 antibodies of the disclosure, does not have a binding affinity to (i) the HLA class I molecule alone; or (ii) the CG or CGI peptide alone. Thus, in some embodiments, the ABP or TCE antibody does not bind to the HLA in the absence of the CGI peptide. In some embodiments, the ABP or TCE antibody does not bind HLA-CG1 peptide in the absence of the HLA. In some embodiments, theABP or TCE antibody binds tumor cells presenting human MHC complexed with the HLA-CG1 peptide, optionally wherein the HLA-CG1 peptide is a tumor antigen characterizing the cancer. In some aspects, the ABP or TCE antibody binds a complex comprising HLA and CGI peptide when naturally presented on a cell such as a tumor cell.

[0205] The HLA-CG1 peptide antigen can be expressed on the surface of any suitable target cell including a tumor cell. In some embodiments, the ABP or TCE antibody specifically binds a complex comprising HLA and a CGI peptide, e.g., derived from a tumor. An ABP or TCE antibody can bind to each portion of an HLA-CG1 peptide complex (i.e., HLA and peptide representing each portion of the complex), which when bound together form a novel target and protein surface for interaction with and binding by the TCE antibody, distinct from a surface presented by the peptide alone or an HLA subtype alone. Generally, the novel target and protein surface formed by binding of HLA to peptide does not exist in the absence of each portion of the HLA-CG1 peptide complex. In some embodiments, the ABP or TCE antibody binds to the HLA-CG1 peptide antigen through at least one contact point with an HLA class I molecule and through at least one contact point with the HLA-CG1 peptide.

[0206] In some aspects, the affinity of an HLA-CG1 peptide TCE antibody for a non-target molecule is less than about 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, or less) 50% of the affinity for HLA-CG1 peptide. In some aspects, the affinity of an HLA-CG1 peptide ABP for a non-target molecule is less than about 40% of the affinity for HLA-CG1 peptide. In some aspects, the affinity of an HLA-CG1 peptide ABP for a non-target molecule is less than about 30% of the affinity for HLA-CG1 peptide. In some aspects, the affinity of an HLA-CG1 peptide ABP for a non-target molecule is less than about 20% of the affinity for HLA- CGI peptide. In some aspects, the affinity of an HLA- CGI peptide ABP for a non-target molecule is less than about 10% of the affinity for HLA-CG1 peptide. In some aspects, the affinity of an HLA-CG1 peptide ABP for a non-target molecule is less than about 1% of the affinity for HLA-CG1 peptide. In some aspects, the affinity of an HLA-CG1 peptide ABP for a non-target molecule is less than about 0.1% of the affinity for HLA-CG1 peptide. In certain aspects, the non-target molecule is CG and / or pHLA-CG, which does not include CGI or pHLA-CGl.

[0207] In some embodiments, the distinct HLA class I molecules are selected from any one of HLA-A*02:01, HLA-A*02:03, HLA-A*02:06, and HLA-A*02:07. In some embodiments, the HLA class I molecule is HLA-A*02:01. In some embodiments, the HLA class I molecule is HLA-A*02:03. In some embodiments, the HLA class I molecule is HLA-A*02:06. In some embodiments, the HLA class I molecule is HLA-A*02:07.

[0208] In certain embodiments, the ABP or TCE antibody binds HLA-CG1 peptide antigen with a higher affinity than its affinity to another HLA-CG1 peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:01-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG1 peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:03-CG1 peptide antigen with a higher affinity than its affinity to an HLA-CG1 peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:06-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG1 peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:07-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG1 peptide.

[0209] In certain embodiments, the ABP or TCE antibody binds HLA-A*02:01-CGl peptide antigen with a higher affinity than its affinity to a CG peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*O2: O3-CG1 peptide antigen with a higher affinity than its affinity to a CG peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:06-CGl peptide antigen with a higher affinity than its affinity to a CG peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:07-CGl peptide antigen with a higher affinity than its affinity to a CG peptide.

[0210] In certain embodiments, the ABP or TCE antibody binds HLA-CG1 peptide antigen with a higher affinity than its affinity to a CGI peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:01-CGl peptide antigen with a higher affinity than its affinity to a CGI peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:03-CGl peptide antigen with a higher affinity than its affinity to a CGI peptide. In certain embodiments, the ABP or TCE antibody binds HLA-A*02:06-CGl peptide antigen with a higher affinity than its affinity to a CGI peptide. In certain embodiments, the ABP or TCR-based mimetic antibody binds HLA-A*02:07-CGl peptide antigen with a higher affinity than its affinity to a CGI peptide.

[0211] In certain embodiments, the ABP or TCE antibody binds HLA-CG1 peptide antigen with a higher affinity than its affinity to any of HLA-A*02:01, HLA-A*02:03, HLA-A*02:06, and HLA-A*02:07 without a complexed peptide (e.g., CGI).

[0212] In some embodiments, the higher affinity is at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 100,000-fold, or at least 1 X 106-fold. In some embodiments, the dissociation constant (KD) of the higher affinity interaction ranges from at least IO’4to IO’5Molar (M), IO'5to IO'6Molar (M), IO'6to IO'7Molar (M), at least IO’7to IO’8Molar (M), at least 10'8to 10'9Molar (M), at least 10'9to IO'10Molar (M), at least IO'10to 10'11Molar (M), at least 10'11to 10'12Molar (M), or at least 10'12to 10’13Molar (M). In certain embodiments, the ABP or TCE antibody binds HLA-CG1 peptide antigen of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HLA class I molecules, where the peptide of the peptide-HLA class I complex shares at least 6 identical amino acids. In some embodiments, the distinct HLA class I molecules are selected from any one of HLA-A*02:01, HLA-A*02:03, HLA-A*02:06, and HLA-A*02:07. In some embodiments, the HLA class I molecule is HL A- A* 02:01. In some embodiments, the HLA class I molecule is HLA-A*02:03. In some embodiments, the HLA class I molecule is HLA-A*02:06. In some embodiments, the HLA class I molecule is HLA-A*02:07. In certain embodiments, the ABP or TCE antibody binds HLA-CG1 peptide antigen of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HLA class I molecules, having a dissociation constant KD) of at least 10’4to 10‘5Molar (M), 10‘5to 10'6Molar (M), 10'6to 10‘7Molar (M), at least 10'7to 10'8Molar (M), at least 10'8to 10'9Molar (M), at least 10'9to IO'10Molar (M), at least IO’10to 10'11Molar (M), at least 10'11to 10'12Molar (M), or at least 10'12to 10'13Molar (M). In certain embodiments, the ABP or TCE antibody binds HLA-CG1 of each distinct HLA class I molecule with a KD of at least 10'4to ICT13Molar (M), wherein the binding of each distinct HLA class I molecule to the ABP or TCE antibody has a distinct KD.

[0213] Affinity differences can be determined by any means known in the art. In some embodiments, such affinity differences are assessed by MSD-ECL, SPR, BLI, or flow cytometry.

[0214] In some embodiments, the ABP or TCE antibody is an ABP or TCE antibody that competes with an illustrative ABP or TCE antibody provided herein. In some aspects, the ABP or TCE antibody that competes with the illustrative ABP or TCE antibody provided herein binds the same epitope as an illustrative ABP or TCE antibody provided herein.

[0215] In some embodiments, the ABPs or TCE antibodies described herein are referred to herein as “variants.” In some embodiments, a variant is derived from any of the sequences provided herein, wherein one or more conservative amino acid substitutions are made. Conservative amino acid substitutions are described herein. In preferred embodiments, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant.In yet embodiments, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent ABP or TCE antibody.

[0216] The ABP or TCE antibody can be isolated and purified by any suitable method known in the art.

[0217] The ABP or TCE antibody can be a multi-specific antibody. The ABP or TCE antibody can bind to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HLA-CG1 peptide antigens. In certain embodiments, the ABP or TCE antibody binds 1, 2, 3, 4, 5 or more antigens that are not peptide-HLA class I molecule complex antigens. In certain embodiments, ABP or TCE antibody can bind to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HLA-CG1 peptide antigens and one additional antigen that is not an HLA-peptide complex antigen. In certain embodiments, TCE antibody can bind to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HLA-CG1 peptide antigens and one or more additional antigens that are not an HLA-peptide complex antigen on an immune cell (e.g., CD3). The immune cell can be a T-cell (e.g., a CD8+ T cell or a cytotoxic T cell). In certain embodiments, the TCE antibody binds an HLA class I CGI peptide complex on a cancer cell. In certain embodiments, the TCE antibody is a multifunctional antibody.

[0218] In certain embodiments, a TCE or other ABP further comprises a conjugated therapeutic moiety. In certain embodiments, the therapeutic moiety is known in the art for use in cancer treatment and / or inducing cancer cell death.

[0219] In certain embodiments, the selective binding of a TCE antibody to the complex comprising the HLA class I molecule and the CGI peptide induces an immune response in a cell. In certain embodiments, the immune response comprises activation of T cells. The T cell can be a CD8+ T cell, cytotoxic T cells (CTLs).

[0220] In certain embodiments, the ABP or TCE antibody or fragment is a murine antibody, a chimeric antibody, a camelid antibody, a humanized antibody, or a human antibody.

[0221] In some embodiments, the ABP or TCE comprises a monovalent antibody fragment comprising a single target molecule binding arm and an Fc region. In some embodiments, the ABP or TCE comprises a fragment antigen-binding region. In some embodiments, the ABP or TCE comprises a single chain antibody or other antibody derivative retaining the antigen specificity and the lower hinge region or a variant thereof. In some embodiments, the ABP or TCE comprises one or more single chain variable fragment antibody or derivative thereof. In some embodiments, theABP or TCE comprises a single chain variable fragment antibody in tandem format. In certain embodiments, the ABP or TCE comprises a single domain antibody. In certain embodiments, the ABP or TCE comprises a humanized single domain antibody.

[0222] In certain embodiments, the TCE molecule comprises at least two antibodies or fragments thereof that bind an immune cell. In certain aspects, at least two antibodies or fragments bind to CD3 and CD28 on an immune cell.

[0223] In certain embodiments, a TCE of the disclosure comprises: i) a trispecific antibody, and optionally ii) a dimerized hinge-CH2-CH3; wherein the trispecific antibody comprises: i) at least one antibody or fragment that binds CD3, (ii) at least one antibody or fragment that binds CD28; and iii) at least one antibody or fragment that selectively binds HLA-CG1 peptide antigen.

[0224] In certain embodiments, the TCE is in a heterodimer format. In certain embodiments, the trispecific antibody is in a heterodimer format, and comprises: (i) an antibody fragment binds CD3; (ii) an antibody fragment binds CD28, and (iii) an antibody fragment that binds HLA-CG1 peptide antigen, and (iv) a heterodimeric hinge-CH2-CH3. In certain embodiments, the antibody comprises (i) an antibody fragment binds CD3; (ii) an antibody fragment binds CD28; (iii) two antibody fragments that bind HLA-CG1 peptide antigen, and (iv) a heterodimeric hinge-CH2-CH3. In certain embodiments, the antibody is in a heterodimer format.

[0225] In certain embodiments, a TCE of the disclosure comprises (i) two antibody fragments that independently bind CD3 and CD28, (ii) two antibody fragments bind HLA-CG1 peptide antigen, and (iii) a heterodimeric hinge-CH2-CH3. In certain embodiments, the antibody is in a single chain format. In certain embodiments, the TCE does not comprise a dimerized hinge-CH2-CH3. In certain embodiments, the TCE comprises an antibody fragment that binds CD3, an antibody fragment that binds CD28, an antibody fragment that binds HLA-CG1 peptide antigen, with or without a 6XHis-tag.

[0226] In certain embodiments, the TCE antibody comprises a first polypeptide chain comprising CD3 and CD28 binding domain, wherein said first chain comprises a first single-chain variable fragment (scFv) that binds CD3 and a second scFv that binds to CD28. In certain aspects, the TCE antibody comprises a second and / or third polypeptide chain that comprise(s) the pHLA-CGl binding domain. In some aspects, the first polypeptide comprises a first hinge-CH2-CH3 and the second polypeptide comprises a second hinge-CH2-CH3; and wherein the first and second hinge-CH2-CH3 form a heterodimer. In certain embodiments, the TCE antibody comprises: i) a firstpolypeptide comprising the first and second scFv, and a second and third polypeptide chain comprising an antibody fragment that binds HLA-CG1 peptide antigen; and ii) a hinge-CH2-CH3.

[0227] In certain embodiments, the second polypeptide does not comprise an antibody fragment that binds CD3, CD28, or an antibody fragment that binds HLA-CG1 peptide antigen. In certain embodiments, the second polypeptide further comprises a third scFv comprising a second antibody fragment that binds HLA-CG1 peptide antigen. In certain embodiments, the TCE antibody comprises two first polypeptides, wherein each first polypeptide comprises the first scFv that binds CD3, a second scFv that binds to CD28 or a second scFv that binds HLA-CG1 peptide antigen. In certain embodiments, the antibody fragment that binds CD3 or CD28 comprises a first Fab.

[0228] In certain aspects, an anti-CD28 binder of the disclosure is included as part of a bispecific antibody. In certain aspects, the bispecific antibody comprises an anti-CD28 binder of the disclosure and a second binder for a target antigen, such as a tumor associated antigen or another cell-specific or disease-specific target antigen.

[0229] In certain embodiments, the antibody fragment that binds CD3 binds human CD3. In certain embodiments, the antibody fragment that binds CD3 binds CD3 with an affinity of a KD of less than 10 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis. In certain embodiments, the antibody fragment that binds CD3 binds CD3 with an affinity of a KD of less than 50 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis. In certain embodiments, the antibody fragment that binds CD3 binds CD3 with an affinity of a KD of greater than 50 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis.

[0230] In certain embodiments, the Fc region (CH2-CH3) is silenced.

[0231] In certain embodiments, the anti-CD28 binders, antibodies, and antibody fragments of the disclosure bind human CD28. In certain embodiments, the anti-CD28 binders, antibodies, and antibody fragments bind human CD28 with an affinity of a KD of less than 10 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis. In certain embodiments, the anti-CD28 binders, antibodies, and antibody fragments bind human CD28 with an affinity of a KD of less than 50 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis. In certain embodiments, the antibody fragment that binds CD28 binds human CD28 with an affinity of a KD of greater than 50 nM as determined by ELISA, SPR, BLI, flow cytometer or equivalent analysis. In certain embodiments, the Fc region (CH2-CH3) is silenced.

[0232] In certain embodiments, a TCE of the disclosure comprises a first polypeptide and a second polypeptide; wherein the first polypeptide and the second polypeptide form a dimer through at least one disulfide bond between a first hinge-CH2-CH3 of the first polypeptide and a second hinge-CH2-CH3 of the second polypeptide. In certain embodiments, the first hinge-CH2-CH3 and / or second hinge-CH2-CH3 comprises one or more amino acid substitutions that increase dimerization of the first polypeptide with the second polypeptide. In certain embodiments, the first polypeptide and second polypeptide form a homodimer through at least one disulfide bond between the first hinge-CH2-CH3 and the second hinge-CH2-CH3. In certain embodiments, the first polypeptide and second polypeptide form a heterodimer through at least one disulfide bond between the first hinge-CH2-CH3 and the second hinge-CH2-CH3. In certain embodiments, the first Fab comprises a disulfide bond between the VH and the VL. In certain embodiments, the hinge region comprises a C220S mutation. In certain aspects, a TCE of the disclosure comprises a third polypeptide chain, wherein the first polypeptide chain and third polypeptide chain form a Fab. In certain embodiments, the antibody fragment that selectively binds the HLA-CG1 peptide antigen comprises a second and third single chain variable fragment (scFv);

[0233] A composition comprising TCE antibodies described herein may be a pharmaceutical composition. Such a composition may comprise multiple TCE antibodies. Exemplary pharmaceutical compositions are described herein. The composition may be capable of eliciting an immune response. The composition may comprise an adjuvant. Suitable adjuvants include, but are not limited to 1018 ISS, alum, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand. 1998; 92:3-11). In certain aspects, alternatively or additionally, cytokines are used. Severalcytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U. S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL- 12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418). HLA surface expression and processing of intracellular proteins into peptides to present on HLA can also be enhanced by interferon-gamma (IFN-y). See, e.g., York IA, Goldberg AL, Mo XY, Rock KL. Proteolysis and class I major histocompatibility complex antigen presentation. Immunol Rev.1999;172:49-66; and Rock KL, Goldberg AL. Degradation of cell proteins and the generation of MHC class I-presented peptides. Ann Rev Immunol. 1999; 17: 12. 739-779, which are incorporated herein by reference in their entirety.

[0234] Also provided herein are host cells comprising an anti-CD28 antibody, ABP or TCE or fragment thereof disclosed herein. In some embodiments, the host cell comprises a polynucleotide encoding an anti-CD28 antibody, ABP TCE or fragment thereof. In some embodiments, the polynucleotide is heterologous to the host cell. In some embodiments, the host cell does not comprise endogenous MHC. In some embodiments, the host cell comprises an exogenous HLA class I molecule. In some embodiments, the host cell is a cultured cell from a tumor cell line. In some embodiments, the tumor cell line expresses an HLA subtype as defined by the HLA-CG1 peptide antigen.

[0235] Also provided herein are cell culture systems comprising a host cell disclosed herein and a cell culture medium. In some embodiments, the host cell expresses the HLA class I subtype as defined by the HLA-CG1 peptide antigen, and the cell culture medium comprises the HLA-CG1 peptide as defined by the HLA- CGI peptide antigen.

[0236] An overview of the present disclosure is to identify TCR like antibodies binding to MHC-1 restricted tumor-specific peptide GG1 derived from Cathepsin G (CG) and engineer into a TCE, as described herein. CG is a myeloid azurophil granule serine protease that is involved in host immunity, cleavage of inflammatory mediators and receptors, and degradation of extracellular matrix components and leukemogenesis. CGI (FLLPTGAEA; SEQ ID NO: 61) is a 9-mer peptide derived from the cathepsin G protein leader sequence, presented by HLA class I, specifically HLA-A*02:01. Cathepsin G is highly expressed in AML, and in particular it has higher expression in leukemia stem cells (LSC) compared to normal hematopoietic stem cells (HSC). Functional CG-CTL can be detected in AML patients following allo-SCT. In addition to AML, CGI is expressed by lymphoid leukemia, specifically ALL and CLL, and possibly lung cancer. Clinical and preclinical data suggest HLA-A2 restricted CGI complex could be a good target in development of antibody based anti-tumor therapy, in particular for cancers and tumors related to hematological or myeloid malignancies.

[0237] Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells. These malignancies can be present in the bone marrow and peripheral blood. They can result from genetic and epigenetic alterations that perturb key processes such as self-renewal, proliferation and impaired differentiation.

[0238] Myeloid malignancies can be categorized as five types: (1) acute myeloid leukemia (AML); (2) myelodysplastic syndromes (MDS); (3) myeloproliferative neoplasms (MPN); (4) myelodysplastic and myeloproliferative (MDS / MPN) neoplasms; and (5) myeloid neoplasms associated with eosinophilia and abnormalities of growth factor receptors derived from platelets or fibroblasts.

[0239] According to the present disclosure, myeloid malignancies that express antigens contemplated by the present disclosure (CGI, for example) include, for example, AML, ALL, and CLL. Compositions and methods according to the present disclosure can detect, diagnose, treat, or otherwise be useful for hematological or myeloid malignancies, in particular those characterized by high expression of CGI (high expression compared to a WT or non-pathogenic subject).

[0240] Additional non-myeloid malignancies, for example lung cancer (which also may express high CGI levels), are also contemplated by the present disclosure as cells expressing antigens of interest relating to hematological or myeloid malignancies may also be expressed by cells involved in non-myeloid pathologies.

[0241] In certain aspects, compositions, pharmaceutical compositions, and methods according to the present disclosure can be utilized, for example, to treat, diagnose, or otherwise detect a hematological or myeloid malignancy. Compositions, pharmaceutical compositions, and methods according to the present disclosure can be utilized, for example, to treat, diagnose, or otherwise detect a non-myeloid malignancy of which antigens presented by hematological or myeloid malignancies are also presented (for example CGI or a derivative thereof, in particular, CGI (FLLPTGAEA) - a 9-mer peptide derived from cathepsin G protein leader sequence, presented by HLA class I, specifically HLA-A*02:01).

[0242] The present disclosure provides anti-CD28 antibodies and binding portions thereof that may be incorporated into larger antibodies, compositions and methods for treating, diagnosing, or otherwise detecting aspects of diseases or disorders such as autoimmune diseases and cancers, including hematological or myeloid malignancies (for example in a subject having or suspected of having a hematological or myeloid malignancy, an animal model, an in vitro tissue culture model, and the like). Antibodies, including TCE antibodies, or antigen binding portions thereof of the disclosure, incorporating the CD28 binders, specifically or selectively bind antigen targets related to cancers, including hematological or myeloid malignancies, are provided herein. In certain aspects, antibodies as described herein are human antibodies. In embodiments of the present disclosure, the provided TCEs incorporate a novel anti-CD28 antibody, and antibody fragments thereof specifically bind to HLA-A2 / CG1 as a tumor antigen. In further embodiments of the present disclosure, TCE antibodies (or antibody fragments) specifically bind to HLA-A2 / CG1 as a tumor antigen, and can specifically bind to antigens that can engage T-cells, for example CD3 and CD28 expressed on the surface of a T-cell. In the context of this disclosure, HLA-A2 / CG1 is a myeloid malignancy tumor antigen. However, it is recognized that HLA-A2 / CG1 may also be a tumor antigen for other types of cancer and, as such, the various compositions and methods provided herein would be useful with respect to other cancers as well.

[0243] As used herein, the terms “specifically binds to”, “specific for”, “selectively binds”, “targets”, “selectively targets”, “selective”, and the like, for example, of a hematological or myeloid malignancy antigen or an epitope on a protein related to a hematological or myeloid malignancy, each mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target

[0244] An antibody, as used herein, can refer to an intact antibody (e., an intact immunoglobulin), a portion of an antibody (including multispecific antibodies such as TCEs described herein) or an antigen binding fragment, including as part of an anti-CD28 binder or TCE. Antigen binding fragments can comprise at least one antigen binding domain. One example of an antigen binding domain is an antigen binding domain formed by a VH-VL or VL-VH dimer.An antibody (including a TCE) or antigen binding fragment can be described by the antigen to which they specifically bind. In some embodiments, the antigen binding fragments provided herein can comprise any of the antigen binding portions (also referred to as antigen binding domains) described below.

[0245] The VH and VL regions can be further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) CDR sequences on the heavy chain (VH) may be designated as CDRH 1, 2, 3, while CDR sequences on the light chain (VL) may be designated as CDRL1, 2, 3.

[0246] Provided herein are antibodies (such as TCEs) and / or antigen binding portions thereof that specifically bind to antigens related to cancers, including hematological or myeloid malignancies.

[0247] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided. In some cases, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0248] In each case, where a specific amino acid sequence is recited, embodiments consisting essentially of a sequence having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided. In some cases, where a specific amino acid sequence is recited, embodiments consisting essentially of a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0249] In each case, where a specific amino acid sequence is recited, embodiments consisting of a sequence having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided. In some cases, where a specific amino acidsequence is recited, embodiments consisting of a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0250] The disclosure provides a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 133, 144-146, or a fragment thereof. The disclosure also provides a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 95% identical (for example, at least 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 133, 144-146, or a fragment thereof. The disclosure also provides a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 133, 144-146, or a fragment thereof. The disclosure also provides a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 133, 144-146, or a fragment thereof.

[0251] The disclosure also provides a TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a or myeloid malignancy antigen), CD3 and CD28, wherein the TCE, antibody, or antigen binding portion thereof comprises a pHLA-CGl binding domain light chain variable region comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 132, 147-149, or a fragment of any thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical) to any of SEQ ID NOS: 132, 147-149, or a fragment of any thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 95% identical (for example, at least 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 132, 147-149, or a fragment of any thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 132, 147-149, or a fragment of any thereof. In certain aspects, the pHLA-CGl binding domain light chain variable region comprises an amino acid sequence that as set forth in any one of SEQ ID NOS: 132, 147-149, or a fragment of any thereof.

[0252] The disclosure also provides a TCE, antibody, antigen binding portion thereof that comprises a CD3 binding domain and a CD28 binding domain. In certain aspects, the CD3 binding domain and CD28 binding domain are on the same peptide chain. In certain aspects, the peptide chain is a different peptide chain from one or more peptide chains comprising the pHLA-CGl binding domain. In certain aspects, the CD3 and CD28 binding domains are scFvs. In certain aspects, the CD3 and CD28 scFvs are joined by a linker. In certain aspects, the TCEs of the disclosure comprise a silent Fc IgGl.

[0253] In some certain aspects, the antibodies, including TCEs of the disclosure comprise a peptide chain comprising the CD3 and CD28 binding domains. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 95% identical (for example, at least 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOS: 150-152, or one or more fragments thereof. In certain aspects, the peptide chain comprising the CD3 and CD28 binding domains comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 150-152, or one or more fragments thereof.

[0254] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 1 -27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 1-27, 133, or 134. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 1-27, 133, or 134.

[0255] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 28-54 or 132.

[0256] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 28-54 or 132.

[0257] In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen (e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 90% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen, e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen, e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs 28-54 or 132. In some embodiments, the TCE, antibody, or antigen binding portion thereof that specifically binds to a cancer antigen, e.g., a hematological or myeloid malignancy antigen), CD3 and CD28, comprises a pHLA-CGl binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to any of SEQ ID NOs 1-27, 133, or 134 and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 28-54 or 132.

[0258] In certain aspects, the antigen is CGI, in particular, a 9-mer peptide derived from cathepsin G protein leader sequence, presented by HLA class I, specifically HLA-A*02:01 (SEQ ID NO:61, for example).

[0259] In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 57, 128, 139, or a fragment of any thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 57, 128, 139, or a fragment of any thereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 57, 128, 139, or a fragment of anythereof. In some exemplary TCEs of the disclosure, the CD3 binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NO: 57, 128, 139, or a fragment of any thereof.

[0260] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 90% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0261] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence having atleast 95% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 95% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0262] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence having at least 99% identity with an amino acid sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0263] Exemplary TCEs of the disclosure that bind to pHLA-CGl on a target cell and CD3 and CD28 on an immune cell may comprise a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises an amino acid sequence as set forth inSEQ ID NO: 144, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 147, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 150, or a fragment thereof; (ii) the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 145, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 148, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 151, or a fragment thereof; or (iii) the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 146, or a fragment thereof, the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 149, or a fragment thereof, and the third peptide chain comprises a sequence as set forth in SEQ ID NO: 152, or a fragment thereof.

[0264] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162.

[0265] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable light chain region comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157.

[0266] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 153. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 158 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 153.

[0267] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 154. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 154.

[0268] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence that is at least 90%identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 159 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 155. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 160 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 155.

[0269] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 156. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 161 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 156.

[0270] In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising anamino acid sequence that is at least 95% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence as set forth in SEQ ID NO: 157. In some exemplary TCEs of the disclosure, the CD28 binding domain comprises a variable heavy chain region comprising an amino acid sequence as set forth in SEQ ID NO: 162 and a variable light chain region comprising an amino acid sequence as set forth in SEQ ID NO: 157.

[0271] The amino acid residue sequences provided herein are set forth in single-letter amino acid code which can be used interchangeably with three-letter amino acid code. An amino acid refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. The twenty natural or genetically encoded alpha-amino acids are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). The structures of these twenty natural amino acids are shown in, e.g., Stryer et al., Biochemistry, 5thed., Freeman and Company (2002). The term amino acid also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs.

[0272] As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the hematological or myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof described herein, for example, in the heavy chain variable region and / or light chain variable region, can occur that do not alter the nature or function of the antibodies or antigen binding fragments thereof. Such modifications include conservative amino acids substitutions, such that each recited sequence optionally contains one or more conservative amino acid substitutions. The list provided below identifies examples of groups that contain amino acids that are conservative substitutions for one another; these groups are exemplary as other conservative substitutions are known to those of skill in the art.1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)

[0273] By way of example, when an aspartic acid at a specific residue is mentioned, also contemplated is a conservative substitution at the residue, for example, glutamic acid. Nonconservative substitutions, for example, substituting a proline with glycine, are also contemplated.

[0274] In some instances, the affinity of antigen-specific antibodies or antigen binding fragments thereof may be optimized through mutations to increase or decrease affinity as desired based on one or more of the known characteristics of the binding interaction with the cognate antigen, the structure of either or both of the antibodies or fragments thereof, or the antigen. In some instances, the mutations permit facile elution of purified antibodies or fragments thereof under desirable elution conditions during isolation and purification.

[0275] Methods of generating and screening for antibodies and antigen binding fragments thereof as provided in this disclosure are described in the Examples and are well-known in the art. Methods of further modifying antibodies for enhanced properties (e g., enhanced affinity, chimerization, humanization) as well as generating antigen binding fragments, as described herein, are also well-known in the art.

[0276] The present disclosure also encompasses antibodies, including TCEs, or fragments thereof that bind to the same epitope of cancer (e g., hematological or myeloid malignancy) antigens as the antibodies disclosed herein. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0277] The present disclosure also encompasses bi-specific and higher multispecific antibodies or fragments thereof that bind to the same epitope of hematological or myeloid malignancy antigens as the antibodies disclosed herein, as well as other antigens. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0278] The term epitope, as used herein, means a component of an antigen capable of specific binding to an antibody or antigen binding fragment thereof. Such components optionally comprise one or more contiguous amino acid residues and / or one or more non-contiguous amino acid residues. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations.

[0279] The present disclosure also provides chimeric antibodies. The term chimeric antibody refers to an antibody in which a component of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0280] A human antibody is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources, genetically modified non-human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0281] In some embodiments, a TCE, antibody, or antigen binding fragment thereof provided herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, a TCE, antibody, or antigen binding fragment thereof includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bi- or tri-valent and bi- or tri-specific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen. A TCE, antibody, or antigen binding fragment thereof can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR-grafted, or an in vitro generated antibody. A TCE, antibody, or antigen binding fragment thereof can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. A TCE, antibody, or antigen binding fragment thereof can also have a light chain chosen from either kappa or lambda light chains.

[0282] As used herein, the term monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are the same or substantially similar and that bind the same epitope(s), except for variants that can normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target, for example, by affinity maturation, to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0283] Antigen binding fragments, e.g., of a TCE or of another antibody molecule, are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH I domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0284] In certain embodiments, a TCE, antibody, or antigen binding fragment thereof, and compositions comprising such TCEs, antibodies, or antigen binding fragments thereof, as provided herein, are distinguishable from naturally occurring antibodies and compositions in one or more respects. Such distinguishable antibodies and compositions may be referred to as “synthetic,” or may be identified by the proviso that the antibody or composition “is not naturally occurring” or affirmatively as “non-naturally occurring.” As used herein the terms “corresponding antibody,” and “corresponding to” describes the relationship between (1) an antibody characterized by six specific CDR sequences of the antibodies described in the Examples below and (2) a synthetic antibody comprising the same six CDR sequences. Synthetic antibodies of this disclosure may differ in structure from naturally occurring antibodies with the same CDRs. That is, synthetic antibodies identified by specified CDRs may be structurally different from antibodies comprising the specified CDRs that are described in the Examples below. Possible differences for synthetic antibodies include variable region sequences that differ corresponding naturally occurring antibodies, different light chain sequences (i.e. lambda type instead of kappa type or vice versa), different isotypes, different allotypes, and different constant domain variants. These differences are discussed in more detail below. In some embodiments, the synthetic antibody is an engineered polypeptide, also referred to as a recombinant polypeptide, that is made using conventional protein and antibody engineering molecular biology, chemical, and biochemical methods as described below, including, but not limited to, those described in the Examples below.

[0285] In one approach, a TCE, antibody, or antigen binding fragment as provided in this disclosure may comprise one or more CDRs of a clone described in Table la and / or Table lb.Table la: Embodiments of Heavy-chain CDRs according to the present disclosureTable lb: Embodiments of Light-chain CDRs according to the present disclosure

[0286] In some embodiments, an antibody comprising an anti-CD28 binder of the disclosure, such as a TCE antibody, comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin producing B cell, and further comprises a kappa or lambda light chain constant region. In some embodiments, the light chain constant region (kappa or lambda) is from the same type of light chain (i.e., kappa or lambda) as the light chain variable region that was derived from the immunoglobulin producing B cell; as a nonlimiting example, if an IgE-producing B cell comprises a kappa light chain, then the antibody that is produced can comprise the light chain variable region from the IgE-producing B cell and further comprises a kappa light chain constant region.

[0287] In some embodiments, an antibody comprising an anti-CD28 binder of the disclosure, such as a TCE antibody, comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin-producing B cell, and further comprises a heavy chain constant region having an IgG isotype (e.g., IgG4), an IgA isotype (e.g., IgAl), an IgM isotype, an IgD isotype, or that is derived from an IgG, IgA, IgM, or IgD isotype (e.g., is a modified IgG4 constant region). It will be appreciated by a person of ordinary skill in the art that the different heavy chain isotypes (IgA, IgD, IgE, IgG, and IgM) have different effector functions that are mediated by the heavy chain constant region, and that for certain uses it may be desirable to have an antibody that has the effector function of a particular isotype (e.g., IgG).

[0288] In some embodiments, an antibody comprising an anti-CD28 binder of the disclosure, such as a TCE antibody, comprises a native (i.e., wild-type) human IgG, IgA, IgM, or IgD constant region. In some embodiments, the antibody comprises a native human IgGl constant region, a native human IgG2 constant region, a native human IgG3 constant region, a native human IgG4 constant region, a native human IgAl constant region, a native human IgA2 constant region, a native human IgM constant region, or a native human IgD constant region. In some embodiments, the antibody comprises a heavy chain constant region that comprises one or more modifications. It will be appreciated by a person of ordinary skill in the art that modifications such as amino acid substitutions can be made at one or more residues within the heavy chain constant region that modulate effector function. In some embodiments, the modification reduces effector function, e.g., results in a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. In some embodiments, the modification (e.g., amino acid substitution) prevents ex vivo Fab arm exchange, which canintroduce undesirable effects and reduce the therapeutic efficacy of the antibody. See, e.g., Silva et al., J Biol Chem, 2015, 280:5462-5469.

[0289] In some embodiments, an antibody comprising an anti-CD28 binder of the disclosure, such as a TCE antibody, comprises a native (i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgGl, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgAl or IgA2 and comprises one or more modifications that modulate effector function. Ini some embodiments the antibody comprises a human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgGl, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgAl or IgA2. In some embodiments, the antibody comprises a native i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgGl, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgAl or IgA2 and comprises one, two, three, four, five, six, seven, eight, nine, ten or more modifications (e.g., amino acid substitutions). In some embodiments the constant regions includes variations (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions) that reduce effector function.

[0290] Synthetic antibodies comprising an anti-CD28 binder of the disclosure, such as a TCE antibody, may comprise variations in heavy chain constant regions to change the properties of the synthetic antibody relative to the corresponding naturally occurring antibody. Exemplary changes include mutations to modulate antibody effector function (e.g., complement-based effector function or FcyR-based effector function), alter half-like, modulate co-engagement of antigen and FcyRs, introduce or remove glycosylation motifs (gly co-engineering). See Fonseca et al., 2018, “Boosting half-life and effector functions of therapeutic antibodies by Fc-engineering: An interaction-function review” Bit J Biol Macromol. 19:306-311; Wang et al., 2018, “IgG Fc engineering to modulate antibody effector functions” Protein Cell 2018, 9(l):63-73; Schlothauer, 2016, “Novel human IgGl and IgG4 Fc-engineered antibodies with completely abolished immune effector functions,” Protein Engineering, Design and Selection 29(10):457-466; Tam et al., 2017, “Functional, Biophysical, and Structural Characterization of Human IgGl and IgG4 Fc Variants with Ablated Immune Functionality” Antibodies 6, 12, each incorporated herein by reference for all purposes.

[0291] In some embodiments, the heavy chain variable region and / or the light chain variable region of an antibody of the disclosure has an identical sequence to the heavy chain variable region and / or the light chain variable region encoded by the immunoglobulin producing single B cell. In some embodiments, the heavy chain variable region and / or the light chain variable region of the antibody comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0292] The heavy chain variable region sequence and / or light chain variable region sequence of an antibody described herein can be engineered to comprise one or more variations in the heavy chain variable region sequence and / or light chain variable region sequence. In some embodiments, the engineered variation(s) improves the binding affinity of the antibody for a disease or disorder (e.g., cancer). In some embodiments, the engineered variation(s) reduces the cross-reactivity of the antibody for a non-targeted endogenous protein, thereby reducing potential side effects.

[0293] In some embodiments, the engineered variation is a variation in one or more CDRs, e.g., an amino acid substitution in a heavy chain CDR and / or a light chain CDR as described herein. In some embodiments, the engineered variation is a variation in one or more framework regions, e.g., an amino acid substitution in a heavy chain framework region and / or a light chain framework region. In some embodiments, the engineered variation is a reversion of a region of the heavy chain and / or light chain sequence to the inferred naive sequence. Methods for determining an inferred naive immunoglobulin sequence are described in the art. See, e.g., Magnani et al., PLoS Negl Prop Dis, 2017, 11:e0005655, doi:10.1371 / joumal.pntd.0005655

[0294] In some embodiments, affinity maturation is used to engineer further mutations that enhance the binding affinity of the antibody for a disease or disorder, e.g., a hematological or myeloid malignancy, or enhance the cross-reactivity of the antibody for a second target, disease or disorder, e.g., a second hematological or myeloid malignancy or other non-myeloid related pathology. Methods for performing affinity maturation are known in the art. See, e.g., Renaut et al., Methods Mol Biol, 2012, 907:451-461.

[0295] Antibody molecules can also be or comprise single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived fromantibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0296] In some embodiments, an antigen binding fragment can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) J. Biol. Chem. 274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is screened, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e g., Hey et al. (2005) TRENDS Biotechnol 23(10): 514-522.

[0297] Synthetic antibodies of this disclosure may differ from naturally occurring compositions in at least one or more of the following respects: (i) composition comprises antibodies that are purified, i.e., separated from tissue or cellular material with which they are associated in the human body, and optionally in an manufactured excipient or medium; and / or (ii) antibody compositions according to the present disclosure contain a single species of antibody (are monoclonal) such that all antibodies in the composition have the same structure and specificity.

[0298] The antigen-specific antibodies (including multispecific antibodies, such as TCEs) or antigen binding fragments thereof disclosed herein may be produced by recombinant expression in a human or non-human cell. Synthetic antibody-producing cells include non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells; heavy chains, light chains, or both heavy and light chains; and human B cells thatproduce heavy chains or light chains, but not both heavy and light chains. Synthetic antibodies of this disclosure may be heterologously expressed, in vitro or ex vivo, in cells other than human B cells, such as non-human cells and human cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.

[0299] The antigen-specific antibodies or antigen binding fragments thereof disclosed herein (e.g., the TCE antibodies disclosed herein) can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding the antibody or antigen binding fragment thereof can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0300] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), CMV, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0301] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPC>4 precipitation, liposome fusion, cationicliposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0302] Appropriate host cells for the expression of antibodies or antigen binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli. fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines, CHO cell lines), as well as primary cell lines.

[0303] In some embodiments, an antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2): 155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2): 147-157.

[0304] The antibodies and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell and are easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see Ausubel et al. (1988) Current Protocols in Molecular Biology Wiley & Sons; and Green and Sambrook (2012) Molecular Cloning— A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells varies depending on a number of factors and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0305] In vitro methods are also suitable for preparing monovalent antibodies, or fragments thereof. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in International Application Publication No. WO 94 / 29348, U. S. Patent No. 4,342,566, and Harlow and Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0306] The Fab fragments produced in antibody digestion can also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0307] One method of producing proteins comprising the provided antibodies or fragments is to link two or more peptides or polypeptides together by protein chemistry techniques (or recombinant DNA techniques). For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). Those of skill in the art readily appreciate that a peptide or polypeptide corresponding to the antibody provided herein, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W. H. Freeman and Co., N. Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer Verlag Inc., NY). Alternatively, the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.

[0308] For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides,or whole protein domains (Abrahmsen et al., Biochemistry, 30:4151 (1991 )). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., Science, 266:776779 (1994)). The first step is the chemosei ective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Baggiolini et al., FEBS Lett. 307:97-101 (1992); Clark et al., J. Biol. Chem. 269:16075 (1994); Clark et al., Biochemistry 30:3128 (1991); Rajarathnam et al., Biochemistry 33:6623-30 (1994)).

[0309] Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (nonpeptide) bond (Schnolzer et al., Science 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

[0310] Recombinant techniques can also be used to modify antibodies or antigen binding fragments thereof. For example, amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity. Insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues can also be made (and are contemplated by the present disclosure), provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody, or antigen binding fragment thereof can be made. Such methods are readily apparent to a skilled practitioner in the art and can include site specific mutagenesis of the nucleic acid encoding the antibody or fragment thereof. (Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)).

[0311] Following expression, the antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) Protein Purification, 3rdedition, Springer- Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof is necessary.

[0312] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

[0313] Any of the antibodies, including anti-CD28 antibodies and TCEs incorporating the anti-CD28 antibodies, or antigen binding fragments thereof described herein can be modified. The modifications can be covalent or non-covalent modifications, and can include one or more amino acid substitutions that change the properties of the antigen-specific antibodies or antigen binding fragments thereof. Such modifications can be introduced into the antibodies or antigen binding fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, or basepair mutations in a nucleotide sequence encoding the antigen-specific antibodies or antigen binding fragments thereof. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments. In some instances, antigen-specific antigen binding fragments may be labeled by a variety of means for use in diagnostic and / or pharmaceutical applications.

[0314] In some embodiments, the antibodies or antigen binding fragments thereof described herein may have a modification comprising one or more amino acid substitutions that provide reduced hydrophobicity and reduce the potential for aggregation, thereby improving the binding or therapeutic capacity of an antibody or an antigen-binding fragment thereof. Such amino acid substitutions can be introduced by changing one or more nucleotides in the polynucleotide encoding the antibody or antigen-binding fragment such that the triplet codon for the amino acid residue position where the modification is to be introduced is replaced with the triplet codonencoding the amino acid substitution. Tn some embodiments, the modification may comprise a single amino acid substitution. In some embodiments, the modification may comprise multiple amino acid substitutions. In some embodiments, the modification may be a substitution of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids. In some embodiments, the amino acid substitution may be located in a CDR.

[0315] In some embodiments, the antibodies or antigen binding fragments thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQ ID NO:58), polyhistidine (6-His; HHHHHH (SEQ ID NO:59)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO:60)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g.,32P,33P,14C,125I,131I,35S, and3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.

[0316] Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages,a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful crosslinkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

[0317] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([123I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or chelate (e.g., to DOTA or DTP A), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules / chelates containing them to the antibodies or antigen binding fragments described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and / or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U. S. Patent No. 6,001,329).

[0318] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein or fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0319] In some embodiments, the antibodies or fragments can be modified, e.g., with a moiety that improves the stabilization and / or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al. (2010) Int J Pharm 387(1-2): 110-119). The stabilization moiety can improve the stability, or retention of, the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0320] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U. S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

[0321] The antibodies and antigen binding fragments thereof and molecules comprising such antibodies and antigen binding fragments thereof discussed above (e.g., Abs, bi-specific Abs) may be produced by recombinant expression in a human or non-human cell. Synthetic antibodyproducing cells include non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells expressing heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy chains or light chains, but not both heavy and light chains. Synthetic antibodies of this disclosure may be heterologously expressed, in vitro or ex vivo, in cells other than human B cells, such as non-human cells and human cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.

[0322] The antibodies and antigen binding fragments thereof and molecules comprising them described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding the antibody or antigen binding fragment thereof can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptionterminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0323] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), CMV, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0324] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0325] Compositions comprising an antigen-specific, including bispecific or multi-specific antibodies (e g., a TCE as described herein) incorporating an anti-CD28 antibody or antigen binding fragment thereof, of the present disclosure and a pharmaceutically acceptable carrier are also provided. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used in a subject with a disease or disorder that would benefit from any of the antigen-specific antibodies or antigen binding fragments thereof described herein.

[0326] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulationmaterial(s) are for s.c. and / or I V. administration. Tn certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the hematological or myeloid malignancy antigen-specific antibody or antigen binding fragment thereof.

[0327] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition comprising a hematological or myeloid malignancy antigen-specific antibody or antigen binding fragment thereof disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington - The Science and Practice of Pharmacy^ 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising a hematological or myeloid malignancy antigen-specific antibody or antigen binding fragment thereof disclosed herein can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some instances, the cryo-preservative may be sucrose or trehalose. In some instances, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0328] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0329] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5-6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5. In an embodiment, the pH is 7.2.

[0330] In certain embodiments when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an antigen-specific (e.g., cancer or disease antigen) antibody or antigen binding fragment thereof in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which a hematological or myeloid malignancy antigen-specific antibody or antigen binding fragment thereof is formulated as a sterile, isotonic solution and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0331] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, an antigen-specific antibody or antigen binding fragment thereof can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising an antigen-specific antibody or antigen binding fragment thereof can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in International Application Publication No. WO / 1994 / 020069, which describes pulmonary delivery of chemically modified proteins.

[0332] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, an antigen-specific antibody or antigen binding fragment thereof that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsulecan be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of an antigenspecific antibody or antigen binding fragment thereof. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0333] In certain embodiments, a pharmaceutical composition can involve an effective quantity of an antigen-specific antibody or antigen binding fragment thereof in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0334] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving an antigen-specific antibody or antigen binding fragment thereof in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, International Application Publication No. WO / 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U. S. Patent No. 3,773,919; U. S. Patent No. 5, 594,091; U. S. Patent No. 8,383,153; U. S. Patent No. 4,767,628; International Application Publication No. WO1998043615, Calo, E. et al. (2015) Eur. Polymer J 65:252-267 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1993) Biopolymers 22:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al. (1981) J Biomed Mater Res. 15: 167-277; and Langer (1982) Chem Tech 12:98-105), ethylene vinyl acetate (Hsu and Langer (1985) J Biomed Materials Res 19(4):445-460) or poly-D(-)-3-hydroxybutyric acid (European Patent No. EP0133988). In certainembodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent No. EP 036,676; and U. S. Patent Nos. 4,619,794 and 4,615,885).

[0335] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0336] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0337] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0338] In certain embodiments, the effective amount of a pharmaceutical composition comprising an antigen-specific antibody or antigen binding fragment thereof to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which an antigen-specific antibody or antigen binding fragment thereof is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0339] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of an antigen-specific antibody or antigen binding fragment thereof in the formulationused. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate doseresponse data.

[0340] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0341] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0342] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising an antigen-specific antibody or antigen binding fragment thereof in an ex vivo manner. In such instances, cells that have been removed from a subject may be exposed to a pharmaceutical composition comprising an antigen-specific antibody or antigen binding fragment thereof after which the cells are subsequently implanted back into the subject.

[0343] In certain embodiments, an antigen-specific antibody or antigen binding fragment thereof can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulated to avoid infdtration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by a subj ect’ s immune system or by other detrimental factors from the surrounding tissues.

[0344] The antigen-specific antibodies, including TCEs, incorporating an anti-CD28 antibody or antigen binding fragment thereof provided in this disclosure are suited for ex vivo use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies or fragments thereof in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize the antibodies according to the present disclosure are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of antigens using the antibodies according to the present disclosure can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0345] The antibodies, including TCEs, incorporating anti-CD28 antibodies and fragments thereof according to the present disclosure can be bound to many different carriers and used to detect the presence of a disease or disorder, e.g., a hematological or myeloid malignancy. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for purposes according to the present disclosure. Those skilled in the art will know of other suitable carriers for binding antibodies according to the present disclosure, or will be able to ascertain such, using routine experimentation.

[0346] For purposes of this disclosure, diseases and disorders may be detected by the provided antibodies fragments thereof when the virus is present in biological fluids and tissues from a subject that may have diseases and disorders. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum or the like; a solid or semi-solid such as tissues, feces, or the like; or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

[0347] Another approach to assessing whether a subject has a disease or disorder is to determine if the B cell receptor (BCR) repertoire of the subject includes a coding sequence for one of the specific antibodies provided in this disclosure. An exemplary method of this type of sequence analysis is described in the art. For example, RNA from whole blood or B cells (PBMCs) can be used as amplifying CDR sequences. CDRs of the heavy chain, light chain, or both may be sequenced, with analysis of either or both of lambda or kappa chain sequences. Primer pools are designed to result in wide-spread amplification of the BCR V(D)JC sequences in the sample. Reverse transcription is then performed to create cDNA sequences corresponding to the BCR coding sequences, which may be amplified for subsequent sequence analysis. Next-generation sequencing of the amplified cDNA library can then be performed. Sequence analysis is used to assess the identity of the BCR V(D)JC sequences in the sample and to determine the percent identity thereof to the antibodies described herein. Various commercial services are also available for performing BCR repertoire analysis (e.g., Magic™ BCR Repertoire Analysis by Creative Biolabs). The presence of BCR sequences encoding the antibodies described in this disclosure in the subject’s sample may be indicative that the subject has a disease or disorder or has previously been affected by a disease or disorder.

[0348] In using the provided antibodies and fragments thereof for the ex vivo detection of target antigen, the detectably labeled antibody or fragment thereof of the disclosure is given in a dose which is diagnostically effective. The term “diagnostically effective” means that the amount of detectably labeled antibody is administered in sufficient quantity to enable detection of the site having cells expressing the antigen and thus the antigen peptide for which the antibodies are specific.

[0349] The concentration of detectably labeled antibody or fragment thereof which is administered should be sufficient such that the binding to cells presenting the target antigen is detectable compared to the background. Further, it is desirable that the detectably labeled antibody or fragment thereof be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

[0350] As a rule, the dosage of detectably labeled antibody or fragment thereof for ex vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of antibody can vary from about 0.01 ng / kg to about 50 mg / kg, preferably 0.1 mg / kg to about 20 mg / kg, most preferably about 0.1 mg / kg to about 2 mg / kg. Such dosages may vary,for example, depending on whether multiple injections are given, on the tissue being assayed, and other factors known to those of skill in the art.

[0351] For ex vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting an appropriate radioisotope. The radioisotope chosen must have a type of decay which is detectable for the given type of instrument. Still another important factor in selecting a radioisotope for ex vivo diagnosis is that the half-life of the radioisotope be long enough such that it is still detectable at the time of maximum uptake by the target, but short enough such that deleterious radiation with respect to the host is acceptable. Ideally, a radioisotope used for ex vivo imaging will lack a particle emission but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras. For ex vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTP A) and ethylenediaminetetra-acetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the antibodies according to the present disclosure are11'in,97Ru,67Ga,68Ga,72As,89Zr and2O1T1.

[0352] The antigen specific antibodies and antigen binding fragments thereof can also be labeled with a paramagnetic isotope for purposes of ex vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually, gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include157Gd,35Mn,162Dy,52Cr and56Fe.

[0353] The provided antibodies and antigen binding fragments can be used / / / vitro and ex vivo to monitor the course of a disease therapy. Thus, for example, by measuring the increase or decrease in the number of cells presenting a target antigen or changes in the concentration of the target antigen present on the cells and / or in the body or in various body fluids, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating the antigen-expression related disease is effective.

[0354] The compositions described herein are useful in, inter alia, methods for treating disorders correlated with antigen expression in a subject. As used herein, the term subject means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs,cats, mice, rats, cows, horses, camels, goats and sheep. Tn some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have cancer. In some embodiments, the subject is diagnosed with a cancer. In some embodiments, the subject is a human that is suspected of having a cancer correlated with elevated antigen expression.

[0355] As used herein, administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a peptide-specific antibody or antigen binding fragment provided herein or a construct encoding same) into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0356] The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavity, oral, intracranial injection, or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion. Techniques for preparing inj ectate or infusate delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those of skill in the art can readily determine the various parameters and conditions for producing antibody injectates or infusates without resort to undue experimentation.

[0357] Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U. S. Patent Application Publication No. 20080241223; U. S. PatentNos. 5,501,856; 5,164,188; 4,863,457; and 3,710,795. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants,electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems. In some embodiments, an antibody or antigen binding fragment of the present disclosure is therapeutically delivered to a subject by way of local administration.

[0358] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and the like.

[0359] Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying or preventing metastasis. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a cancer in a subject by administering an antibody as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0360] The principal symptoms can include (without intending to be limiting) bone pain, nausea, constipation, loss of appetite, mental fogginess or confusion, fatigue, frequent infections, weightloss, weakness or numbness in the legs, excessive thirst, easily fractured or broken bones, anemia, leukopenia, thrombocytopenia, excessive urination, hypercalcemia, spinal cord compression, kidney dysfunction, hyperviscosity, and the like.

[0361] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of an antigen-specific antibody or antigen binding fragment thereof that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease are ameliorated, or the likelihood of the disease developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating a cancer in a subject, can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole cancer antigen-specific antibody may be required to treat a subject with a cancer as compared to the dose of a fragment of a cancer antigen-specific antibody (e.g., Fab’ antibody fragment) required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or extent of the cancer. For example, a subject that has had a previous cancer may require administration of a different dosage of cancer antigen-specific antibody or antigen binding fragment thereof than a subject who has not previously had the cancer. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some instances, a therapeutically effective amount may vary from about 0.001 ng / kg to about 50 mg / kg, preferably from about 0.001 ng / kg to about 20 mg / kg, most preferably from about 0.001 ng / kg to about 2 mg / kg, in one or more dose administrations daily, atleast once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, bi-weekly, or monthly. In some instances, the cancer antigen-specific antibody or antigen binding fragment thereof is administered for 2 to 5 or more consecutive days, weeks or months in order to avoid “rebound” of a disease or other pathology being treated from occurring.

[0362] A pharmaceutical composition can include a therapeutically effective amount of a antigenspecific antibody or antigen binding fragment thereof described herein. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the administered antigen-specific antibody or antigen binding fragment thereof, or the combinatorial effect of the antigen-specific antibody or antigen binding fragment thereof with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition. In certain aspects, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01, or about 0.005, or about 0.0001 ng / kg, or any intervening dose between about 0.001 ng / kg and 1 mg / kg.

[0363] Suitable human doses of any of the antigen-specific antibody or antigen binding fragment thereof described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1 ): 523 -531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0364] Toxicity and therapeutic efficacy of such antigen-specific antibodies or antigen binding fragments thereof can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. An antigen-specific antibody or antigen binding fragment thereof that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0365] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage generally within a range ofcirculating concentrations of the antigen-specific antibody or antigen binding fragment that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the ECso(i.e., the concentration of the construct - e.g., antibody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0366] In some embodiments, an antibody or antigen binding fragment thereof described herein can be administered to a subject as a monotherapy. Alternatively, the antibody or antigen binding fragment thereof can be administered in conjunction with other therapies for a disease or disorder (combination therapy). For example, the composition can be administered to a subject at the same time, prior to, or after, a second therapy. In some embodiments, the antibody or antigen binding fragment thereof and one or more additional active agents are administered at the same time. Optionally, the antibody or antigen binding fragment thereof is administered first in time and one or more additional active agents are administered second in time. In some embodiments, one or more additional active agents are administered first in time, and the antibody or antigen binding fragment thereof is administered second in time. Optionally, the antibody or antigen binding fragment thereof and one or more additional agents are administered simultaneously in the same or different routes. For example, a composition comprising the antibody or antigen binding fragment thereof optionally contains one or more additional agents.

[0367] An antibody or antigen binding fragment thereof described herein can replace or augment a previously or currently administered therapy. For example, upon treating with an antibody or antigen binding fragment thereof, administration of one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the antibody or antigen binding fragment thereof reaches a level sufficient to provide a therapeutic effect.

[0368] Monitoring a subject (e.g., a human patient) for an improvement, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in one or more symptoms of cancer exhibited by the subject. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a disease or disorder described herein.

[0369] In some instances, the antibody or antigen binding fragment thereof can be administered via virus-like particles. Virus-like particles (VLPs) comprise viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0370] In some instances, the antibody or antigen binding fragment thereof can be administered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0371] In some instances, the antibody or antigen binding fragment thereof can be administered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006 / 110728.

[0372] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient cells that have been genetically engineered, using methods such as those described herein, to express and secrete an antibody or antigen binding fragment thereof as described in this disclosure.

[0373] In another aspect, provided is a method of treating a subject with disease or disorder, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding a antibody or antigen binding fragment thereof as described in this disclosure.

[0374] There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or ex vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.

[0375] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding expression of the nucleic acid molecule and / or adapter polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61: 1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al., J. Virology 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, ex vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. In some instances, the nucleic acid molecules encoding the antibody or antigen binding fragment thereof can be delivered via virus-like particles.

[0376] Non-viral based delivery methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding the adapter polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La lolla, CA), andInvitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0377] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., 0-actin promoter or EFla promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the P-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0378] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0379] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40promoter, the beta-actin promoter, the EFla promoter, and the retroviral long terminal repeat (LTR).

[0380] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

[0381] In certain embodiments, the effective amount of a pharmaceutical composition comprising an antibody or antigen binding fragment thereof to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a antibody or antigen binding fragment thereof is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0382] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the antibody or antigen binding fragment thereof in the formulation used. Such pharmacokinetic parameters are well known in the art, i.e., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo- Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). In certainembodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0383] In some cases, the dosage (of the active component) ranges from about 0.0001 ng / kg to 100 mg / kg, and more usually 0.0002 ng / kg to 20 mg / kg, of the patient’s body weight. For example, dosages can be 0.0003 mg / kg body weight, 0.0001 mg / kg body weight, 0.0003 mg / kg body weight, 0.0005 mg / kg body weight, 0.001 mg / kg body weight or within the range of 0.0001 ng / kg -20 mg / kg. In certain examples, the antibody or antigen binding fragment thereof can be administered at a dose of 0.0001 mg / kg, 0.0002 mg / kg, 0.0003 mg / kg, 0.0004 mg / kg, or 0.0005 mg / kg once every other day, week, or month at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering antibody or antigen binding fragment thereof according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen resumes. For example, a patient may be on a antibody or antigen binding fragment thereof -specific antibody dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for antibodies or antigen binding fragments thereof of this disclosure include 0.0001 mg / kg body weight, 0.0003 mg / kg body weight, 0.0002 mg / kg body weight, 0.0004 mg / kg body weight, or 0.001 mg / kg via intravenous administration, with the antibodies or antigen binding fragments thereof being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by up to 0.001 mg / kg body weight every three weeks.

[0384] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously,intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0385] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0386] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising an antibody or antigen binding fragment thereof in an ex vivo manner. In such instances, cells that have been removed from a subject may be exposed to a pharmaceutical composition comprising an antibody or antigen binding fragment thereof after which the cells are subsequently implanted back into the subject.

[0387] In some instances, the provided methods may include administering to the subject an antibody or antigen binding fragment thereof that is conjugated to a therapeutic agent. The therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, or an immunosuppressive agent. Such therapeutic agents are described below.

[0388] In some instances, the provided methods may include administering an antibody or antigen binding fragment thereof and a second form of therapy to the subject. The second form of therapy may include a cytotoxic agent, a chemotherapeutic agent, an immune-activating agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of therapy is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies which may be administered as a second form of therapy include, but are not limited to, rituximab (e.g., for treatment of B-cell lymphomas), trastuzumab (e.g., for treatment of breast cancer), and cetuximab (e.g., for treatment of lung cancer).

[0389] In some embodiments, the antibody or antigen binding fragment thereof is conjugated to a moiety that specifically binds to an immune cell. In some embodiments, provided is a bispecific antibody comprising an antibody or antigen binding fragment thereof as described herein and an antibody or antigen binding fragment thereof that specifically binds to an immune cell. In someembodiments, the bispecific antibody comprises an antibody or antigen binding fragment thereof and an antibody moiety that specifically binds to T cells. Such a molecule is referred to as a bispecific T cell engager and may induce T cell-mediated cytotoxicity of hematological malignancy antigen-expressing cancer cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38).

[0390] In certain aspects, antibody or antigen binding fragment thereof according to the present disclosure can be administered as a co-therapy with other therapeutic agents. Other examples of therapeutic agents include chemotherapeutic agents, radiotherapeutic agents, and immunotherapeutic agents, as well as combinations thereof. In this way, the antibody or peptide complex delivered to the subject can be multifunctional, in that it exerts one therapeutic effect by binding to the antigen protein and a second therapeutic effect by delivering a supplemental therapeutic agent.

[0391] The therapeutic agent can act extracellularly, for example by initiating or affecting an immune response, or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling. The therapeutic agent is optionally cleavable from the antibody or antigen binding fragment thereof. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.

[0392] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of disease or disorder. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm ), bortezomib (such as VELCADE®, Millenium Pharm.), fulvestrant (such as FASLODEX®, AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA®, Novartis), imatinib mesylate (such as GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (such as ELOXATIN®, Sanofi), 5 -fluorouracil (5-FU), leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYK. ERB®, GSK572016, GlaxoSmithKline), lonafarnib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine (such as XELODA®, Roche), docetaxel (such as TAXOTERE®), and gefitinib (such as IRESSA®, Astrazeneca), AG1478, AG1571 (such as SU 5271; Sugen Inc.), alkylating a...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An isolated anti-CD28 antibody or antigen binding portion thereof comprising:(a) a heavy chain variable region (VH) having at least 90% identity to any one of SEQ ID NOS: 158-162; and(b) a light chain variable region (VL) having at least 90% identity to any one of SEQ ID NOS: 153-157.

2. The isolated anti-CD28 antibody or antigen biding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises:(a) a heavy chain variable region (VH) having at least 95% identity to any one of SEQ ID NOS: 158-162; and(b) a light chain variable region (VL) having at least 95% identity to any one of SEQ ID NOS: 153-157.

3. The isolated anti-CD28 antibody or antigen biding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises:(a) a heavy chain variable region (VH) having an amino acid sequence as set forth in any one of SEQ ID NOS: 158-162; and(b) a light chain variable region (VL) having an amino acid sequence as set forth in any one of SEQ ID NOS: 153-157.

4. The isolated anti-CD28 antibody or antigen biding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises:i) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 153, or a fragment thereof;ii) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 154, or a fragment thereof;iii) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 160 or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 155, or a fragment thereof;iv) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 156, or a fragment thereof; or v) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 157, or a fragment thereof.

5. The isolated anti-CD28 antibody or antigen biding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises:i) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 153, or a fragment thereof;ii) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 154, or a fragment thereof;iii) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 160 or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 155, or a fragment thereof;iv) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 156, or a fragment thereof; or v) a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 157, or a fragment thereof.

6. The isolated anti-CD28 antibody or antigen biding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises:i) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 158, or a fragment thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 153, or a fragment thereof;ii) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 159, or a fragment thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 154, or a fragment thereof;iii) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 160 or a fragment thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 155, or a fragment thereof;iv) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 161, or a fragment thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 156, or a fragment thereof; orv) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 162, or a fragment thereof, and a light chain variable region comprising an amino acid sequence t as set forth in SEQ ID NO: 157, or a fragment thereof.

7. The isolated anti-CD28 antibody or antibody fragment of any one of claims 1-6, wherein the antibody or antibody fragment is a humanized antibody or humanized antibody fragment.

8. The isolated anti-CD28 antibody or antibody fragment of any one of claim 1-7, wherein the antibody or antibody fragment is a multispecific antibody that comprises the anti-CD28 antibody or antigen portion thereof.

9. The isolated anti-CD28 antibody or antibody fragment of claim 8, wherein the multispecific antibody comprises an antibody or antibody fragment that binds to a target antigen.

10. The isolated anti-CD28 antibody or antibody fragment of claim 8, wherein the multispecific antibody is a bispecific antibody.

11. The isolated anti-CD28 antibody or antibody fragment of claim 10, wherein the target antigen is expressed on an immune cell.

12. The isolated anti-CD28 antibody or antibody fragment of claim 10, wherein the target antigen is expressed on the surface of a target cell.

13. The isolated anti-CD28 antibody or antibody fragment of claim 10, wherein the target antigen is a soluble target.

14. The isolated anti-CD28 antibody or antibody fragment of claim 9, wherein the multispecific antibody further comprises at least one additional antibody or antibody fragment that binds to at least one additional target antigen.

15. The isolated anti-CD28 antibody or antibody fragment of claim 14, wherein the multispecific antibody further comprises at least one antibody or antibody fragment that binds to a second additional target antigen.

16. The isolated anti-CD28 antibody or antibody fragment of claim 14 or claim 15, wherein the target antigen, the additional target antigen, or the second additional target antigen is expressed on an immune cell.

17. The isolated anti-CD28 antibody or antibody fragment of any one of claims 14-16, wherein the target antigen, the additional target antigen and / or the second additional target antigen is expressed on the surface of a target cell.

18. The isolated anti-CD28 antibody or antibody fragment of any one of claims 14-17, wherein the target antigen, the additional target antigen and / or the second additional target antigen is a soluble target.

19. The isolated anti-CD28 antibody or antibody fragment of claim 13 or claim 18, wherein the soluble target comprises one or more soluble proteins, peptides, carbohydrates, lipids,lipoproteins, steroids, other small molecules, cytokines, soluble receptors, antibodies, antigens, prostaglandins, vitamins, metabolites (such as sugars, amino acids, etc.), drugs and drug metabolites, DNA and RNA strands in their different forms, polysaccharides, chromosomes, genes, cells, cell membranes or cell parts (e.g., nucleus, mitochondria), bacteria, viruses, or other microorganisms, natural metabolites, hormones, pollutants, pesticides, and complexes or components of any of the preceding.

20. The isolated anti-CD28 antibody or antibody fragment of any one of claims 1-19, wherein the antibody fragment comprises a monovalent scFv (single chain fragment variable), divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.

21. The isolated anti-CD28 antibody or antibody fragment of any one of claims 8-20, wherein multispecific antibody is selected from a T cell engager, a TCE, a bispecific T cell engager (BiTE), tandAb, or Dual-affinity Re-targeting Antibody (DART), a bispecific killer engager (BiKE), a trispecific killer engager (TriKE), Fab-Fc-scFv, “bottle-opener”, Mab-scFv, Mab-Fv, Dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, common light chain-IgG ross-Mab, SEED, BEAT, TrioMab, and DuetMab.

22. The isolated antibody or antibody fragment of any one of claims 8-21, wherein the additional target antigen and / or second additional target antigen is selected from one or more of 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha- 1 -antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL1 7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD1 la, CD1 lb, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, ED AR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, Enkephalinase, eNOS, Eot, eotaxinl, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcRI, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP- 14, CDMP-1), GDF-6 (BMP- 13, CDMP-2), GDF-7 (BMP- 12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF- 15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alphal, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gpl20,heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF -beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / betal, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / betal, integrin alpha5 / beta3, integrin alpha6, integrin betal, integrin beta2, interferon gamma, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein LI, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin Beta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1 -alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Muellerian-inhibitin substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGGI, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PC AD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PEC AM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PPM, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76, RPA2, RSK, SI 00, SCF / KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD,SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-betaPan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRI, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF1 IB (OPG OCIF, TRI), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM AT AR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNF RII CD 120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (0X40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4- IBB CD 137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11 (TRANCE / RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSFIA (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (0X40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD 153), TNFSF9 (4- IBB Ligand CD 137 Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-Rl, TRAIL-R2, TRANCE, transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VC AM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrands factor, WIF-1, WNT1, WNT2,WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT1OB, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

23. The isolated anti-CD28 antibody or antibody fragment of claim 12 or claim 17, wherein the target cell is a cancer cell.

24. The isolated anti-CD28 antibody or antibody fragment of claim 23, wherein the cancer is selected from a carcinoma, a lymphoma, a blastoma, a sarcoma, a leukemia and lymphoid malignancies.

25. The isolated anti-CD28 antibody or antibody fragment of claim 23, wherein the cancer is selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell / histocyte-rich large B-cell lymphoma, multiple myeloma, myeloid leukemia-protein 1 (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular tumor, breast cancer, colon cancer, hepatocellular cancer (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngolaryngeal cancer, hepatobiliary cancer, central nervous system cancer, esophagus cancer, malignant pleural mesothelioma, systemic light chain amyloidosis,lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine tumor, Merkel cell cancel, testicular cancer, and skin cancer.

26. The isolated anti-CD28 antibody or antibody fragment of claim 12 or claim 17, wherein the target cell is associated with an infection or autoimmune disorder.

27. The isolated anti-CD28 antibody or antibody fragment of any one of claims 1-26, wherein said antibody is an IgG antibody or a recombinant IgG antibody or antibody fragment.

28. A T cell engager (TCE) antibody or fragment thereof, wherein said TCE antibody comprises:the isolated anti-CD28 antibody or antibody fragment of any one of claims 1-7; a first binding domain that binds to an HLA-CG1 peptide complex on a target cell; anda second binding domain that binds to CD3,wherein the first antigen binding domain comprises:a heavy chain (HC) variable region sequence, wherein the HC variable region comprises an amino acid sequence having at least 85% similarity with an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134; and a light chain (LC) variable region sequence, wherein the LC variable region comprises an amino acid sequence having at least 85% similarity with an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 35, 36, 42, 43, or 56, or 132.

29. The TCE or fragment thereof of claim 28, wherein the first antigen-binding domain HC CDR2 sequence comprises an amino acid sequence as set forth in SEQ ID NO: 131.

30. The TCE or fragment thereof of claim 28 or claim 29, wherein the first antigen binding domain HC variable region sequence comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 134.

31. The TCE or fragment thereof of claim 28 or claim 29, wherein the first antigen binding domain HC variable region sequence comprises SEQ ID NO: 134.

32. The TCE or fragment thereof of claim 28, wherein the first antigen binding domain HC CDR2 sequence comprises an amino acid sequence as set forth in SEQ ID NO: 130.

33. The TCE or fragment thereof of claim 28 or claim 32, wherein the first antigen binding domain HC variable region sequence comprises an amino acid sequence having at least 85% similarity to SEQ ID NO: 133.

34. The TCE or fragment thereof of claim 28 or claim 32, wherein the first antigen binding domain HC variable region sequence comprises SEQ ID NO: 133.

35. The TCE or fragment thereof of any one of claims 28-34, wherein the first antigen binding domain comprises a light chain (LC) variable region, wherein said LC variable region comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 132.

36. The TCE or fragment thereof of any one of claims 28-35, wherein the first antigen binding domain first antigen binding domain comprises a light chain (LC) variable region, wherein said LC variable region comprises an amino acid sequence having at least 95% similarity to SEQ ID NO: 132.

37. The TCE or fragment thereof of any one of claims 28-36, wherein the first antigen binding domain first antigen binding domain comprises a light chain (LC) variable region, wherein said LC variable region comprises an amino acid as set forth in SEQ ID NO: 132.

38. A pharmaceutical preparation comprising:(a) a pharmaceutically acceptable carrier; and(b) the isolated anti-CD28 antibody or antibody fragment of any one of claims 1-37.

39. A diagnostic preparation comprising:(a) a pharmaceutically acceptable carrier; and(b) the isolated anti-CD28 antibody or antibody fragment of any one of claims 1-37.

40. A method for treating a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical preparation of claim 38.

41. A method for treating a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a TCE of any one of claims 28-40.

42. The method of claim 41, wherein the cancer is a hematological or myeloid malignancy.

43. The method of claim 42, wherein the hematological or myeloid malignancy is AML, ALL, CLL, CML, CMML, or MDS.

44. A method for detecting a cancerous cell, comprising:(a) administering to a subject in need thereof an effective amount of the diagnostic preparation of claim 39, and(b) detecting binding of the anti-CD28 antibody or fragment thereof as a determination of the presence of a cancerous cell.

45. A method of inducing T-cell dependent cellular cytotoxicity (TDCC) or Redirected T cell cytotoxicity (RTCC) in a subject in need thereof, comprising: administering to the subject an effective amount of the pharmaceutical composition of claim 38.

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