TCR mimetic t cell engaging antibodies targeting human leukocyte antigen cathepsin g peptide complex and CD3 and related methods

Abstract

Provided are TCR mimetic T cell engaging antibodies or antigen binding portions thereof that specifically bind antigens relating to myeloid malignancies, various compositions of such antibodies or antigen binding portions thereof, and methods of their use. The disclosure provides such antibodies, fragments of such antibodies retaining myeloid malignancy antigen-binding ability, pharmaceutical compositions including such antibodies or antigen binding fragments thereof, and diagnostic compositions including such antibodies or antigen binding fragments thereof. This disclosure further provides for isolated nucleic acids encoding such antibodies amino acid sequences of such antibodies, and host cells transformed therewith. Additionally, this disclosure provides for therapeutic and diagnostic methods employing the antibodies and nucleic acids of the disclosure. Finally, the present disclosure provides for recombinant bispecific antibodies capable of specifically binding CG1 and CD3.

Description

TCR MIMETIC T CELL ENGAGING ANTIBODIES TARGETING HUMAN LEUKOCYTE ANTIGEN CATHEPSIN G PEPTIDE COMPLEX AND CD3 AND RELATED METHODSSEQUENCE LISTING

[0001] The present application contains a sequence listing wh ch 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-004-01WQ-Sequence-Listing.xml and is 158 KB in size.BACKGROUND

[0002] Treatment for myeloid malignancies, for example in leukemia, have made progress in recent years. Stem cell transplant and other therapies (e.g., small molecule-based inhibitor therapies) could benefit some patients. However, for acute myeloid leukemia (AML) patients, there exists a large patient group that will be refractory and relapsed from the standard of care. Successes in developing immunotherapeutic approaches for the treatment of AML have been limited. This has been due, in part, to a lack of reliable, tumor-specific surface targets (e.g., CD33, CD 123), which also avoid undesirable toxicities due to their expression on normal hematopoietic cells, which become targets themselves due to this expression. Concurrently, while avoiding off-target toxicity, it would be advantageous for an AML therapy to provide “bystander killing”, particularly in the bone marrow and extra-medullary sites, which may help overcome potential resistance mechanisms that arise due to heterogeneous tumor antigen expression.

[0003] The therapeutic potential of immunotherapies for tumors has been limited by a paucity of known tumor-specific membrane proteins (Yarmarkovich et al. Nature, Nov 32021). The majority of oncogenic drivers or tumor-specific targets are intracellular proteins. Thus, immunotherapeutic targeting of peptides derived from intracellular oncogenes is advantageous for the treatment of tumors.

[0004] 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 AMLblasts 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.

[0005] 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. Consequently, there is an urgent need for novel therapies, the present disclosure could provide a new therapeutic avenue for many patients suffering from the effects of myeloid malignancies.BRIEF SUMMARY

[0006] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0007] Described herein are antibodies that are capable of specifically binding to CGI and their incorporation into novel T cell receptor mimetic T cell engagers as described herein. Also described herein are bispecific recombinant antibodies that are capable of specifically binding both CGI and CD3. More preferred aspects of the disclosure include, in certain embodiments, novel T cell receptor (TCR) mimetic (TCRm) T cell engager (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; and (ii) at least one immune cell (e.g., via CD3 expressed on the surface of an immune cell). In certain aspects, the distinct complexes comprise HLA class I molecule-CGl complexes. As described herein, these TCRm TCE antibodies provide promising therapeutic efficacy, even when compared to other bispecific antibodies target the same antigens.

[0008] The presently disclosed TCRms 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.

[0009] 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.

[0010] 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 directly into 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.

[0011] 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 TCRms may be designed to overcome this MHC-restriction, which allows certain disclosed TCRms to bind to more than one MHC allele-peptide complex for a given peptide derived from aprotein of interest, such as CG / CG1. This, in turn, allows such TCRms to provide increased therapeutic activity for diverse groups expressing various MHC alleles. By identifying and targeting CG / CG1 as targets for AML using TCRm 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.

[0012] The TCR mimetic antibodies of the disclosure include those referred to herein as TCRm, TCR mimetic TCE, and TA-# (e.g., TA-1). TCRm TCEs are characterized in that, in addition to targeting tumor associated peptides / antigens (TAAs) presented by the MHC, they also have one or more binding domains that target an immune cell associated protein, e.g., an immune cell receptor (e.g., T cell receptor) protein, an immune cell activator, an immune cell engager, and the like, as described in detail herein and / or known in the art.

[0013] Surprisingly, the present inventors have found that the presently disclosed TCRm TCEs have demonstrated (i) “peptide-centric” binding for target antigens; (ii) desirable levels of restriction breaking for the target antigens across HLA alleles; (iii) selective activation of T cells only in the presence of target peptide antigen; (iv) a clear cytotoxicity window; and (v) specific cytotoxicity in in vitro and ex vivo assays, including demonstrating potent killing of primary AML cells, both ex vivo (evaluated using Patient-derived Xenograft mouse models) and ex vivo.

[0014] Furthermore, the presently disclosed TCRm 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 receptorindependent 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 TCRms of the disclosure. As a result, bystander-activated CD8+ 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 toimmunotherapy. 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 cellactivating receptors and cytolytic molecules.

[0015] In certain preferred aspects, the present disclosure includes methods, compositions, formulations, and various techniques for producing TCR-mimetic (TCRm) bispecific T Cell Engager (TCE) antibodies (Ab) that bind to the CGl / HLA-A*02:01 pHLA complex with high affinity. The TCRm-based molecules of the disclosure, including the specific TCRm-based TCE referred to herein as TA-1, induce potent T cell mediated killing in vitro of leukemia cell lines, with varying levels of target expression, at sub-nanomolar EC50 levels. Further the TCRm-based TCEs have exhibited highly effective tumor control ex vivo against multiple AML cell line (CDX) murine models at doses as low as 0.005 mg / kg. The TCRm-based TCEs of the disclosure, especially, TA-1 demonstrate potent efficacy, reduced tumor burden, and a clear survival benefit versus a control arm in an aggressive primary AML patient derived (PDX) tumor mouse model that harbors FLT3-ITD, DNMT3a, and NPM1 mutations. Consistent with these results, the disclosure herein shows that the TCRm-based TCEs of the disclosure, e.g., TA-1, are able to induce significant levels of IFNy release from several primary AML patient samples co-cultured with donor effector T cells versus a negative control.

[0016] In certain aspects, the TCRm-based TCEs (e.g., TCRm 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 an immune cell associated protein (e.g., an immune cell engager such as an anti-CD3 binding domain). In preferred aspects, the second binding domain binds to CD3 on an immune cell.

[0017] 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.

[0018] Described herein are also antibodies, antigen binding regions, and fragments thereof, that may be incorporated into a TCRm-based molecule 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 bispecific recombinant antibodies, antigen bindingregions, and fragments thereof that are capable of specifically binding both CGI and CD3 and incorporated into a TCRm-based molecule as described herein.

[0019] In certain aspects, described herein are TCRm-based antibodies, isolated antibodies, and / or antigen binding portions thereof, which bind to CGI. In certain aspects, the CGI binding TCRms, 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 and 172. In certain aspects, the CGI binding TCRm-based antibodies, 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, or 129-131 and / or a CDR3 sequence comprising one of SEQ ID NOs: 88-95. TCRm-based antibodies, 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.

[0020] Isolated antibodies or antibody fragments as described herein can be a human antibody or human antibody fragment.

[0021] In certain aspects, described herein are TCRm-based antibodies, isolated antibodies, and / or antigen binding portions thereof comprising a heavy chain variable region (VH) having at least 90% identity to any one of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134; and / or (b) a light chain variable region (VL) having at least 90% identity to any one of SEQ ID NOs: 24, 35, 36, 42, 43, 56, or 132.

[0022] In certain aspects, described herein are TCRm-based antibodies, isolated antibodies, or antigen binding portions thereof. In certain aspects, the TCRm-based antibodies, isolated antibodies or antigen binding portions thereof according to the present disclosure can 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 and 172. Isolated antibodies or antigen binding portions thereof according to the present disclosure can further comprise, in the HC variable region, a CDR2 sequence comprising one of SEQ ID NOs: 79-87, or 129-131 and / or a CDR3 sequence comprising one of SEQ ID NOs: 88-95. Isolated 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.

[0023] Isolated antibodies or antibody fragments as described herein can be a human antibody or human antibody fragment.

[0024] In certain aspects, the TCRm-based antibodies, 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.

[0025] In certain aspects, the TCRm-based TCE comprises an Fc domain. In certain aspects, the Fc domain comprises one or more amino acid substitutions.

[0026] In preferred aspects, the TCRm-based TCE is TA-1, TA-2, or TA-3. In more preferred aspects, the TCRm-based TCE is TA-1.

[0027] In certain aspects, the TCRm can be or comprise a chimeric antibody, bispecific antibody, trispecific or other multi-specific antibody, or BiTE. In certain aspects, the TCRm can be an IgG antibody or a recombinant IgG antibody or antibody fragment.

[0028] In certain aspects, the TCRm-based antibodies or antibody fragments thereof can exhibit increased binding affinity for CGI presented by HLA-A*02:01 compared to sample from a wildtype subject or subject not having a cancer.

[0029] In certain aspects, antibodies, including TCRms, 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 ofZr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, orPb-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.

[0030] In certain aspects, the TCRm antibody or antibody fragment can further comprise an amino acid having at least 90% identity with SEQ ID NO:57, 128 and 139. In certain aspects, the TCRm antibody or antibody fragment thereof can comprise amino acid sequences having at least 90% similarity with any one of SEQ ID NOs: 55, 56, 57, 128, 139-143. In certain aspects, the TCRm antibody or antibody fragment thereof can comprise any one of SEQ ID NOs: 55-57, 128 and 139-143. In certain aspects, the TCRm antibody or antibody fragment thereof comprises an amino acidsequence consisting essentially of any one of SEQ ID NOs: 55-57, 128 and 139-143. In certain aspects, the TCRm antibody or antibody thereof can comprise an amino acid sequence consisting essentially of (a) a heavy chain variable region (VH) having at least 90% identity to any one of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134; and (b) a light chain variable region (VL) having at least 90% identity to any one of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In certain aspects, the TCRm antibody or antibody fragment can further comprise an amino acid having at least 95% identity with SEQ ID NO: 57, 128 and 139. In certain aspects, the TCRm antibody or antibody fragment thereof can comprise amino acid sequences having at least 95% similarity with any one of SEQ ID NOs: 55, 56, 57, 128, 139-143. In certain aspects, the TCRm antibody or antibody fragment thereof can comprise any one of SEQ ID NOs: 55-57, 128 and 139-143. In certain aspects, the TCRm antibody or antibody fragment thereof comprises an amino acid sequence consisting essentially of any one of SEQ ID NOs:. 55-57, 128 and 139-143. In certain aspects, the TCRm antibody or antibody thereof can comprise an amino acid sequence consisting essentially of (a) a heavy chain variable region (VH) having at least 95% identity to any one of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134; and (b) a light chain variable region (VL) having at least 95% identity to any one of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In certain aspects, the TCRm antibody or antibody fragment thereof comprises an amino acid sequence consisting essentially of: (a) any one of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134; and (b) any one of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In certain aspects, described herein is an isolated nucleic acid encoding the TCRm antibody heavy and / or light chain variable region of the TCRm antibody or antibody fragment or other amino acid of any aspect as described herein. Described herein are also expression vectors comprising a nucleic acid encoding any amino acid sequence as described herein.

[0031] In certain aspects, described herein are hybridoma or engineered cells comprising a nucleic acid encoding any of the TCRm-based antibodies or antibody fragments thereof. In certain aspects, described herein is a hybridoma or engineered cell comprising a nucleic acid encoding any amino acid sequence, antibody or antibody fragment thereof, as described herein.

[0032] In certain aspects, also described herein are pharmaceutical preparations. In some aspects, pharmaceutical preparations according to the present disclosure comprise (a) a pharmaceutically acceptable carrier; and (b) a TCRm-based antibody or antigen binding portion thereof of any embodiment as described herein. In certain aspects, also described herein are pharmaceuticalpreparations. In some aspects, pharmaceutical preparations according to the present disclosure comprise (a) a pharmaceutically acceptable carrier; and (b) TA-1. In certain aspects, also described herein are pharmaceutical preparations. In some aspects, pharmaceutical preparations according to the present disclosure comprise (a) a pharmaceutically acceptable carrier; and (b) TA-2. In certain aspects, also described herein are pharmaceutical preparations. In some aspects, pharmaceutical preparations according to the present disclosure comprise (a) a pharmaceutically acceptable carrier; and (b) TA-3.

[0033] In certain aspects, also described herein are diagnostic preparations. As described herein, diagnostic compositions according to the present disclosure can comprise (a) a pharmaceutically acceptable carrier; and (b) a TCRm-based antibody or antigen binding portion thereof of embodiment as described herein.

[0034] In certain aspects, also described herein are methods for treating a subject having cancer. Methods according to the present disclosure can comprise administering to a subject in need thereof a therapeutically effective amount of any pharmaceutical preparation or any TCRm-based antibody or any TCRm antibody fragment as described herein. In certain aspects, the cancer can be a hematopoietic malignancy, for example, AML, ALL, or CLL.

[0035] In certain aspects, described herein are methods for diagnosing a cancer. Diagnostic methods for cancer as described herein can comprise (a) administering to a subject in need thereof an effective amount of any diagnostic preparation as described herein, and (b) detecting binding of the TCRm-based antibody or antigen binding portion thereof as a determination of the presence of the cancer. In certain aspects, the cancer of which diagnosis is sought in the subject can be a hematopoietic malignancy, for example, AML, ALL, or CLL.

[0036] In certain aspects, described herein are methods of detecting the presence of a cancer or malignant cell in a biological sample. Such methods can comprise (a) contacting said sample with any diagnostic preparation as described herein, and (b) detecting an amount of binding of the TCRm-based antibody or antigen binding portion thereof as a determination of the presence of said cancer or malignant cell. In certain aspects, the cancer or malignant cell of which diagnosis is sought in the subject can be a hematopoietic malignancy, for example, AML, ALL, or CLL.

[0037] Also described herein are methods of making TCRm-based antibodies 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 TCRm-basedantibody or a fragment thereof and, optionally, isolating the TCRm-based antibody or fragment from the culture.

[0038] In certain aspects, the TCR mimetic-based antibody comprises a first and second antigen binding domain as a bispecific antibody in a 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 TCRm-based antibody comprises a third binding domain that comprises a single-chain variable fragment (scFv) that binds to CD3. In preferred 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 preferred aspects, the first and second binding domains comprise an scFv that binds to the plurality of HLA class I CG / CG1 peptide antigens.

[0039] In certain TCRm-based antibodies (i.e., TCRm-based T-cell engagers), the mimetic comprises: a first polypeptide comprising the first binding domain; and a second polypeptide comprising the second and third binding domains. In certain aspects, the second polypeptide include the scFvs of the second and third binding domains are in a tandem format. In certain aspects, the first and second polypeptide each comprise a hinge-CH2-CH3 and form a heterodimer.

[0040] In certain embodiments, the TCR mimetic-based T cell engager protein is in a heterodimer format. In certain embodiments, the bispecific antibody is in a heterodimer format, and comprises: (i) an antibody fragment that binds CD3, (ii) an antibody fragment that binds HLA-CG1 peptide antigen, and (iii) a heterodimeric hinge-CH2-CH3. In certain aspects, TCR mimetic-based T cell engager protein comprises an anti-CD3 antibody fragment is a single chain variable fragment (scFv). In certain aspects, the anti-CD3 antibody fragment is an scFv and the antibody fragment that selectively binds HLA-CG1 is a Fab. In certain aspects, the TCR mimetic-based T cell engager protein comprises a first heavy chain, a second heavy chain, and a light chain. In some aspects, the first heavy chain comprises the anti-CD3-scFv-Fc, the second heavy chain comprises the anti-HLA-CG1 Fab VH-CHl-Fc, and the light chain comprises the anti-HLA-CGl Fab VL-Ck.

[0041] Provided herein are T-cell-receptor-mimic (TCRm) antibodies and antigen-binding fragments comprising three peptide chains, referred to herein as a first, second, and third polypeptide.

[0042] In some aspects, the first polypeptide comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 139; in other aspects the first peptide chain has at least 95%similarity to SEQ ID NO: 139; and in still other aspects the first peptide chain is identical to SEQ ID NO: 139.

[0043] In further aspects, the second peptide chain independently comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 140; in other aspects the second peptide chain comprises amino acid sequence that has at least 95% similarity to SEQ ID NO: 140; and in still other aspects the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 140.

[0044] In additional aspects, the third peptide chain independently comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143; in other aspects the third peptide chain comprises an amino acid sequence having at least 95% similarity to any of SEQ ID NOs: 141-143; and in still other aspects the third peptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 141-143.

[0045] In exemplary aspects, the TCRm antibody comprises a first peptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 139, a second peptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 140, and a third peptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 141.

[0046] In other exemplary aspects, the first and second peptide chains comprise an amino acid sequence as set forth in of SEQ ID NOs: 139 and 140, respectively, while the third peptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 142.

[0047] In yet other exemplary aspects, the first and second peptide chains comprise an amino acid sequence as set forth in SEQ ID NOs: 139 and 140, respectively, while the third peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 143. In certain aspects, the three-chain architecture supports assembly into an antigen-binding TCRm format or fragment thereof, including IgG, IgG-like, Fab, Fab', F(ab')2, scFv-containing formats, or other engineered multispecific configurations that maintain the binding determinants encoded by SEQ ID NOs: 139-143.

[0048] In some embodiments, the TCRm-based antibody 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 TCRm-based antibody is or comprises a single domain antibody, a human single domain antibody, or a humanized single domain antibody. In some embodiments, the TCRm-based antibody is or comprises a murine antibody, a chimeric antibody, a camelid antibody, a humanizedantibody, or a human antibody. In some embodiments, the antibody is part of a multispecific antibody or a multifunctional antibody.

[0049] In some embodiments, selective binding of a TCR mimetic antibody disclosed herein to the complex comprising the HLA class I molecule and the peptide induces a cellular response; optionally wherein the cellular response is selected from: an immune response, cancer cell death, virally-infected cell death, antibody dependent cytotoxicity, immune cell proliferation, and cytokine release. In some embodiments, the immune response comprises activation of T cells. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the immune response comprises activation of cytotoxic T cells (CTLs). In some embodiments, the TCR mimetic antibody binds a peptide-HLA class I molecule complex on a cancer cell. In some embodiments, the TCR mimetic antibody binds an HLA class I molecule-peptide complex on a virally infected cell.

[0050] Described herein, in certain embodiments, are nucleic acids encoding the TCR mimetic antibody or fragment thereof described herein, or a therapeutic fusion polypeptide described herein.

[0051] In some embodiments, the TCR mimetic-based antibody is a bispecific antibody in a heterodimeric format. In some embodiments, the TCRm-based antibody comprises a heterodimeric hinge-CH2-CH3. In some embodiments, the TCR mimetic antibody is a bispecific antibody comprising a homodimeric or heterodimeric format. In some embodiments, the TCRm-based antibody comprises at least one antibody or fragment thereof that binds an immune cell.

[0052] Described herein, in certain embodiments, are pharmaceutical compositions comprising the TCR mimetic-based antibody or fragment thereof described herein, the therapeutic fusion polypeptide described herein, the nucleic acid described herein, or the cell engager described herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise a plurality of T Cell Receptor (TCR) mimetic-based antibodies or fragments thereof described herein, wherein each member of such plurality has a first binding affinity for a first complex comprising a first peptide-HLA class I molecule and a second binding affinity for a second complex comprising a second, distinct peptide-HLA class I molecule, wherein the first complex and the second complex comprise the same peptide. In some embodiments, the plurality of TCR mimetic antibodies or fragments thereof are operably connected in a single polypeptide or a polypeptide complex. In some embodiments, the pharmaceutical compositionscomprise an amalgam of polypeptides each comprising the TCR mimetic antibodies or fragments thereof. In some embodiments, the first binding affinity is at least 10% to 1000% greater than the second binding affinity. In some embodiments, the pharmaceutical compositions are formulated for administration to a human subject having been identified as having AML, and are HLA-A*02:01, HLA-A*02:06, or HL A- A* 02: 07 positive.

[0053] Described herein, in certain embodiments, are methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition described herein.

[0054] Described herein, in certain embodiments, are methods 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 described herein. In some embodiments, the method further comprises administering to the subject a checkpoint inhibitor prior to, subsequent to, or simultaneously with administration of the TCR mimetic antibody or fragment thereof.

[0055] Described herein, in certain embodiments, are pharmaceutical compositions comprising in one or more polypeptide chains or polypeptide complexes a plurality of T cell receptor (TCR) mimetic-based antibodies or fragment thereof, each member of the plurality specifically binds a plurality of complexes comprising an HLA class I molecule and a CG or CGI peptide. In some embodiments, each member of such plurality has a first binding affinity for a first peptide-HLA class I molecule and a second binding affinity for a second, distinct peptide-HLA class I molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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.

[0057] FIG. 1 is an overview of neutrophil granule proteases. Cathepsin G (CG), for example, is a myeloid azurophil granule serine protease that is involved in host immunity, cleavage of inflammatory mediators and receptors, degradation of extracellular matrix components and leukemogenesis.

[0058] FIG. 2 depicts an overview of aspects of an amino acid sequence of an antigen target according to certain aspects of the present disclosure. CGI is a 9-mer peptide derived from the cathepsin G (CG) protein leader sequence, presented by human leukocyte antigen (HLA) class I, specifically HLA-A*02:01. CG is highly expressed in acute myeloid leukemia (AML), with a higher expression in leukemia stem cells (LSC) compared to normal hematopoietic stem cells (HSC). It is further noted that: (1) CG anti-leukemia immunity can be elicited in vitro and ex vivo; (2) functional CG-CTL can be detected in AML patients following allogeneic hematopoietic stem cell transplantation (allo-SCT); and (3) in addition to AML, CGI is expressed by lymphoid leukemia, specifically acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) and may also be detectable in lung cancer.

[0059] FIG. 3 depicts a visual overview of aspects of an antigen target according to certain aspects of the present disclosure.

[0060] FIG. 4 provides a schematic of an exemplary TCRm TCE of the disclosure.

[0061] FIG. 5 provides a schematic of an exemplary TCRm TCE of the disclosure that includes an Fc domain modification and an additional antigen binding domain.

[0062] FIG. 6 provides a schematic of a TCRm TCE of the disclosure that includes two additional antigen binding domains.

[0063] FIG. 7 depicts a visual overview of aspects of an antigen target according to certain aspects of the present disclosure.

[0064] FIG. 8 provides affinity data for TA-1 and TA-2.

[0065] FIGS. 9A shows detection of CG1-HLA peptide complex of varying target levels using TA-1.

[0066] FIG. 9B provides T-cell activation data for TA-1 and TA-2 in target-negative and targetpositive cells using Jurkat activation assay.

[0067] FIGS. 10A-10C provide T-cell activation data in the form of CD69 and CD25 activation markers as well as IFNy levels following TA-1 treatment across cells presenting varying levels of endogenous pHLA.

[0068] FIGS. 11A-11B show data from cytotoxicity assays using leukemic cells and TA-1.

[0069] FIG. 12 provides a schematic of bystander killing using TCRms of the disclosure.

[0070] FIG. 13 provides data demonstrating bystander killing using TCRms of the disclosure.

[0071] FIGS. 14A-14B shows results of murine model studies using TCRms of the disclosure.

[0072] FIG. 15 shows in vivo results showing primary, patient-derived AML tumor control by TCRms of the disclosure.

[0073] FIG. 16 shows a schematic of an ex vivo primary AML efficacy study used to evaluate TCRms of the disclosure.

[0074] FIG. 17 summarizes characteristics of nine primary AML models used in the ex vivo study using TA-1.

[0075] FIGS. 18A-18F provides results from the ex vivo study using TA-1.

[0076] FIG. 19 shows results showing that the TCRms of the disclosure do not provide an off-target response in the presence of CG-expressing neutrophils that do not present CGI pHLA.

[0077] FIG. 20 shows CG protein expression in neutrophils.

[0078] FIG. 21 provides data showing that TA-1 does not activate T cells in the presence of neutrophils.

[0079] FIG. 22 provides data showing that TA-1 does not induce IFNy release in activated polarized macrophages or target inflammatory sites.

[0080] FIGS. 23A-23B provides data comparing cytotoxicity of TA-1 versus other TCE, which are not TCRms, to different target cells.

[0081] FIG. 24 provides data showing that TA-1 displays no toxicity towards normal hematopoietic stem cells.

[0082] FIG. 25 shows data indicating that TA-1 does not deplete any cell type, including monocytes and other CD 123+ cells from PBMCs.

[0083] FIGS. 26A-26B shows bone marrow colony forming unit assay results for TA-1.

[0084] FIG. 27 shows data indicating that TA-1 does not induce cytokine release in the absence of target-positive cells.

[0085] FIGS. 28A-28B provide data showing no polyreactivity is caused by TA-1 or TA-4 (a TA-1 analogue).

[0086] FIGS. 29A-29B provide cytotoxicity results showing that TA-1 does not kill targetnegative cancer cell lines in the absence of cells expressing the target pHLA complex.

[0087] FIG. 30 provides data showing that TA-1 requires 3 amino acid residues for binding to CGI pHLA TA-1.

[0088] FIG. 31 outlines a computational, experimentally guided cross-reactivity assessment performed for TA-1 / TA-2.

[0089] FIGS. 32A-32B provide results of a computational, experimentally guided cross-reactivity assessment.

[0090] FIGS. 33A-33B provide results for TA-1 potential interactions with normal primary tissues and cross-HLA interactions.

[0091] FIGS. 34A-34B provide a summary of the baseline stress and developability criteria met by TA-1.

[0092] FIGS. 35A-35B show data from PK studies using IV and SC administration of antibodies of the disclosure.

[0093] FIG. 35C shows immunogenicity data for an antibody of the disclosure.

[0094] FIG. 36 summarizes a binding profile assessment of an antibody of the disclosure.DETAILED DESCRIPTION

[0095] 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.

[0096] 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.

[0097] 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”).

[0098] 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 notmaterially affect the basic and novel characterise c(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.”

[0099] 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.

[0100] 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.

[0101] 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 differentpolynucleotide 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 particular polynucleotide 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.

[0102] 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.

[0103] 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 (i.e., two) or multimer (i.e., three or more) of single chainpolypeptides. 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.

[0104] 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.

[0105] 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.

[0106] The term “identity” or “substantial identity,” 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.

[0107] 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 calculatesthe percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0108] 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. Sci. (U. S. A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0109] 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 scoring sequence 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, anexpectation (E) of 10, M=1, 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)).

[0110] 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 10-20.

[0111] The term “antigen binding protein” or “ABP” is used herein in its broadest sense and includes certain types of molecules such as TCR mimetic-based antibodies comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.

[0112] 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.

[0113] 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-CG 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.

[0114] The term “Test Article” (“TA”), “TCR mimetic antibody”, “TCRm-based antibody”, “TCRm-based molecule”, “T Cell Receptor mimetic antibody”, “TCRm”, “TCRm antibody”, “TCR mimetic T cell engager (TCE)” or “TCR mimetic binding protein”, and the like, as used herein, refers to an ABP that binds at least one peptide MHC-Class I molecule complex on a target cell and a moiety expressed or presented on an immune cell.

[0115] The term “T cell engager” or “TCE”, as used herein, refers to a molecule, preferably a multi-specific antibody, that binds an immune cell and at least one peptide on a different cell. A TCRm-based antibody, as disclosed herein is a TCE that binds to a peptide MHC-Class I molecule complex on a target cell and that includes an immune cell engager or other binding protein (e.g., an immune cell activator or co-stimulatory molecule) that binds to an immune cell (e.g., a T cell).

[0116] A “HLA-CG TCR mimetic antibody”, “Anti-HLA-CG TCR mimetic antibody”, “HLA-CG peptide ABP,” “anti-HLA-CG peptide ABP,” or “HLA-CG 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 TCR mimetic antibody”, “Anti-HLA-CGl TCR mimetic antibody”, “HLA-CG1 peptide ABP,” “anti-HLA-CGl peptide ABP,” or “HLA-CG1 peptidespecific ABP” is an ABP, as provided herein, which specifically binds to the peptide-HLA class I molecule complex bound to a CGI peptide.

[0117] 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.

[0118] 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 TCR CDRs, e g, aCDRl, aCDR2, aCDR3, CDR1, CDR2, and £CDR3. TCR CDRs are described herein.

[0119] The amino acid sequence boundaries of a CDR, including an antibody or TCR 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(ll):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.

[0120] “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 bindingaffinity, 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®).

[0121] With regard to the binding of a TCR mimetic antibody, T cell engager protein or ABP 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.

[0122] 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.

[0123] The term “ka” ( T' / 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.

[0124] 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.

[0125] The term “KA” (M-1), as used herein, refers to the association equilibrium constant of a particular ABP-antigen interaction. KA = ka / kd.

[0126] An “immunoconjugate” is an ABP conjugated to one or more heterologous molecule(s), such as a therapeutic (cytokine, for example) or diagnostic agent.

[0127] 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-CG peptide). In one exemplary assay, HLA-CG peptide is coated on a surface and contacted witha first HLA-CG peptide ABP, after which a second HLA-CG peptide ABP is added. In another exemplary assay, a first HLA- CG peptide ABP is coated on a surface and contacted with HLA-CG peptide, and then a second HLA-PEPTIDE ABP is added. If the presence of the first HLA-CG peptide ABP reduces binding of the second HLA-CG 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-CG 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.gov / 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.

[0128] 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.

[0129] 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 algorithmsdescribed 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.

[0130] 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).

[0131] 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 the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0132] 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.

[0133] 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.”

[0134] 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.

[0135] 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.

[0136] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an TCRm-based antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[0137] 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 or condition that can be treated with an TCRm-based antibody provided herein. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[0138] 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.

[0139] 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.

[0140] 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 asubject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0141] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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-CG peptide antigens.

[0148] 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-CG peptide neoantigens.

[0149] 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 polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.

[0150] As used herein the term “candidate antigen” is a mutation or other aberration giving rise to a sequence that may represent an antigen.

[0151] As used herein the term “coding region” is the portion(s) of a gene that encode protein.

[0152] As used herein the term “coding mutation” is a mutation occurring in a coding region.

[0153] As used herein the term “ORF” means open reading frame.

[0154] As used herein the term “NEO-ORF” is a tumor-specific ORF arising from a mutation or other aberration such as splicing.

[0155] As used herein the term “missense mutation” is a mutation causing a substitution from one amino acid to another.

[0156] 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.

[0157] As used herein the term “frameshift mutation” is a mutation causing a change in the frame of the protein.

[0158] As used herein the term “indel” is an insertion or deletion of one or more nucleic acids.

[0159] As used herein the term “non-stop or read-through” is a mutation causing the removal of the natural stop codon.

[0160] In an aspect, provided herein are ABPs and TCR mimetic antibodies (the TCRm-based TCE antibodies disclosed herein) or fragments thereof that bind HLA-CG, preferably HLA-CG1, peptide antigens disclosed herein. In certain aspects, the TCRms 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 preferred aspects, an ABP or TCR mimetic-based antibody disclosed herein specifically binds to an HLA-CG peptide comprising CGI peptide complexed with an HLA class I molecule.

[0161] In certain embodiments, the HLA-CG or HLA-CG1 peptide is located in the peptide binding groove of an al / a2 domain of the HLA class I molecule heavy chain.

[0162] In certain aspects, described herein are TCR mimetic-based antibodies or fragments thereof that specifically bind a plurality of complexes comprising an HLA class I molecule and a CGI peptide.

[0163] In certain embodiments, the ABP or TCR mimetic-based antibody 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 TCR mimetic-based antibody does not bind to the HLA in the absence of the CGI peptide. In some embodiments, the ABP or TCR mimetic-based antibody does not bind HLA-CG1 peptide in the absence of the HLA. In some embodiments, the ABP or TCR mimeticbased 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 TCR mimetic-based antibody binds a complex comprising HLA and CGI peptide when naturally presented on a cell such as a tumor cell.

[0164] The HLA-CG! peptide antigen can be expressed on the surface of any suitable target cell including a tumor cell. In some embodiments, the ABP or TCR mimetic-based antibody specifically binds a complex comprising HLA and a CGI peptide, e g., derived from a tumor.

[0165] An ABP or TCR mimetic-based 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 TCR mimetic-based antibody, distinct from a surface presented by the peptide alone or an HLA subtypealone. 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 TCR mimetic-based 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.

[0166] In some aspects, the affinity of an HLA-CG1 peptide TCR mimetic-based 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 preferred aspects, the non-target molecule is CG and / or pHLA-CG, which does not include CGI or pHLA-CGl.

[0167] 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.

[0168] In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-CG1 peptide antigen with a higher affinity than its affinity to another HLA-CG peptide. In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-A*02:01-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG peptide. In certain embodiments, the ABP or TCR mimeticbased antibody binds HLA-A*02:03-CG1 peptide antigen with a higher affinity than its affinity to an HLA-CG peptide. In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-A*02:06-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG peptide.In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-A*02:07-CGl peptide antigen with a higher affinity than its affinity to an HLA-CG peptide.

[0169] In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-A*02:01-CG1 peptide antigen with a higher affinity than its affinity to a CG peptide. In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-A*02:03-CG1 peptide antigen with a higher affinity than its affinity to a CG peptide. In certain embodiments, the ABP or TCR mimetic-based 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 TCR mimetic-based antibody binds HLA-A*02:07-CGl peptide antigen with a higher affinity than its affinity to a CG peptide.

[0170] In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-CG1 peptide antigen with a higher affinity than its affinity to a CGI peptide. In certain embodiments, the ABP or TCR mimetic-based 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 TCR mimetic-based 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 TCR mimetic-based antibody binds HLA-A*02:06-CG1 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.

[0171] In certain embodiments, the ABP or TCR mimetic-based 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).

[0172] 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 (XD) of the higher affinity interaction ranges from 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 10’10Molar (M), at least 10’10to 10'11Molar (M), at least 10'11to 10'12Molar (M), or at least 10'12to 10’13Molar (M). In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-CG1 peptide antigen of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct HL A 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 moleculesare 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. In certain embodiments, the ABP or TCR mimetic-based 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 10'10to 10'11Molar (M), at least 10’11to 10'12Molar (M), or at least 10‘12to 10'13Molar (M). In certain embodiments, the ABP or TCR mimetic-based antibody binds HLA-CG1 of each distinct HLA class I molecule with a KD of at least 10-4to 10-13Molar (M), wherein the binding of each distinct HLA class I molecule to the ABP or TCR mimetic-based antibody has a distinct KD.

[0173] 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.

[0174] In some embodiments, the ABP or TCR mimetic-based antibody is an ABP or TCR mimetic-based antibody that competes with an illustrative ABP or TCR mimetic-based antibody provided herein. In some aspects, the ABP or TCR mimetic-based antibody that competes with the illustrative ABP or TCR mimetic-based antibody provided herein binds the same epitope as an illustrative ABP or TCR mimetic-based antibody provided herein.

[0175] In some embodiments, the ABPs or TCR mimetic-based 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 nonconservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. In yet more preferred 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 TCR mimeticbased antibody.

[0176] The ABP or TCR mimetic-based antibody can be isolated and purified by any suitable method known in the art.

[0177] The ABP or TCR mimetic-based antibody can be a multi specific antibody. The ABP or TCR mimetic-based 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 TCR mimetic-based 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 TCR mimetic-based 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, TCR mimetic-based 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 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 TCR mimetic-based antibody binds an HLA class I CGI peptide complex on a cancer cell. In certain embodiments, the TCR mimetic-based antibody is a multifunctional antibody.

[0178] In certain embodiments, the AB or TCR mimetic-based antibody 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.

[0179] In certain embodiments, the selective binding of the TCR mimetic-based 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).

[0180] In certain embodiments, the ABP or TCR mimetic-based antibody or fragment is a murine antibody, a chimeric antibody, a camelid antibody, a humanized antibody, or a human antibody.

[0181] In some embodiments, the ABP or TCR mimetic-based antibody comprises a monovalent antibody fragment comprising a single target molecule binding arm and an Fc region. In some embodiments, the ABP or TCR mimetic-based antibody comprises a fragment antigen-binding region. In some embodiments, the ABP or TCR mimetic-based antibody 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 TCR mimetic-based antibody comprises a single chain variable fragment antibody or derivative thereof. In some embodiments, the ABP or TCR mimetic-based antibody comprises a single chain variable fragment antibody in tandem format. In certain embodiments, the ABP or TCR mimetic-based antibody comprises a singledomain antibody. In certain embodiments, the ABP or TCR mimetic antibody comprises a humanized single domain antibody.

[0182] In certain embodiments, the TCR mimetic-based antibody comprises: i) a bispecific antibody, and optionally ii) a dimerized hinge-CH2-CH3; wherein the bispecific antibody comprises: i) at least one antibody fragment that binds CD3; and ii) at least one antibody fragment that selectively binds HLA-CG1 peptide antigen. In certain aspects, the anti-CD3 antibody fragment is a single chain variable fragment (scFv). In certain aspects, the anti-CD3 antibody fragment is an scFv and the antibody fragment that selectively binds HLA-CG1 is a Fab.

[0183] In certain embodiments, the TCR mimetic-based T cell engager protein is in a heterodimer format. In certain embodiments, the bispecific antibody is in a heterodimer format, and comprises: (i) an antibody fragment that binds CD3, (ii) an antibody fragment that binds HLA-CG1 peptide antigen, and (iii) a heterodimeric hinge-CH2-CH3. In certain aspects, TCR mimeticbased T cell engager protein comprises an anti-CD3 antibody fragment is a single chain variable fragment (scFv). In certain aspects, the anti-CD3 antibody fragment is an scFv and the antibody fragment that selectively binds HLA-CG1 is a Fab. In certain aspects, the TCR mimetic-based T cell engager protein comprises a first heavy chain, a second heavy chain, and a light chain. In some aspects, the first heavy chain comprises the anti-CD3-scFv-Fc, the second heavy chain comprises the anti-HLA-CGl Fab VH-CHl-Fc, and the light chain comprises the anti-HLA-CGl Fab VL-Ck. In certain embodiments, the bispecific antibody comprises (i) an antibody fragment that binds CD3, (ii) two antibody fragments bind HLA-CG1 peptide antigen, and (iii) a heterodimeric hinge-CH2-CH3. In certain embodiments, the bispecific antibody is in a homodimer format.

[0184] In certain embodiments, the bispecific antibody comprises (i) two antibody fragments bind CD3, (ii) two antibody fragments bind HLA-CG1 peptide antigen, and (iii) a homodimeric hinge-CH2-CH3.

[0185] In certain embodiments, the bispecific antibody is in a single chain format. In certain embodiments, the TCR mimetic T cell engager protein does not comprise a dimerized hinge-CH2-CH3. In certain embodiments, the TCR mimetic T cell engager protein comprises an antibody fragment that binds CD3, an antibody fragment that binds HLA-CG1 peptide antigen, and a 6XHis-tag.

[0186] In certain embodiments, the antibody fragment that binds CD3 comprises a first singlechain variable fragment (scFv). In certain embodiments, the TCR mimetic T cell engager proteincomprises a first polypeptide comprising the first scFv and a second polypeptide comprising a second scFv comprising the antibody fragment that binds HLA-CG1 peptide antigen. In certain embodiments, 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 TCR mimetic T cell engager protein comprises: i) a first polypeptide comprising the first scFv and a second scFv comprising the antibody fragment that binds HLA-CG1 peptide antigen; and ii) a second polypeptide comprising a hinge-CH2-CH3.

[0187] In certain embodiments, the second polypeptide does not comprise an antibody fragment that binds CD3 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 TCR mimetic T cell engager protein comprises two first polypeptides, wherein each first polypeptide comprises the first scFv that binds CD3 and a second scFv that binds HLA-CG1 peptide antigen. In certain embodiments, the antibody fragment that binds CD3 comprises a first Fab.

[0188] In certain embodiments, the TCR mimetic-based antibody comprises i) a first polypeptide comprising the first Fab and ii) a second polypeptide fragment comprising an scFv that binds HLA-CG1 peptide antigen. In certain embodiments, the TCR mimetic T cell engager protein comprises two first polypeptides, wherein each of the first polypeptides comprise the first Fab and an scFv that binds HLA-CG1 peptide antigen.

[0189] 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.

[0190] In certain embodiments, the Fc region (CH2-CH3) is silenced. In certain aspects, the Fc region comprises a C-terminal lysine.

[0191] In certain embodiments, the TCR mimetic-based antibody 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 asecond 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.

[0192] In certain embodiments, the antibody fragment that selectively binds the HLA-CG1 peptide antigen comprises a second and third single chain variable fragment (scFv).

[0193] A composition comprising TCR mimetic-based antibodies described herein may be a pharmaceutical composition. Such a composition may comprise multiple TCR mimetic-based 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. Several cytokines 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 asimmunoadjuvants (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.

[0194] Also provided herein are host cells comprising an ABP or TCR mimetic-based antibody or fragment thereof disclosed herein. In some embodiments, the host cell comprises a polynucleotide encoding an ABP or TCR mimetic-based antibody 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.

[0195] 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-CG1 peptide antigen.

[0196] 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 TCR mimetic (TCRm) T-cell engager (TCE), as defined 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 restrictedCGI complex could be a good target in development of antibody based anti-tumor therapy, in particular for cancers and tumors related to myeloid malignancies.

[0197] 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.

[0198] Myeloid proliferations and neoplasms have been categorized to include 9 sub-categories (families): 1) Myeloid precursor lesions, 2) Myeloproliferative neoplasms (MPNs), 3) Mastocytosis, 4) Myelodysplastic neoplasms (MDNs, previously known as myelodysplastic syndrome, MDS), 5) MDN / MPNs, 6) Acute myeloid leukemia (AML), 7) Secondary myeloid neoplasms, 8) Myeloid / lymphoid neoplasms with eosinophilia and defining gene rearrangement, 9) Acute leukemias of mixed or ambiguous lineage.

[0199] According to the present disclosure, hematological and / or myeloid malignancies that express antigens contemplated by the present disclosure (CGI, for example) include, for example, AML, CML, CMML, ALL, and CLL. Compositions and methods according to the present disclosure can detect, diagnose, treat, or otherwise be useful for myeloid and other heme malignancies, in particular those characterized by high expression of CGI (high expression compared to a WT or non-pathogenic subject).

[0200] Additional non-myeloid malignancies, for example ALL, CLL, and lung cancer (which also may express high CGI levels), are also contemplated by the present disclosure as cells expressing antigens of interest relating to myeloid malignancies may also be expressed by cells involved in non-myeloid pathologies.

[0201] Compositions, pharmaceutical compositions, and methods according to the present disclosure can be utilized, for example, to treat, diagnose, or otherwise detect a 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 myeloid malignancies are also presented (for example CGI or a derivative thereof, in particular, CGI (FLLPTGAEA; SEQ ID NO:61) - a 9-mer peptide derived from the cathepsin G protein leader sequence, presented by HLA class I, specifically HLA-A*02:01).

[0202] The present disclosure provides compositions and methods for treating, diagnosing, or otherwise detecting aspects of hematological and / or myeloid malignancies (for example in a subject having or suspected of having a hematological and / or myeloid malignancy, an animal model, an in vitro tissue culture model, and the like). TCRm-based antibodies or antigen binding portions thereof of the disclosure that specifically or selectively bind antigen targets related to hematological and / or myeloid malignancies are provided herein. In certain aspects, antibodies as described herein are human antibodies. In embodiments of the present disclosure, the provided TCRm-based antibodies and fragments thereof that specifically bind to HLA-A2 / CG1 as a tumor antigen. In further embodiments of the present disclosure, novel TCRm-based antibodies (or antibody fragments) specifically bind to HLA-A2 / CG1 as a tumor antigen, and can specifically bind to an antigen that can engage T-cells, for example CD3 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.

[0203] As used herein, the terms “specifically binds to”, “specific for”, “selectively binds”, “targets”, “selectively targets”, “selective”, and the like, for example, of a myeloid malignancy antigen or an epitope on a protein related to a 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.

[0204] An antibody, as used herein, can refer to an intact antibody (e.g., an intact immunoglobulin), a portion of a TCRm-based antibody, and antibody fragment, for example, an antigen binding fragment, or a bispecific antibody, including as part of a TCRm-based antibody. 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 dimer. A TCRm-based antibody, or antigen binding fragment can be described by the antigen to which they specificallybind. 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.

[0205] 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 CDRH1, 2, 3, while CDR sequences on the light chain (VL) may be designated as CDRL1, 2, 3.

[0206] Provided herein are TCRms, antibodies, and / or antigen binding portions thereof that specifically bind to antigens related to hematological and / or myeloid malignancies.

[0207] 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 (e.g., SEQ ID NOs: 7-9, 15-16, 34-36, 42-43, 55-57, 128, 132-134, 139-143) 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0208] 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 (e.g., SEQ ID NOs: 7-9, 15-16, 34-36, 42-43, 55-57, 128, 132-134, 139-143) 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0209] 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 (eg., SEQ ID NOs: 7-9, 15-16, 34-36, 42-43, 55-57, 128, 132-134,139-143) are also provided. In some cases, 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0210] The disclosure also provides a TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm, antibody, or antigen binding portion thereof comprises a 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: 7, 8, 9, 15, 16, 55, 133, or 134 and a 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Sequence details can be found in Example 2 below. In some embodiments, the TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a 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 1-27, 133, or 134 and a 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 28-54, 145-171 or 132.

[0211] The disclosure also provides a TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm, antibody, or antigen binding portion thereof comprises a 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: 7, 8, 9, 15, 16, 55, 133, or 134 and a light 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In some embodiments, the TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a 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 1-27, 133, or 134 and a light chain variableregion 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 28-54, 145-171, or 132.

[0212] The disclosure also provides a TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm, antibody, or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In some embodiments, the TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a heavy chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% 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 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs 28-54, 145-171, or 132.

[0213] The disclosure also provides a TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm, antibody, or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In some embodiments, the TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a heavy chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% 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 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs 28-54, 145-171, or 132.

[0214] The disclosure also provides an TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm, antibody, or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acidsequence that is at least 99% identical to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. In some embodiments, the TCRm, antibody, or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises 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 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, 145-171, or 132.

[0215] In preferred 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).

[0216] In some embodiments, the TCRm, antibody, or antigen binding portion thereof comprises the heavy 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: 7, 8, 9, 15, 16, 55, 133, or 134. In some embodiments, the TCRm, antibody, or antigen binding portion thereof comprises the heavy chain variable region 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: 1-27, 133, or 134.

[0217] Provided herein are TCRms and antigen binding portions thereof that specifically bind to antigens related to myeloid malignancies as well as another antigen, for example, CD3.

[0218] In each case of a TCRm, 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 (e.g., SEQ ID NOs: 7-9, 15, 16, 34-36, 42-43, 55-57, 128, 132-134, 139-143) are also provided. In some cases of the TCRm, 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0219] In each case of a TCRm, 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 (e.g., SEQ ID NOs: 7-9, 15-16, 34-36, 42-43, 55-57, 128, 132-134, 139-143) are also provided. In some cases of the TCRm, where aspecific 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0220] In each case of a TCRm, 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 (e.g., SEQ ID NOs: 7-9, 15-16, 34-36, 42-43, 55-57, 128, 132-134, 139-143) are also provided. In some cases of the TCRm, 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 (e.g., SEQ ID NOs: 1-57, 128, 132-134, 139-143) are also provided.

[0221] The disclosure also provides a TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen, wherein the TCRm or antigen binding portion thereof comprises a 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: 7, 8, 9, 15, 16, 55, 133, or 134 and a 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Such TCRms also comprise an antibody or antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:57, 128, 139 (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical). Sequence details can be found in Example 2 below. In some embodiments, the TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a 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: 1-27, 133, or 134 and a 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: 28-54, 145-171, 56 or 132.

[0222] The disclosure also provides a TCRm or antigen binding portion thereof that specifically binds to a hematological malignancy antigen, wherein the TCRm or antigen binding portionthereof comprises a 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: 7, 8, 9, 15, 16, 55, 133, or 134 and a light 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Such TCRms also comprise an antibody or antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:57, 128, 139 (for example, at least 95%, 96%, 97%, 98% or 99% identical). In some embodiments, the TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a 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: 1-27, 133, or 134 and a light 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: 28-54, 145-171, 56 or 132.

[0223] The disclosure also provides a TCRm or antigen binding portion thereof that specifically binds to a hematological malignancy antigen, wherein the TCRm or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Such TCRm antibodies also comprise an antibody or antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:57, 128, 139 (for example, at least 97%, 98% or 99% identical). In some embodiments, the TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a heavy chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% 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 97% identical (for example, at least 97%, 98% or 99% identical) to any of SEQ ID NOs: 28-54, 145-171, 56 or 132.

[0224] The disclosure also provides a TCRm or antigen binding portion thereof that specifically binds to a hematological malignancy antigen, wherein the TCRm or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Such TCRms also comprise an antibody or antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:57, 128, 139 (for example, at least 98% or 99% identical). In some embodiments, the TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises a heavy chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% 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 98% identical (for example, at least 98% or 99% identical) to any of SEQ ID NOs: 28-54, 145-171, 56 or 132.

[0225] The disclosure also provides a TCRm or antigen binding portion thereof that specifically binds to a hematological malignancy antigen, wherein the TCRm or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 99% identical to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 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: 34, 151, 35, 152, 36, 153, 42, 159, 43, 160, 56, or 132. Such TCRms also comprise an antibody or antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:57, 128, 139. In some embodiments the TCRm or antigen binding portion thereof that specifically binds to a myeloid malignancy antigen comprises 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 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, 145-171, 56 or 132.

[0226] In examples, 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). In examples, the non-myeloid malignancy antigen can be CD3.

[0227] In some embodiments of a TCRm, the TCRm or an antigen binding portion thereof comprises the heavy 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: 7, 8, 9, 15, 16, 55, 133, or 134. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the antibody or antigen binding portion thereof 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 SEQ ID NO:57, 128, 139. In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises a 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: 1-27, 133, or 134.

[0228] In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises the heavy 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 SEQ ID NOs: 7, 55, 133, or 134. and a 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 SEQ ID NOs: 34, 151, 56, or 132. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof 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 SEQ ID NO:57, 128, 139.

[0229] In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises the heavy chain variable region comprises an amino acid sequence that is at least 95% identical (for example, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOs: 7, 8, 9, 15, 16, 55, 133, or 134. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the TCRm or antigen binding portion thereof 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 SEQ ID NO:57, 128, 139. Insome embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical (for example, 95%, 96%, 97%, 98% or 99% identical) to any of SEQ ID NOs: 1-27, 133, or 134.

[0230] In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises the heavy 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 SEQ ID NOs: 7, 55, 133, or 134 and a light 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 SEQ ID NOs: 34, 151, 56, or 132. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the antigen binding portion thereof comprises an amino acid sequence that is at least 95% identical (for example, at least 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NO:57, 128, 139.

[0231] In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises the heavy chain variable region comprises an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to SEQ ID NOs: 7, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to SEQ ID NOs: 34, 151, 56, or 132. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the antigen binding portion thereof comprises an amino acid sequence that is at least 97% identical (for example, at least 97%, 98% or 99% identical) to SEQ ID NO:57, 128, 139.

[0232] In some embodiments of a TCRm, the TCRm or antigen binding portion thereof comprises the heavy chain variable region comprises an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to SEQ ID NOs: 7, 55, 133, or 134 and a light chain variable region comprising an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to SEQ ID NOs: 34, 151, 56, or 132. Such TCRms also comprise an antigen binding portion thereof that specifically binds to a non-myeloid malignancy antigen, such as CD3, wherein the antigen binding portion thereof comprises an amino acid sequence that is at least 98% identical (for example, at least 98% or 99% identical) to SEQ ID NO:57, 128, 139.

[0233] 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 refersto 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 (Ile 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 (Val 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.

[0234] The terms identical or percent identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 90%, or 95% or greater identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0235] Identity or similarity with respect to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0236] As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof described herein, for example, inthe 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).

[0237] 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.

[0238] In some instances, the affinity of myeloid malignancy 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 hematological malignancy antigen, the structure of either or both of the antibodies or fragments thereof, or the hematological malignancy antigen. In some instances, the mutations permit facile elution of purified antibodies or fragments thereof under desirable elution conditions during isolation and purification.

[0239] 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.

[0240] The present disclosure also encompasses TCRms, antibodies or fragments thereof that bind to the same epitope of hematological malignancy antigens as the antibodies disclosed herein. Suchantibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0241] The present disclosure also encompasses bi-specific antibodies or fragments thereof that bind to the same epitope of hematological 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] In some embodiments, a TCRm, 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 halfantibody). In another example, a TCRm, 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) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen. A TCRm, 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 TCRm, antibody, or antigen binding fragment thereof can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. A TCRm, antibody, or antigen binding fragment thereof can also have a light chain chosen from either kappa or lambda light chains.

[0246] 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.

[0247] Antigen binding fragments, e.g., of a TCRm or of an 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 CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (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.

[0248] In certain embodiments, a TCRm, antibody, or antigen binding fragment thereof, and compositions comprising TCRms 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.

[0249] In one approach, a TCRm, antibody, or antigen binding fragment as provided in this disclosure comprises 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

[0250] In some embodiments, the TCRm antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin producing human 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 human B cell; as a non-limiting example, if an IgE-producing human 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.

[0251] In some embodiments, the TCRm antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin-producing human 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).

[0252] In some embodiments, the TCRm 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 can introduce undesirable effects and reduce the therapeutic efficacy of the antibody. See, e.g., Silva et al., J Biol Chem, 2015, 280:5462-5469.

[0253] In some embodiments, the TCRm 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. In 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., wildtype) 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.

[0254] Synthetic TCRm antibodies of this disclosure 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-life, 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” Int 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.

[0255] In some embodiments, the heavy chain variable region and / or the light chain variable region of the TCRm antibody 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 from thehuman subject having a myeloid malignancy. In some embodiments, the heavy chain variable region and / or the light chain variable region of the TCRm antibody comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0256] The heavy chain variable region sequence and / or light chain variable region sequence of a TCRm 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 TCRm antibody for a hematological and / or myeloid malignancy. In some embodiments, the engineered variation(s) improves the cross-reactivity of the TCRm antibody for a second hematological and / or myeloid malignancy.

[0257] 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 Trop Dis, 2017, ll:e0005655, doi:10.1371 / journal.pntd.0005655

[0258] In some embodiments, affinity maturation is used to engineer further mutations that enhance the binding affinity of the antibody for a myeloid malignancy or enhance the crossreactivity of the antibody for a second 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.

[0259] TCRm 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 from antibodies. 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 domainantibodies 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.

[0260] 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.

[0261] Synthetic TCRm 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.

[0262] The hematological malignancy antigen-specific antibodies 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 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 andhuman cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.

[0263] The hematological malignancy antigen-specific antibodies or antigen binding fragments thereof disclosed herein (e.g., the TCRm 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.

[0264] 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 Tn 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 A cad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0265] 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, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0266] 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. coll, 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.

[0267] 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.

[0268] 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 vary 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., Kaszubskaet al. (2000) Protein Expression and Purification 18:213-220).

[0269] 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 Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with asingle 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.

[0270] 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.

[0271] 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 ex vivo. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.

[0272] 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 orpolypeptides 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 chemoselective 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)).

[0273] 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)).

[0274] 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)).

[0275] 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 include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, anantibody 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.

[0276] 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).

[0277] Any of the TCRm 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 TCRm 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 base-pair 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 TCRm antibodies or fragments. In some instances, the TCRm or the hematological malignancy antigen-specific antigen binding fragments may be labeled by a variety of means for use in diagnostic and / or pharmaceutical applications.

[0278] In some embodiments, the TCRm 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 a TCRm 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 TCRm 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 codon encoding the amino acid substitution. In some embodiments, the modification may comprise a single amino acid substitution. In some embodiments, themodification may comprise multiple amino acid substitutions. Tn 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.

[0279] For example, the amino acid substitution may be located in heavy chain variable region CDR2 sequence of a TCRm antibody or antigen binding fragment provided in this disclosure. In some embodiments, the amino acid substitution can be at the 5thor 6thposition (from N terminus to C terminus) of the heavy chain variable region CDR2 sequence of a TCRm antibody or antigen binding fragment provided in this disclosure. In some embodiments, the amino acid substitution can be at the 5thor 6thposition (from N terminus to C terminus) of the heavy chain variable region CDR2 sequence of SEQ ID NO:82 of a TCRm antibody or antigen binding fragment provided in this disclosure. In some embodiments, the amino acid substitution can be at the 5thor 6thposition (from N terminus to C terminus) of the heavy chain variable region CDR2 sequence of SEQ ID NO: 82 of a TCRm or antigen binding fragment provided in this disclosure, wherein the TCRm antibody or antigen binding fragment further comprises a heavy chain variable region CDR1 of SEQ ID NO:71 and a heavy chain variable region CDR3 of SEQ ID NO:90. In some embodiments, the amino acid substitution can be at the 5thor 6thposition (from N terminus to C terminus) of the heavy chain variable region CDR2 sequence of SEQ ID NO:82 of a TCRm antibody or antigen binding fragment provided in this disclosure, wherein the heavy chain variable region comprises at least 90% identity to SEQ ID NO: 7. In some embodiments, the amino acid substitution can be at the 5thor 6thposition (from N terminus to C terminus) of the heavy chain variable region CDR2 sequence of SEQ ID NO: 82 of a TCRm antibody or antigen binding fragment provided in this disclosure, wherein the heavy chain variable region comprises at least 90% identity to SEQ ID NO:7, and wherein the TCRm antibody or antigen binding fragment comprises a light chain variable region comprising at least 90% identity to SEQ ID NO:34 or SEQ ID NO: 151. In some embodiments, the amino acid mutations may include aspartic acid to serine modification. In some embodiments, the modification may include an alanine to glycine. One skilled in the art may understand that any modification to the antibody sequence that reduced the hydrophobicity of an amino acid sequence may fall under the scope of such modifications. Described above modifications are provided herein not to limit the scope of the invention but merely provided as examples of modifying an amino acid sequence for altering the hydrophobicity.

[0280] In some embodiments, the TCRm 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.

[0281] 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-oc-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 thatcross-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).

[0282] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the TCRm antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]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).

[0283] 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.

[0284] In some embodiments, the TCRm 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 Reviews54:459-476, 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.

[0285] In some embodiments, the TCRm 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.

[0286] The myeloid malignancy antibodies and antigen binding fragments thereof and molecules comprising such antibodies and antigen binding fragments thereof discussed above (e.g., Abs, bispecific Abs,) 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 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.

[0287] The myeloid malignancy 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, 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.

[0288] 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).

[0289] 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, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0290] Compositions comprising a hematological malignancy antigen-specific TCRm 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 malignancy that would benefit from any of the antigen-specific antibodies or antigen binding fragments thereof described herein.

[0291] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and / or I. V. administration. In 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 ex vivo release and / or rate of ex vivo clearance of the myeloid malignancy antigen-specific antibody or antigen binding fragment thereof.

[0292] 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 pHcontrolling buffer such phosphate-buffered saline or acetate-buffered saline. Tn certain embodiments, a composition comprising a 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 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 cryopreservative, 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.

[0293] 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.

[0294] 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.

[0295] 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 a myeloid malignancy antigen-specific 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 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.

[0296] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, a hematological malignancy antigen-specific antibody or antigen binding fragment thereof can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising a myeloid malignancy 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.

[0297] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, a hematological malignancy 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 capsule can 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 a myeloid malignancy antigen-specific 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.

[0298] In certain embodiments, a pharmaceutical composition can involve an effective quantity of a hematological malignancy 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.

[0299] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a hematological malignancy 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., fdms, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U. S. Patent No. 3,773,919; U. S. PatentNo. 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 765:252-267 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 PatentNo. EP0133988). In certain embodiments, 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 PatentNo. EP 036,676; and U. S. Patent Nos. 4,619,794 and 4,615,885).

[0300] The pharmaceutical composition to be used for ex vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by fdtration 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.

[0301] 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.

[0302] 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.

[0303] In certain embodiments, the effective amount of a pharmaceutical composition comprising a hematological malignancy 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 a hematological malignancy 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.

[0304] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the hematological malignancy antigen-specific antibody or antigen binding fragment thereof in the formulation used. 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, forexample, 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.

[0305] 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.

[0306] 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.

[0307] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising a hematological malignancy 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 a myeloid malignancy antigen-specific antibody or antigen binding fragment thereof after which the cells are subsequently implanted back into the subject.

[0308] In certain embodiments, a myeloid malignancy 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 decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the protein product(s) but prevent the destruction of the cells by a subject’s immune system or by other detrimental factors from the surrounding tissues.

[0309] The hematological malignancy antigen-specific TCRm antibody or antigen binding fragment thereof provided in this disclosure are suited for in vitro 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.

[0310] The TCRm antibodies according to the present disclosure can be bound to many different carriers and used to detect the presence of hematological 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.

[0311] For purposes of this disclosure, hematological malignancy may be detected by the provided antibodies fragments thereof when the virus is present in biological fluids and tissues from a subj ect that may have myeloid malignancy. Any sample containing a detectable amount of myeloid malignancy can be used. 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.

[0312] Another approach to assessing whether a subject has a hematological malignancy is to determine if the B cell receptor (BCR) repertoire of the subject includes a coding sequence for one of the hematological malignancy specific antibodies provided in this disclosure. An exemplary method of this type of sequence analysis is described in the art. For example, RNA from wholeblood or B cells (PBMCs) can be used as the template for the amplification of 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 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 myeloid malignancy or has previously been affected by hematological malignancy.

[0313] In using the provided antibodies and fragments thereof for the ex vivo detection of hematological malignancy antigens, the detectably labeled TCRm antibody or fragment thereof 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 the hematological malignancy antigen for which the antibodies are specific.

[0314] The concentration of detectably labeled TCRm antibody or fragment thereof which is administered should be sufficient such that the binding to hematological malignancy 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.

[0315] As a rule, the dosage of detectably labeled TCRm 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 mg / 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.

[0316] 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 whichis 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 are111In,97Ru,67Ga,68Ga,72As,89Zr and201Tl.

[0317] The hematological malignancy 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,5’Mn,162Dy,52Cr and56Fe.

[0318] The provided antibodies and antigen binding fragments can be used in vitro and ex vivo to monitor the course of hematological malignancy disease therapy. Thus, for example, by measuring the increase or decrease in the number of hematological malignant cells or changes in the concentration of hematological malignancy present in the body or in various body fluids, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating hematological malignancy disease is effective.

[0319] As described herein, the present disclosure provides a method of treating a subject with a hematological malignancy, comprising administering to the subject a therapeutically effective amount of a hematological malignancy-antigen-specific TCRm antibody or antigen binding fragment thereof of the present disclosure. In some embodiments, the subject has or is determined to have a hematological malignancy.

[0320] The compositions described herein are useful in, inter alia, methods for treating a hematological malignancy in a subject. As used herein, the term subject means a mammaliansubject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have a hematological malignancy. In some embodiments, the subject is diagnosed with a hematological malignancy. In some embodiments, the subject is a human that is suspected of having a myeloid malignancy, for example, a hematological malignancy as described herein.

[0321] 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 hematological malignancy antigen-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.

[0322] 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, 8th 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.

[0323] 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 sikalastic 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 ApplicationPublication No. 20080241223; U. S. Patent Nos. 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, a hematological malignancy antigen-specific antibody or antigen binding fragment of the present disclosure is therapeutically delivered to a subject by way of local administration.

[0324] 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.

[0325] 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., a hematological malignancy, 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 hematological malignancy 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 inbetween 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.

[0326] The principal symptoms of hematological malignancy can include (without intending to be limiting) bone pain, nausea, constipation, loss of appetite, mental fogginess or confusion, fatigue, frequent infections, weight loss, 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.

[0327] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of a hematological malignancy 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 hematological malignancy disease are ameliorated, or the likelihood of hematological malignancy 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 hematological malignancy 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 hematological malignancy antigen-specific antibody may be required to treat a subject with a hematological malignancy as compared to the dose of a fragment of a hematological malignancy 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 hematological malignancy. For example, a subject that has had a previous hematological malignancy may require administration of a different dosage of hematological malignancy antigenspecific antibody or antigen binding fragment thereof than a subject who has not previously had a hematological malignancy. 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 understoodthat 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 mg / kg to about 50 mg / kg, preferably from about 0001 mg / kg to about 20 mg / kg, most preferably from about 0.002 mg / kg to about 2 mg / kg, in one or more dose administrations daily, at least 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 hematological malignancy 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.

[0328] A pharmaceutical composition can include a therapeutically effective amount of a hematological malignancy antigen-specific 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 hematological malignancy antigen-specific antibody or antigen binding fragment thereof, or the combinatorial effect of the hematological malignancy 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 mg / kg, or any intervening dose between about 0.001 mg / kg and 1 mg / kg.

[0329] Suitable human doses of any of the hematological malignancy 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.

[0330] Toxicity and therapeutic efficacy of such hematological malignancy 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 cancersdescribed herein). These procedures can be used, e g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (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. A hematological malignancy 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.

[0331] 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 of a hematological malignancy antigen-specific antibody or antigen binding fragment thereof lies generally within a range of circulating concentrations of the hematological malignancy antigen-specific antibody or antigen binding fragment that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For hematological malignancy antigen-specific antibodies or antigen binding fragments thereof described herein, 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 EC50 (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.

[0332] In some embodiments, a hematological malignancy antigen-specific antibody or antigen binding fragment thereof described herein can be administered to a subject as a monotherapy. Alternatively, the hematological malignancy antigen-specific antibody or antigen binding fragment thereof can be administered in conjunction with other therapies for cancer (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 hematological malignancy antigen-specific antibody or antigen binding fragment thereof and the one or more additional active agents are administered at the same time. Optionally, the hematological malignancy antigen-specificantibody or antigen binding fragment thereof is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the hematological malignancy antigenspecific antibody or antigen binding fragment thereof is administered second in time. Optionally, the hematological malignancy antigen-specific antibody or antigen binding fragment thereof and the one or more additional agents are administered simultaneously in the same or different routes. For example, a composition comprising the hematological malignancy antigen-specific antibody or antigen binding fragment thereof optionally contains one or more additional agents.

[0333] A hematological malignancy antigen-specific antibody or antigen binding fragment thereof described herein can replace or augment a previously or currently administered therapy. For example, upon treating with a hematological malignancy antigen-specific antibody or antigen binding fragment thereof, administration of the 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 hematological malignancy antigen-specific antibody or antigen binding fragment thereof reaches a level sufficient to provide a therapeutic effect.

[0334] Monitoring a subject (e.g., a human patient) for an improvement of hematological malignancy, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in one or more symptoms of hematological malignancy 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 hematological malignancy described herein.

[0335] In some instances, the hematological malignancy antigen-specific 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 andusing virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0336] In some instances, the hematological malignancy antigen-specific 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).

[0337] In some instances, the hematological malignancy antigen-specific 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.

[0338] 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 a hematological malignancy antigen-specific antibody or antigen binding fragment thereof as described in this disclosure.

[0339] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient immune cells that express a CAR comprising a hematological malignancy antigen-specific antibody or antigen binding fragment thereof as described herein.

[0340] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding a hematological malignancy antigen-specific antibody or antigen binding fragment thereof as described in this disclosure.

[0341] 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.

[0342] 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. Alsopreferred 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 hematological malignancy antigen-specific antibody or antigen binding fragment thereof can be delivered via virus-like particles.

[0343] 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 Jolla, CA), and Invitrogen / 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.

[0344] 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.. -actin promoter or EFla promoter),or from hybrid or chimeric promoters (e.g., CMV promoter fused to the 0-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0345] 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.

[0346] 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 SV40 promoter, the beta-actin promoter, the EFla promoter, and the retroviral long terminal repeat (LTR).

[0347] 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), andluciferase. 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.

[0348] In certain embodiments, the effective amount of a pharmaceutical composition comprising a hematological malignancy 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 a hematological malignancy 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.

[0349] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the hematological malignancy antigen-specific 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), / . 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) Enr. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra'). 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.

[0350] In some cases, the dosage (of the active component) ranges from about 0.0001 to 100 mg / kg, and more usually 0.0002 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 0.0001-20 mg / kg. In certain examples, the hematological malignancy antigen-specific 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 hematological malignancy antigen-specific 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 hematological malignancy antigen-specific 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 hematological malignancy antigen-specific 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 hematological malignancy antigen-specific 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.

[0351] 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.

[0352] 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.

[0353] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising a hematological malignancy 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 a hematological malignancy antigenspecific antibody or antigen binding fragment thereof after which the cells are subsequently implanted back into the subject.

[0354] In some instances, the provided methods may include administering to the subject a hematological malignancy antigen-specific 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.

[0355] In some instances, the provided methods may include administering a hematological malignancy antigen-specific antibody or antigen binding fragment thereof and a second form of cancer therapy to the subject. The second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies which may be administered as a second form of cancer 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).

[0356] In some embodiments, the hematological malignancy antigen-specific 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 a hematological malignancy antigen-specific 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 a hematological malignancy antigen-specific 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).

[0357] In certain aspects, hematological malignancy antigen-specific 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, a radiotherapeutic agent, and immunotherapeutic agent, 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 hematological malignancy antigen protein and a second therapeutic effect by delivering a supplemental therapeutic agent.

[0358] 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 hematological malignancy antigen-specific antibody or antigen binding fragment thereof. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.

[0359] In some instances, the therapeutic agent is a cytotoxic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof. Additional examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, col chicin, dihydroxy anthracin di one, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e. g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents e.g., vincristine and vinblastine).

[0360] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. 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 ELOXATFN®, Sanofi), 5 -fluorouracil (5-FU), leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYKERB®, 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 agents such as thiotepa and cyclosphosphamide (such as CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin yi1and calicheamicin Oi1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (such as ADRIAMYCIN®, including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Trametes Versicolor polysaccharide-K (Krestin, PSK) (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (cytosine arabinoside, “Ara-C”); cyclophosphamide; thiotepa; taxoids, e g., paclitaxel (such as TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N. J.), ABRAXANE™ (a Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)), and doxetaxel (such as TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0361] Chemotherapeutic agents, as used herein, also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON®); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestanie, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3 -di oxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) VEGF receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN-7® vaccine (plasmid / lipid complex containing the DNA sequences encoding HLA-B7 and 132 microglobulin), LEUVECTIN® vaccine (plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID® vaccine (patient-specific naked DNA vaccine); IL-2 or aldesleukin (such as PROLEUKIN®); topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropin-releasing hormone antagonists (such as ABARELIX®); (x) anti-angiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0362] In some instances, the treatment methods provided herein may further comprise administering one or more immunomodulatory agent, such as one or more immunosuppressive agent, for example checkpoint inhibitors and antagonists, as part of the method. In certain aspects, the immunomodulatory agent is immunosuppressive. Exemplary immunosuppressive agents are agents targeting PD-1 (such as nivolumab and pembrolizumab), agents targeting PD-L1 (such as atezolizumab, durvalumab, and avelumab), and agents targeting CTLA-4 (such as ipilimumab). In one example, the second form of cancer therapy comprises an inhibitor or immunomodulatory agent, including but not limited to a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor. In some instances, the PD-L1 targeting agent is a PD-L1 inhibitor, the PD-1 targeting agent is a PD-1 inhibitor, and / or the CTLA4 targeting agent is a CTLA4 inhibitor. In some instances,combinations of such immunomodulatory agents can be administered, e.g., two different immunomodulatory agents each targeting a different target (e.g., PD-1 and CTLA4). In some instances, the immunomodulatory agent is a multispecific agent, which is specific for two or more immunomodulatory targets, e.g., PD-1 and CTLA4.

[0363] In some instances, the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Systemic radiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells.EXAMPLES EXAMPLE 1: Development of bispecific T cell receptor (TCR)-like antibodies targeting A2 / CG1 for hematological malignancy.

[0364] FIG. 1 is an overview of neutrophil granule proteases. Cathepsin G (CG), for example, is a myeloid azurophil granule serine protease that is involved in host immunity, cleavage of inflammatory mediators and receptors, degradation of extracellular matrix components and leukemogenesis.

[0365] Briefly, anti-HLA-A2 / CGl binder sequences were produced and incorporated into antibodies. Assays revealed that these anti-HLA-A2 / CGl compounds recognize their target on leukemia cell lines: U937-A2, MV4-11-A2, THP-1, MOLM-13-A2, OCI-AML3, EM2, SKM-1, and ML-2.

[0366] Assays were performed to characterize the specificities of the top lead HLA-A2-CG1 binders using various techniques. In silico docking data from MOE identified a variable region fully covering the CGI peptide and HLA-A2 surface. Hydrogen Deuterium Exchange (HDX) mass spectrometry at amino acids resolution confirmed binding an epitope covering most of CGI peptide.

[0367] Top performing anti-HLA-A2 / CGl binders were incorporated into a bispecific antibody for HLA-A2 / CG1 and CD3e.

[0368] Assays were performed to assess in vitro T-cell engaged killing of U937-A2, EM2, THP1, AML-14 and ML2 cell lines by the CGlxCD3e bispecific antibody. Assays assessing activation of T-cells after TDCC to U937-A2, EM2, THP1, AML-14 and ML2 were performed by detecting early activation marker CD69+ T-cells and later activation marker CD25+ T cells. T-cell engaged induction of IFNy levels by CGlxCD3e to U937-A2, EM2, and ML2 were detected by immunoassay. Cytokine release in the presence of TA-1 was detected using a cytokine cytometric bead array (CBA) human inflammatory cytokine kit. The efficacies of the CG1XCD3 bispecific antibody were assessed in the ML2 xenograft model. Female NSG mice at 6-10 weeks old received tail vein injection with 0.1 M of ML2 / Luc human AML cells per mouse. 11-days after tumor inoculation, 9M PBMC / mouse were IV injected for humanization. Mice were either treated with a dummy TCE or CGlxCD3 at different doses.

[0369] The anti-tumor activity of CGlxCD3 BsAb in SIMP92 AML patient derived xenograft model was assessed across donors. Sublethally irradiated female NSG mice at 6-10 weeks old received tail vein injection with 6M of SIMP92 cells per mouse. 3M PBMC / mouse were IV injected for humanization. Mice were either treated with dummy TCE control or CG1-CD3 at O.lmg / kglV.

[0370] The Pharmacokinetics of 1B7 / CD3 BsAb were also assessed. Briefly, single dose of CG1-16xCD3 PK in human CGI & HLA-A*02:01 double transgenic mice was assessed at 1, 0.1 or 0.01 mg / kg doses. Blood was collected at indicated time points post injection through serial sampling and processed to plasma for bioanalytical and PK analyses with coated CGI monomer and detecting antibody GG-5 conjugated sulfo-tag using MSD methods. PK analyses were performed according to standard non-compartmental analysis using WinNonlin.

[0371] Sequences used in this Example are found below.

[0372] Based on the results of these assays, top lead anti-HLA-A2 / CGl candidates were discovered, and their specific, therapeutic activities revealed. Top performing candidates were brought forward for a further variant discovery campaign and for possible inclusion in a TCRm-based T-cell engager as described herein.Example 2: TCR Mimetic T Cell Engagers Targeting pHLA-CGl

[0373] Based on results from above, lead anti-CGl-pHLA candidates were chosen for inclusion in a T-Cell Receptor Mimetic (TCRm) T-Cell Engager (TCE) specific for both the CGl-pHLA peptide complex and CD3 expressed by an immune cell. The lead candidate TCRms are referredto herein as TA-1 and TA-2. TA-2 differs from TA-1 in the use of a different CGl-pHLA binder. FIGS. 4-6 provide schematics of TCRms of the disclosure, including TA-l / TA-2. FIG. 4 provides an example of a TCRm, such as TA- 1 or TA-2 (right), which is an immune cell engager that targets the peptide MHC-1 complex (in the case of TA-1 / TA-2, the CGl-pHLA complex) on a target cell while engaging an immune cell via the immune cell receptor CD3 (left) and contains an Fc domain with silencing mutations. As shown in FIGS. 4-6, additional TCRm formats are disclosed herein.

[0374] FIGS. 4-5 diagram exemplary TCRms of the disclosure. FIG. 5 provides another preferred TCRm embodiment 601. This exemplary TCRm includes a first antigen binding domain 603 on a first peptide and a second binding domain 605 and third binding domain 607 on a second peptide. The antigen binding domains are bound to an Fc region 609 via a hinge. The Fc region may include modifications and substitutions (611) as described herein, including a label, linker, or other point of attachment. Preferably, the modifications and / or substitutions provide Fc silencing.

[0375] The first and second antigen binding domains (603; 605) target an antigen (e.g., on a target cell / an HLA-peptide complex), while the third antigen binding domain binds to an immune cell (e.g., as an immune cell engager or activator). For example, the first and second antigen binding domains may bind to different CG peptides presented by HLA, different HLA-CG peptide complexes (e.g., presented by different HLA alleles), and / or bound or unbound CG peptides, and the like, while the third antigen binding domain binds to CD3 on a T cell.

[0376] As shown in FIG. 7, in certain malignancies, such as AML, CG is overexpressed. CGI is an HLA-A*02-restricted CG peptide. CGI binds HLA-A*02 with a high affinity. Of particular interest, the CG1 / HLA-A*O2 complex has been detected in 75% of tested HLA-A*02 AML patients and in 50% of HLA-A*02 ALL patients, which includes across patients with various cytogenetics and FAB subtypes. Further, recently, functional CGl / HLA-A*02:01-specific cytotoxic T lymphocytes was detected in peripheral blood post allo-SCT in 100% of tested (8 / 8) AML patients.

[0377] Consequently, the TCRm was designed to target both CGI and CD3. For the CD3 arm, TA-1 used a binder having an amino acid sequence of SEQ ID NO: 128. TA-1 incorporated an anti-CGl / HLA-A*02 binder with a variable light chain sequence as found in SEQ ID NO: 132, a heavy chain variable sequence as found in SEQ ID NO: 133 and a full antibody sequence comprising three peptide chains as set forth in SEQ ID NOS: 139-141.

[0378] To assess the potency of TA-1, its binding affinity for the CGl-pHLA complex was assayed as was its affinity for CD3. FIG. 8 provides affinity data for TA-1 and TA-2. Affinities were measured via Surface Plasmon Resonance (SPR), using a Carterra instrument. Desirably, the data shows that TA-1 / TA-2 have sub-nM affinity for the CGl-pHLA complex, and bind to the pHLA 4-5x more potently than to CD3. As such, when TA-1 / TA-2 bind to an immune cell, its higher affinity for the CGl-pHLA peptide complex will help assure it engages an immune cell along with a target cell expressing the CGl-pHLA complex. Moreover, this potent binding to CGl-pHLA maintained at pH=6, which mitigates risk that the TCRm would not be active at lower pH (e.g., diseased bone marrow, extramedullary sites).

[0379] FIGS. 9A shows detection of TA-1 on cells with varying CG1-HLA peptide complex levels. The following cells were contacted with TA-1, ML2 (cells have -3,700 copies of CG1-HLA), U937-A2 cells, which are U937 cells (which are HLA-A*03:01 -positive), transduced with HLA-A*02:01 (-2,300 copies of CG1-HLA per cell), U937 cells, and U937-A2-CTSG knockout cells. As shown in FIG. 9A, despite the pHLA targets in this assay generally having lower target copy numbers per cell, on-cell binding for CG1-HLA was readily detectable. The height of the curves correlates with the amount of antigen present on the cells: CD3e on Jurkat cells > CG1-HLA on ML2 > CG1-HLA on U937-A2, which is an avidity measurement. The slope of the curves correlates with the binding strength of TA-1 to the antigen on the cell: CG1-HLA binding is more potent than CD3e binding (consistent with Carterra data), which represents an affinity measurement. On-cell binding data predicts TA-1 binding affinity for CG1-HLA is ~2x more potent than for CD3e. Similar data not shown was measured for TA-2. As shown in Jurkat NF AT Activation Assay data provided in FIG. 9B, with a positive control (U937-A2) and negative control (U937 without HLA-A*02:01), contact with TA-1 or TA-2 causes an HLA-CG1 -specific T-cell activation.

[0380] Next, assays were performed to assess in vitro T-cell engaged killing of U937-A2, EM2, and ML2, and AML-14 cell lines by TA-1 at varying concentrations. Each cell line represents a leukemia cell with a varied CG1-HLA expression level. The data across all cell lines is found in FIG. HA. As shown in FIG. HA and 1 IB, TA-1 mediates potent killing of leukemic cells with a single digit EC50 and sub-nM EC90 across target cell lines with varying CG1-HLA copy numbers.

[0381] As shown in FIGS. 10A-10C, TA-1 mediates a strong T-cell activation on target-positive cells, including due to binding endogenous pHLA complexes found on AML cells, and when thoseendogenous pHLA complexes are expressed at relatively low per-cell amounts. Equivalent results were obtained for TA-2, for which the data is not shown. The negative control cells, as expected, did not experience substantial T cell activation until the concentration of TA-1 rose to at least 1 nM. Even then, as shown in FIG. IOC, the T cells in the presence of non-target-expressing cells did exhibit meaningful IFNy cytokine release, which is a cytokine involved in upregulating bystander killing. Thus, the presently disclosed TCRms do not induce bystander killing in the absence of the target pHLA, even at high concentrations that otherwise seem to cause some T cell activation.

[0382] To confirm the ability of the presently disclosed TCRms to induce killing of target-positive leukemia cells, TA-1 was added to a 3:1 culture of leukemia cells and PBMC effector cells in an in vitro killing assay. For target-positive cells, U937-A2 cells were used, which are positive for CG and HLA-A*02:01, ML2 cells, EM2 cells, and AML-14 cells. As shown in FIG. 1 IB, TA-1 caused a strong cytotoxic response on cells with a pHLA per cell number as low as 33. For targetnegative cells, U937-A2- CG-knock-out (KO)cells were used (also referred to as U937-A2-KO), which are negative for Cathepsin G (CRISPR knock-out (KO)), positive for HLA-A*02:01 (see FIG 29A).

[0383] A novel feature of the presently disclosed TCRm is their ability to cause “bystander killing”, which leads to more complete and effective clearance of diseased cells, particularly, cancer cells. FIG. 12 provides a schematic of the bystander killing produced by the TCRms of the disclosure. As shown, upon contact with TA-1, T cells are directly activated and rapidly kill targetpositive (e.g., CGl-pHLA-A*-02:01) cells. The activated T cells upregulate FAS-L, express LFA-1 with a high affinity, and secrete cytokines. Secreted IFNy and TNFa act on nearby targetnegative cells, thereby inducing upregulation of FAS and ICAM-1. Through the FAS-L / FAS and LFA-l / ICAM-1 interactions with the activated T cells, bystander cells are rendered susceptible to (delayed) killing by activated T cells. This additional cytotoxic activity, which occurs proximal to target-activated T cells, is critical to overcoming heterogeneous tumors and mounting a more systemic immune response. This is particularly relevant in treating AML, in which eliminating target-negative, yet cancerous, cells in bone marrow and extramedullary sites are required for full clearance of the disease.

[0384] To confirm the ability of the presently disclosed TCRms to induce bystander killing of target-negative cells, TA-1 was co-cultured with target-positive and target-negative cells atincreasing concentrations in an in vitro killing assay. For target-positive cells, U937-A2 cells were used, which are positive for CG and HLA-A*02:01. For target-negative cells, U937-A2-KO cells were used, which are negative for Cathepsin G (CRISPR KO), positive for HLA-A*02:01. As shown in the results provided in FIG. 13, the TCRm non-pHLA expressing AML cells were nonetheless targeted in response to T-cell activation by the TCRms when the activation is caused in response to contact with pHLA-expressing target cells.

[0385] To confirm the efficacy of the TCRms in vivo, TA-1 was provided to an ML2 cell line-derived xenograft CDX murine model at various concentrations. As a control, a “dummy” TCRm was prepared, which lacked the pHLA binding site. As shown in FIG. 14B, by day 14, the mice that received the “dummy” TCRm had uncontrolled tumor spread. In contrast, mice that received TA-1 exhibited clear tumor control over the course of the study, indicating a potent tumor controlling activity. Further, as shown, in a murine model (FIG. 14A), TA-1 exhibited a long halflife across all dose levels administered in human FcRn mice.

[0386] FIG. 15 provides results from further in vivo assays. As shown, TA-1 provided a clear survival benefit to tumor-infected mice, while no benefit was obtained using the “dummy” TCRm (isotype TA). Further, immunophenotyping results show that the TCRm effectively targets AML cells in the blood, bone marrow, lung, liver and spleen while remaining non-brain-penetrant.

[0387] Based on the aforementioned results, the TA-1 was tested across nine HLA-A*02:01 positive primary AML models. A schematic of these tests is provided in FIG. 16. Frozen primary un-passaged (Pl) AML samples were obtained using autologous patient T cells in each model as effector cells. The goals of these tests included assessing primary model E: T ratios at baseline (Day 0) and assessing T-cell activation and efficacy caused by TA-1 on AML blast populations using flow cytometry following 3-day incubation with TA-1 (Day 3 endpoint). FIG. 17 provides pertinent information on the nine relevant models.

[0388] FIGS. 18A-18F provide results from the study evaluating autologous T cell activation and cytotoxicity mediated by TA-1 towards primary AML cells, measured in ex vivo assays. As shown in FIGS. 18A-B, the TCRms induce T cell expansion and IFNy secretion in a dose-dependent manner. Expansion of CD8+ T cells was observed in 5 / 9 models and IFNy upregulation observed in 6 / 9 models. As shown in FIGS. 18C-D, the TCRms of the disclosure induce T cell activation in a dose-dependent manner. CD25 upregulation was observed in all 9 models and CD69 upregulation observed in 6 / 9 models. As shown in FIGS. 18E-F, TA-1 provided dose-dependentcytotoxicity in AML Blasts and leukemic stem cells (LSCs). As shown, 6 / 9 models exhibit TA-1 dose-dependent cytotoxicity in AML monoblast populations. LSC % cytotoxicity could only be observed in 5 models which had sufficient LSC absolute counts / events for evaluation.

[0389] As these results have demonstrated, the TCRms of the disclosure, including TA-1, provide a clear, target-specific therapeutic effect. As the following results demonstrate, the TCRms of the disclosure, including TA-1, demonstrate a unique and effective safety profile.

[0390] One concern when using CG-specific therapies is the risk that they will induce a cytotoxic response against neutrophils. Cathepsin G forms early in normal granulocyte development and is a serine protease stored in azurophilic granules in neutrophils. As shown in FIG. 19, TA-1 displays no activity, and thus presumably no or little toxicity, towards neutrophils. As shown in FIG. 20, this is because, while neutrophils contain CG protein, they do not present CGI. CG is a bactericidal serine protease stored in neutrophil azurophilic granules. HLA-A*02:01-postive neutrophils have HLA-A*02:01-levels lower than those of AML cell lines, but are able to present peptide. In spite of this, the CGI peptide is not detected on the surface of neutrophils. Thus, as shown, the TCRms of the disclosure are specific for CGI and / or the pHLA complex, not the peptide on its own. Further, as shown in FIG. 21, the TCRms of the disclosure do not activate T cells in the presence of neutrophils. As shown, when HLA-A*02:01 neutrophils are co-cultured with PBMC no T-cell activation is detected (assessed via NFAT reporter assay) or IFNy production by T cells is observed. Only when pulsing the HLA-A*02:01 neutrophils with CGI peptide, is there a small but dose-dependent T-cell activation, yet no IFNY production. Further, HLA-A* 11:01 neutrophils are unable to activate T cells, even when pulsed with CGI. As shown in FIG. 22, TA-1 does not induce IFNy release in activated polarized macrophages or target inflammatory sites.

[0391] To assess the specificity of the TCRm-based TCE of the disclosure, including any potential pHLA interaction beyond the designed binding site, a cytotoxicity assay was performed using TA-1, the “dummy” TCRm-based TCE, and a TCE designed with a CD123 binding site in the place of the pHLA-CGl TCRm binding site. The target cells expressed varying levels of the pHLA-CGl complex and CD123. As shown in FIGS. 23A-23B, TA-1 displays cytotoxicity towards AML cell lines on par with the CD123 TCRm TCE. As shown in FIG. 24, although CG protein is expressed at low levels in HSCs (much less than in LSCs), which is consistent with its generation in early myeloid development, TA-1 displays no toxicity towards normal hematopoietic stem cells. As also shown, the CD 123 TCE induces IFNy release in the presence of PBMC s alone and PBMCs +CD34+ HSCs. FIG. 25 shows data indicating that TA-1, unlike the CD 123 -targeted TCE does not deplete any specific cell population, including monocytes and other CD123+ cells from PBMCs. Furthermore, as shown in the bone marrow colony formation unit (CFU) assay (described in FIG.26A) FIG., TA-1 does not impact the formation of bone marrow colonies in this assay (FIG. 26B) and therefore it is anticipated that TA-1 will possess a wide therapeutic index and will not impair normal hematopoiesis. As shown in the TNFa, IFNy, IL-6, and IL- 13 secretion results in FIG. 27, TA-1 does not induce cytokine release in the absence of target-positive cells. As shown in FIG.28A, TA-1 and TA-2 did not show poly-reactivity, nor did TA-3, which is an analogue of TA-1 or TA-2, that has the same format but a different CGI binder. As shown in FIG. 28B, TA-1 analogue, TA-4, did not display poly-reactivity. FIGS. 29A-29B provide cytotoxicity results showing that TA-1 does not kill target-negative cancer cell lines in the absence of cells expressing the target pH LA complex.

[0392] As shown in FIG. 30, the Ala / Gly scan reveals that for TA-1, 3 positions in the CGI peptide are critical for interaction with TA-1.

[0393] To further assess the safety profde of TA-l / TA-2 and their pHLA-CGl binders, a computational, experimentally guided cross-reactivity assessment was undertaken. FIG. 31 outlines this assessment. FIG. 32A shows mean percent of normalized activation or cytotoxicity following coculture of Jurkat cells or human PBMCs with A375 shown. Each dot below the 10% dotted line represents putative cross-reactive peptide with data value below 10% (percentage refers to fraction of Jurkat activation observed or cytotoxicity observed when pulsing cells with CGI). Each dot between the 10% and 60% dotted lines represents putative cross-reactive peptide with data value above 10% but below 60%. The one dot above 60% represents putative cross-reactive peptide with data value above 60%. Control target peptide (CGI) is indicated As shown in the results provided in FIGS. 32A-32B, the only identified putative cross-reactive peptide was from a GDF5 protein. However, as shown in the cytotoxicity assay results in FIG. 32B, TA-1 does not target cells expressing this protein sequence. Thus, it does not represent a cross-reactive peptide that would impact the safety profile of TA-1 / TA-2. FIGS. 33A-33B provide results for TA-1 potential interactions with normal primary tissues and cross-HLA interactions. As shown in FIG.33A, TA-1 has no detectable interactions with normal primary tissues in the panel of 23 HLA-A*02+ normal primary cell types across vital organs and human PBMCs with TA-1. As shown in FIG. 33B, TA-1 has no detectable cross-HLA interactions in the panel of 10 B-LCL cells selectedcovering 37 unique HLA haplotypes (all HLA-A*02 negative). This panel includes all HLA-types which occur in >5% of the US population.

[0394] Finally, as shown in FIGS. 34A-34B, TA-1 met all baseline and stress developability criteria for a therapeutic targeting the pHLA-CGl peptide complex as a T cell engager.

[0395] To assess in vivo pharmacokinetics (PK) of TA-1, naive male cynomolgus monkeys were administered a single dose of TA-1 either intravenously (IV) or subcutaneously (SC) at 0.01 mg / kg, 0.1 mg / kg or 1 mg / kg. Serum samples were assessed between 0 and 672 hours post-dose, and half-life, area under the curve, and CO were calculated for both IV and SC routes and are shown in FIGS. 35A-35B. Both routes of administration confirm the IgG-like half-life and PK profile of TA-1. Serum samples for assessment of anti-drug antibodies (ADA) were collected from cynomolgus monkeys at 0, 336, 504, and 672 hours post-dose and are shown in FIG. 35C. Anti-TA-1 antibodies were exhibited in one animal at 336 hours until the end of study (672 hours) at the lowest detectable level of ADA, suggesting a minimal or "borderline" positive response to the drug, where the antibodies are present but at a very low concentration. Together, these suggest that there are no immunogenicity risks for TA-1.

[0396] The Retrogenix Cell Microarray Technology platform was used to screen for specific off-target binding interactions of 20 ug / ml TA-1 using a library of 6111 individual full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins, as well as a further 403 human heterodimers (FIG. 36). TA-1 showed a significant specific interaction with one of its primary targets, CD3. TA-1 showed an additional significant specific interaction with FCGR2A (isoforms 1 and 2). The interaction with FCGR2A was presumably Fc domain mediated and was observed under these assay conditions which utilized high concentrations of TA-1, far above therapeutically relevant concentrations. Notably, at lower concentrations in the Octet BLI study there was no binding observed for TA-1 to FcyRs suggesting no potential for Fc effector functions. These data indicate high specificity of TA-1 for one of its primary targets, CD3, and no significant off target interactions by its other CGl / HLA-A*02:01 pHLA binding arm.ADDITIONAL SEQUENCES OF THE PRESENT DISCLOSUREFor SEQ ID NOs.: 1-56 CDRs 1-3 are in bold, FRs 1-4 are underlined, and when shown, start of constant region are shown in italic characters.> CG1-R-17 VH amino acid sequence (SEQ ID NO:1) EVOLVESGGGLVOPGGSLRLSCGASGFTFSSYWMSWVRQAPGKGLEWVANIKODGSE KYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASALYYYYYYGMDVWGO GTTVTVS> CG1-R-18 VH amino acid sequence (SEQ ID NO:2) OVOLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIROPPGKGLEWIGYIYYSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSLYYYYYYGMDVWGOGTTV TVS> CGl-6 VH amino acid sequence (SEQ ID NO:3) OVHLQESGPGLVKPSETLSLTCTVSGGSVSSYYWNWIROPPGKGLEWIGYIYYSESTNY NTSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARSLYYYYYYGMDVWGOGTM VTVSS> CG1-1O VH amino acid sequence (SEQ ID NO:4) EVOLVESGGGLVQPGGSLRLSCAASGFTFNNYWMTWVROAPGKGLEWVANIKQDGN EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWYWFDPWGOG TL VTVSS> CG1-12 VH amino acid sequence (SEQ ID NO:5) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINODGSE KNYVDSVKGRFTISRDNANNSLHLQMNSLRAEDTAVYYCTKSLYYYYYYGMDVWGO GTTVTVSS> CG1-15 VH amino acid sequence (SEQ ID NO:6) OVHLQESGPGLVKPSETLSLTCTVSGGSVSSYYWNWIROPPGKGLEWIGYIYYSESTNY NTSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARSLYYYYYYGMDVWGOGTM VTVSS> CG1-16 and TA-3 VH amino acid sequence (SEQ ID NO:7) EVOLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVROAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGOG TL VTVSS> CGl-23 VH amino acid sequence (SEQ ID NO:8) EVOVVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVROAPGKGLEWVANIKHDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDRAVYYCVREGGWYWFDPWGOG TL VTVSS> CGl-27 VH amino acid sequence (SEQ ID NO:9) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMSWVROAPGKGLEWVANIKODGSE KYYVDSVKGRFT1SRDNAKNSOYLQMNSLRAEDTAVYYCAREGGWYWLDPWGQGT LVTVSS> CGl-33 VH amino acid sequence (SEQ ID NO:10) OVOLVESGGGVVOPGGSLRLSCAASGFIFSSYWMSWVRQAPGKGLEWVANINODGSE KYYVDSVKGRFTISRDNANNSLFLQMNSLRAEDTAVYYCTKSLYYYYYYGMDVWGO GTTVTVSS> CG1-41 VH amino acid sequence (SEQ ID NO:11) EVOLVECGGGLVQPGGSLRLSCAASGFTFSDYWMIWVRPAPGKGLEWVANIKODGSE KYYVDSVKGRFTISRDNAKNSLNMQMNSLRAEDTAVYYCAREGGWYWFDPWGOGT LVTVSS> CGl-44 VH amino acid sequence (SEQ ID NO: 12) OVHLQESGPGLVKPSETLSLTCTVSGGSVSNYYWNWIROPPGKGLEWIGYIYYSESTNY NTSLKSRVTISLDTSKNEFSLKVTSVTAADTAVYYCARSLYYYYYYGMDVWGOGTTV TVSS> CGl-46 VH amino acid sequence (SEQ ID NO: 13) OVHLQESGPGLVKPSETLSLTCTVSGGSVSNYYWNWIROPPGKGLEWIGYIYYSESTNY NTSLKSRVTISLDTSKNEFSLKVTSVTAADTAVYYCARSLYYYYYYGMDVWGOGTTV TVSS> CGl-55 VH amino acid sequence (SEQ ID NO:14) EVOVVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVROAPGKGLEWVANIKHDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDRAVYYCVREGGWYWFDPWGOG TLVTVSS> CG1-6O VH amino acid sequence (SEQ ID NO:15) EVOLVESGGGLVOPGGSLRLSCAAAGFTFSNYWMSWVRQAPGKGLEWVANIKODGS EKYYVDSVKGRFTISRDNAKNSOYLQMNSLRVEDTAVYYCAREGGWYWLDPWGOG TLVTVSS> CG1-61 VH amino acid sequence (SEQ ID NO:16) OVOLVESGGGVVOPGRSLRLSCAASGFTFSNYWMSWVROAPGKGLEWVANIKODGS EKYYVDSVKGRFTISRDNAKNSOYLOMNSLRVEDTAVYYCAREGGWYWLDPWGOG TLVTVSS> CGl-65 VH amino acid sequence (SEQ ID NO:17) OVHLQESGPGLVKPSETLSLTCTVSGGSVSSYYWNWIROPPGKGLEWIGYIYYSESTNY NTSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARSLYYYYYYGMDVWGOGTTV TVSS> CGl-68 VH amino acid sequence (SEQ ID NO:18) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMSWVROAPGKGLEWVANIKODGSE KYYVDSVKGRFTISRDNAKNSQYLQMNSLRAEDTAVYYCAREGGWYWLDPWGQGT LVTVSS> CGl-74 VH amino acid sequence (SEQ ID NO:19) EVOLVESGGGLVOPGGSLRLSCAAAGFTFSNYWMSWVRQAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSOYLQMNSLRVEDTAVYYCAREGGWYWLDPWGOG TLVTVSS> CGl-78 VH amino acid sequence (SEQ ID NO:20) EVOLVESGGGLVQPGGSLRLSCAASGFTFNNYWMTWVROAPGKGLEWVANIKODGN EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWYWFDPWGOG TLVTVSS> CG1-1O4 VH amino acid sequence (SEQ ID NO:21) OVOLVESGGGVVHPGRSLRLSCAASGFTFSHYVMHWVRQAPGKGLEWVAVIWYDGS NINYAESLKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARGPAPTIFWGOGTLVTV SS> CGl-110 VH amino acid sequence (SEQ ID NO:22) EVOLLESGGGLVQPGGSLRLSCAASGFTFSSNAMSWVROAPGKGLEWVSAISGRGGN TYYADSVKGRFTISRDNSKNTLYLOMLSLRAEDTAVYYCARDSYYDVLTAYYDFLTT GAREPWSPSR> CG1-112 VH amino acid sequence (SEQ ID NO:23) EVOLVESGGGLVOPGGSLRLSCAASGFIFSSYWMSWVRQAPGKGLEWVANINQDGSE KYYVDSVKGRFTISRDNANDSLFLQMNSLRAEDTAVYYCTKSLYYYYYYGMDVWGO GTPVTVSS> CG1-176 VH amino acid sequence (SEQ ID NO:24) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKODGSE KYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCAREGGWYWFDPWGOGTL VTVSS> CG1-177 VH amino acid sequence (SEQ ID NO:25) EVOLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVROAPGKGLEWVANIKODGS EKYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGOG TMVTVSS> CG1-179 VH amino acid sequence (SEQ ID NO:26) EVOVVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVROAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCAREGGWYWFDPWGQG TLVTVSS> CG1-189 VH amino acid sequence (SEQ ID NO:27) EVOVVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVROAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSQYLQMNSLRAEDTAVYYCAREGGWYWFDPWGQG TLVTVSS> CG1-R-17 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:28) DIVMTQSPLSLPVTPGEPASISCRSSOSLLHSNGYNYLDWYLOKPGOSPQLLIYLGSIRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTRLEIKRZ> CG1-R-18 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:29) DIVMTOSPLSLPVTPGEPASISCRSSOSLLYSHGYNYLDWYLQKPGOSPOLLIYLGSIRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM0AL0TPWTFG0GTKVEIKA7 > CGl-6 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:30) DIVMTOSPLSLPVTPGEPASISCMSSOSLLOSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALOTPWTFGOGTKVDIKAY> CGl-10 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:31) DIOMTOSPSSLSASVGDRVTITCRASQSISSFLNWYOQKPGKAPKLLIYTASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGOGTKVEIKRT> CG1-12 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:32) DIVMTOSPLSLPVTPGEPASISCRSSQSLLLSHGYNYLDWYLQKPGOSPOLLIYLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOGLOTPWTFGQGTKVDIKRZ> CG1-15 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:33) DIVMTOSPLSLPVTPGEPASISCMSSQSLLQSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVDIKAZ> CG1-16 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:34) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLLYAASSFLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDIKRT> CGl-23 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:35) DIOMTOSPSSLSASVGDRVTITCRASOIISSFLNWYHQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIK E> CGl-27 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:36) DIOMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOOKPGKAPKLLIYTASSLQGGVPS RFSGSGSGTDFTLT1SSLOPEDFATYYCOOSYSTPYTFGOGTRLE1KAZ> CGl-33 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:37) DIVMTQSPLSLPVTPGEPASISCRSSOSLLLSHGYNYLDWYLOKPGOSPQLLIYLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPWTFGQGTKVDIKAZ> CG1-41 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:38) DIQMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLLYAASSFLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVEIKAZ> CGl-44 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:39) DIVMTOSPLSLPVTPGEPASISCRSSOSLLOSHGFNHLDWYLQKPGOSPOLLIYLVSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALOTPWTFGQGTKVDIKAZ> CGl-46 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:40) DIVMTOSPLSLPVTPGEPASISCRSSQSLLOSHGFNHLDWYLQKPGOSPOLLIYLVSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVEIKAT> CGl-55 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:41) DIOMTOSPSSLSASVGDRVTITCRASQIISSFLNWYHQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGOGTKVDIKZT> CG1-6O VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:42) DLOMTOSPSSLSASVGDRVTITCRASQSIASFLNWYOQKPGKAPKLLIYTASSFQGGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCOQSYSTPYTFGOGTKVDIKZZ> CG1-61 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:43) DLOMTOSPSSLSASVGDRVTITCRASQSIASFLNWYOQKPGKAPKLLIYTASSFQGGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDIKZZ> CGl-65 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:44) DIVMTOSPLSLPVTPGEPASISCRSSQSLLOSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKTSRVEAEDVGVYYCMOALOTPWTFGOGTKVDIKZZ> CGl-68 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:45) DIOMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYTASSLOGGVPS RFSGSGSGTDFTLT1SSLQPEDFATYYCOOSYSTPYTFGOGTKLE1KA7 4AP> CGl-74 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO: 46) EIQMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYTASSFOGGVPS RFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPYTFGOGTKVEIKA7 4AP> CGl-78 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO: 47) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYTASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDIK r> CG1-1O4 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO: 48) DIOMTOSPSTPSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIYKASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCOOYNSYSYTFGOGTKLESNVA> CGl-110 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:49) DVLMTOSPSSLSASLGERVSLTCOASODISNNLNWYOQTPGKAPRLLIYDTSKLEDGVP SRFSGTGYRTDFIFTISSLEEEDVATYFCLQHRYLPWTFGGGTKLEIKAT> CG1-112 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:50) DIVMTOSPLSLPVTPGEPASISCRSSOSLLLSHGYNYLDWYLQKPGOSPOLLIYLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOGLOTPWTFGOGTKVEIKA7 > CG1-176 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:51) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYAASSFOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQOSYSTPYTFGOGTKVDIKRT> CG1-177 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:52) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLLYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGOGTRLEIKRT> CG1-179 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:53) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYAASSFQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIKfE> CG1-189 VL amino acid sequence + start of constant region (constant region amino acids are in italic letters) (SEQ ID NO:54) EIOMTOSPSSLSASVGDRVTITCRASQSIASFLNWYOQKPGKAPKLLIYAASSLOSGVPS RFSGSGSGTDFTLT1SSLOPEDFATYYCOOSYSTPYTFGOGTRLE1KAZAmino Acid Sequences for VL without constant region amino acids: for SEQ ID NOs.: 145-171 CDRs 1-3 are in bold, FRs 1-4 are underlined.> CG1-R-17 VL amino acid sequence (SEQ ID NO: 145) D1VMTOSPLSLPVTPGEPAS1SCRSSOSLLHSNGYNYLDWYLQKPGOSPQLL1YLGSIRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPYTFGQGTRLEIK> CG1-R-18 VL amino acid sequence (SEQ ID NO: 146) DIVMTOSPLSLPVTPGEPASISCRSSOSLLYSHGYNYLDWYLOKPGOSPOLLIYLGSIRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVEIK> CGl-6 VL amino acid sequence (SEQ ID NO:147) DIVMTOSPLSLPVTPGEPASISCMSSOSLLOSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVDIK> CG1-1O VL amino acid sequence (SEQ ID NO: 148) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOOKPGKAPKLLIYTASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVEIK> CG1-12 VL amino acid sequence (SEQ ID NO: 149) DIVMTOSPLSLPVTPGEPASISCRSSOSLLLSHGYNYLDWYLOKPGOSPOLLIYLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOGLOTPWTFGQGTKVDIK> CG1-15 VL amino acid sequence (SEQ ID NO: 150) DIVMTOSPLSLPVTPGEPASISCMSSOSLLOSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVDIK> CG1-16 VL amino acid sequence (SEQ ID NO: 151) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOOKPGKAPKLLLYAASSFLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVDIK> CGl-23 VL amino acid sequence (SEQ ID NO: 152) DIOMTOSPSSLSASVGDRVTITCRASOIISSFLNWYHOKPGKAPKLLIYAASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTRLEIK> CGl-27 VL amino acid sequence (SEQ ID NO: 153) DIOMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYTASSLOGGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIK> CGl-33 VL amino acid sequence (SEQ ID NO: 154) DIVMTOSPLSLPVTPGEPASISCRSSOSLLLSHGYNYLDWYLOKPGOSPOLLIYLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOGLQTPWTFGQGTKVDIK> CG1-41 VL amino acid sequence (SEQ ID NO:155) DIQMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLLYAASSFLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVEIK> CGl-44 VL amino acid sequence (SEQ ID NO: 156) DIVMTOSPLSLPVTPGEPASISCRSSOSLLOSHGFNHLDWYLQKPGOSPOLLIYLVSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVDIK> CGl-46 VL amino acid sequence (SEQ ID NO: 157) DIVMTOSPLSLPVTPGEPASISCRSSOSLLOSHGFNHLDWYLQKPGOSPOLLIYLVSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVEIK> CGl-55 VL amino acid sequence (SEQ ID NO: 158) DIOMTOSPSSLSASVGDRVTITCRASOIISSFLNWYHQKPGKAPKLLIYAASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDIK> CG1-6O VL amino acid sequence (SEQ ID NO: 159) DLQMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOOKPGKAPKLLIYTASSFQGGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVDIK> CG1-61 VL amino acid sequence (SEQ ID NO: 160) DLOMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYTASSFOGGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVDIK> CGl-65 VL amino acid sequence (SEQ ID NO: 161) DIVMTOSPLSLPVTPGEPASISCRSSOSLLOSHGYNHLDWYLQKPGOSPOLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALOTPWTFGQGTKVDIK> CGl-68 VL amino acid sequence (SEQ ID NO: 162) DIQMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYTASSLOGGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKLEIK> CGl-74 VL amino acid sequence (SEQ ID NO: 163)EIQMTQ SP S SL S AS VGDRVTITCRASOSIASFLNW YQQKPGK APKLLIYTASSFQGGVP S RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGQGTKVEIK> CGl-78 VL amino acid sequence (SEQ ID NO: 164) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYTASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDTK> CG1-1O4 VL amino acid sequence (SEQ ID NO: 165) DIOMTOSPSTPSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIYKASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCOQYNSYSYTFGQGTKLESNVIll> CG1-11O VL amino acid sequence (SEQ ID NO:166) DVLMTOSPSSLSASLGERVSLTCOASODISNNLNWYOQTPGKAPRLLIYDTSKLEDGVP SRFSGTGYRTDFIFTISSLEEEDVATYFCLQHRYLPWTFGGGTKLEIK> CG1-112 VL amino acid sequence (SEQ ID NO: 167) D1VMTOSPLSLPVTPGEPAS1SCRSSOSLLLSHGYNYLDWYLOKPGOSPOLL1YLGSSRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOGLOTPWTFGQGTKVEIK> CG1-176 VL amino acid sequence (SEQ ID NO: 168) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYAASSFOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTKVDIK> CG1-177 VL amino acid sequence (SEQ ID NO: 169) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLLYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIK> CG1-179 VL amino acid sequence (SEQ ID NO: 170) DIOMTOSPSSLSASVGDRVTITCRASOSISSFLNWYOQKPGKAPKLLIYAASSFOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIK> CG1-189 VL amino acid sequence (SEQ ID NO: 171) EIOMTOSPSSLSASVGDRVTITCRASOSIASFLNWYOQKPGKAPKLLIYAASSLOSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCOOSYSTPYTFGOGTRLEIK CGI-16 x CD3 bispecific Ab which contains three chains:> CG1-16 bispecific He (SEQ ID NO:55) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSDTKVDKKVEPKSCDKTHTCPPCPAP PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKP REEEYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKL TVDK SRWEQGD VF SC S VMHE ALHNHYTQKSL SLSPG> CG1-16 Lc (SEQ ID NO:56) DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLLYAASSFLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHKVYACEVTHQGL S SP VTKSFNRGEC> CD3 bispecific (SEQ ID NO:57) EVQLVESGGGLVQPGGSLRLSCAASGFITSTYAMNWVRQAPGKGLEWVGRIRSKYNN YATYYADSMCGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAY WGQGTLVTVSSGKPGSGKPGSGKPGSGKPGSQAVVTQEPSLTVSPGGTVTLTCGSSTGA VTTSNYANWVQQKPGKSPRGLIGGTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEAD YYCALWYSNHWVFGGGTKLTVLEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLM1 SRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGA> FLAG (SEQ ID NO:58)DYKDDDDK>poly-histidine (6-His; (SEQ ID NO:59))HHHHHH>hemagglutinin (HA; (SEQ ID NO:60))YPYDVPDYA> CG1 (SEQ ID NO:61)FLLPTGAEA> CMA1 (SEQ ID NO:62)FLLCSRAEA> PI3 (SEQ ID NO: 63)FLLPTGVYL> MIF (SEQ ID NO:64)FLLPTGLSSL> CMV (SEQ ID NO: 65)NLVPMVATV> PAP135 (SEQ ID NO:66)ILLWQPIPV> Signal Peptide (SEQ ID NO: 124)MQPLLLLAFLLPTGAEA> C-peptide (SEQ ID NO: 125)FKLLDQMETPL> Fab chain 1 of Exemplary TCRm (SEQ ID NO: 126);CGI TA-1 VH-CH1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQSGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS S VVT VPS S SLGTQT YICNVNHKP SNTK VDKKV> Fab chain 1 of Exemplary TCRm (SEQ ID NO: 127);> CGI TA-3 VH-CH1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS SWT VPS S SLGTQT YICNVNHKP SNTK VDKKV> Full TA-1 / TA-2 / TA-3 sequence HC1 (SEQ ID NO: 139);> HC1 AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGG GGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGL EWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYG DSDWYFDVWGQGTLVTVSSGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL VKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVF SC SV MHEALHNHYTQKSLSLSPG> Full TA-1 / TA-2 / TA-3 sequence LC1 (SEQ ID NO: 140);> LC DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLLYAASSFLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KAD YEKHKVYACE VTHQGL S SP VTKSFNRGEC> Full TA-1 sequence HC2 (SEQ ID NO: 141);> HC2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQSGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVC TLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG> Full TA-2 sequence HC2 (SEQ ID NO: 142);> HC2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDASE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVC TLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG> Full TA-3 sequence HC2 (SEQ ID NO: 143);> HC2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVC TLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG> TA-l / TA-2 / TA-3 CD3 binder sequence (SEQ ID NO: 128);AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGG GGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGL EWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYG DSDWYFDVWGQGTLVTVSS> CGI TA-3 heavy chain CDR2 region fragment (SEQ ID NO: 129, IMGT CDR annotation) IKQDGSEK> CGI TA-1 heavy chain CDR2 region fragment (SEQ ID NO: 130, IMGT CDR annotation) IKQSGSEK> CG1 TA-2 heavy chain CDR2 region fragment (SEQ ID NO: 131, IMGT CDR annotation) IKQDASEK> CG1 TA-1, TA-2 and TA-3 light chain variable region (SEQ ID NO: 132) DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLLYAASSFLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIK> CGI TA-1 heavy chain variable region (SEQ ID NO: 133) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQSGSE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSS> CGI TA-2 heavy chain variable region (SEQ ID NO: 134) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDASE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSS> Fab chain 2 of Exemplary TCRm (SEQ ID NO: 135);> CGI TA-1, TA-2 and TA-3 LC DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLLYAASSFLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS K AD YEKHKVYACEVTHQGL S SP VTK SFNRGEC> Fab chain 1 of Exemplary TCRm (SEQ ID NO: 136);> CGI TA-2 VH-CH1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMTWVRQAPGKGLEWVANIKQDASE KYYVDSVKGRFTISRDNAKNSLNLQMNSLRAEDTAVYYCAREGGWYWFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS S VVT VPS S SLGTQT YICNVNHKP SNTK VDKKV> Silent Fc hole chain (SEQ ID NO: 137) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ VCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSilent Fc (Eu numbering) - L234A& L235A (LALA mutation) to reduced effector function of the IgGl antibody.K322A to reduces or eliminates the ability of the antibody binding to Clq and activate the complement systemHole chain for heterodimerization (Eu numbering) - Y349C, T366S, L368A, Y407V> Silent Fc knob chain (SEQ ID NO: 138) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVF SC S VMHEALHNHYTQK SL SLSPGK322A to reduces or eliminates the ability of the antibody binding to Clq and activate the complement system.Silent Fc (Eu numbering) - L234A& L235A (LALA mutation) to reduced effector function of the IgGl antibody.Knob chain for heterodimerization (Eu numbering) - S354C, T366W

[0323] Disclosed are materials, compositions, and ingredients that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0324] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further nonlimiting examples of the disclosed compositions and methods.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A T-cell receptor (TCR) mimetic antibody or fragment thereof, wherein said TCR mimetic (TCRm) antibody comprises:a first binding domain that binds to an HLA-CG1 peptide complex on an targetpresenting cell and a second binding domain that binds to an immune cell receptor, wherein the first antigen binding domain comprises:a heavy chain (HC) variable region sequence, wherein the HC variable region comprises a CDR2 sequence comprising SEQ ID NO: 131 or SEQ ID NO: 130.

2. The TCRm or fragment thereof of claim 1, wherein the HC CDR2 sequence of the first antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 131.

3. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 134.

4. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence having at least 95% similarity to SEQ ID NO: 134.

5. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 134.

6. The TCRm or fragment thereof of claim 1, wherein the HC CDR2 sequence of the first antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 130.

7. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 133.

8. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence having at least 95% similarity to SEQ ID NO: 133.

9. The TCRm or fragment thereof of claim 1 or claim 2, wherein the HC variable region of the first antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 133.

10. The TCRm or fragment thereof of any one of claims 1-9, 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.

11. The TCRm or fragment thereof of any one of claims 1-10, 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 95% similarity to SEQ ID NO: 132.

12. The TCRm or fragment thereof of any one of claims 1-11, wherein the 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.

13. A T-cell receptor (TCR) mimetic antibody or fragment thereof, wherein said TCR mimetic (TCRm) antibody, or fragment thereof, comprises:a first binding domain that binds to an HLA-CG1 peptide complex on a target presenting cell and a second binding domain that binds to an immune cell receptor,wherein the first antigen binding domain comprises:a heavy chain (HC) variable region sequence, wherein the HC variable region comprises a CDR1 sequence comprising one of SEQ ID NOs: 67-78; anda light chain (LC) variable region sequence, wherein the LC variable region comprises a CDR1 sequence comprising one of SEQ ID NOs: 96-105 and 172.

14. The TCRm antibody or fragment thereof of claim 13, wherein the first antigen binding domain HC variable region further comprises a CDR2 sequence comprising one of SEQ ID NOs: 79-87, or 129-131.

15. The TCRm antibody or fragment thereof of claim 13 or claim 14, wherein the first antigen binding domain LC variable region further comprises a CDR2 sequence comprising one of SEQ ID NOs: 106-117.

16. The TCRm antibody or fragment thereof of any one of claims 13-15, wherein the first antigen binding domain HC variable region further comprises a CDR3 sequence comprising one of SEQ ID NOs: 88-95.

17. The TCRm antibody or fragment thereof of any one of claims 13-16, wherein the first antigen binding domain LC variable region further comprises a CDR3 sequence comprising one of SEQ ID NOs: 118-123.

18. The TCRm antibody or fragment thereof of any one of claims 1-17, wherein the immune cell receptor is CD3.

19. The TCRm antibody or fragment thereof of any one of claims 1-18, wherein the second antigen binding domain comprises an amino acid sequence having at least 90% to SEQ ID NO: 57 or SEQ ID NO. 128 or SEQ NO. 139.

20. The TCRm antibody or fragment thereof of any one of claims 1-18, wherein the second antigen binding domain comprises an amino acid sequence having at least 95% to SEQ ID NO: 57 or SEQ ID NO. 128 or SEQ NO. 139.

21. The TCRm antibody or fragment thereof of any one of claims 1-18, wherein the second antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 57 or SEQ ID NO. 128 or SEQ NO. 139.

22. The TCRm antibody or fragment thereof of any one of claims 1-21, wherein the first antigen binding domain has an 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.

23. The TCRm antibody or fragment thereof of any one of claims 1-22, wherein the TCRm has an 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.

24. The TCRm antibody or fragment thereof of any one of claims 1-23, wherein the TCR mimetic antibody does not have a substantial binding affinity to the HLA class I molecule absent CGI.

25. The TCRm antibody or fragment thereof of any one of claims 1-24, wherein the TCR mimetic antibody has at least 2-fold lower binding affinity to the HLA class I molecule absent CGI.

26. The TCRm antibody or fragment thereof of any one of claims 1-25, wherein the TCR mimetic antibody has a reduced binding affinity for CGI in the absence of a bound HLA class I molecule as compared to the HLA class I molecule-CGl peptide complex.

27. The TCRm antibody or fragment thereof of any one of claims 1-26, wherein the comprises 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.

28. The TCRm antibody or fragment thereof of any one of claims 1-27, wherein said antibody is a chimeric antibody, bispecific antibody, trispecific or other multi-specific antibody, or BiTE.

29. The TCRmc antibody or fragment thereof of any one of claims 1-28, wherein said antibody is an IgG antibody or a recombinant IgG antibody or antibody fragment.

30. A pharmaceutical preparation comprising:(a) a pharmaceutically acceptable carrier; and(b) a TCRm antibody of any one of claims 1-29.

31. A diagnostic preparation comprising:(a) a pharmaceutically acceptable carrier; and(b) a TCRm antibody of any one of claims 1-29.

32. 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 30.

33. The method of claim 32, wherein the cancer is a myeloid malignancy.

34. The method of claim 32, wherein the cancer is a hematologic malignancy is AML, ALL, CLL, CML. CMML, orMDS.

35. A method for treating a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a TCR antibody or fragment thereof of any one of claims 1-29.

36. The method of claim 35, wherein the cancer is a myeloid malignancy.

37. The method of claim 36, wherein the cancer is a hematologic malignancy is AML, ALL, CLL, CML, CMML, orMDS.

38. 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 31, and(b) detecting binding of the TCRm antibody or fragment thereof as a determination of the presence of a cancerous cell.

39. The method of claim 38, wherein the cancer is a myeloid malignancy.

40. The method of claim 38, wherein the cancer is a hematologic malignancy is AML, ALL, CLL, CML, CMML, orMDS.

41. 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 any one of claims 1-29.

42. The method of claim 41, further comprising administering to the subject a checkpoint inhibitor prior to, subsequent to, or simultaneously with administration of the pharmaceutical composition.

43. The method of claim 41, wherein the pharmaceutical composition induces bystander killing of cells that do not express the HLA-CG1 peptide antigen complex.

44. The TCRm antibody or fragment thereof of claim 1, wherein the TCRm comprises a first, second, and third peptide chain:wherein the first peptide chain comprises an amino acid sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 139.

45. The TCRm antibody or fragment of claim 44, wherein the TCRm antibody comprises a first, second, and third peptide chain:wherein the first peptide chain comprises an amino acid sequence having at least 95% similarity to the amino acid sequence set forth in SEQ ID NO: 139.

46. The TCRm antibody or fragment of claim 44, wherein the TCRm antibody or fragment thereof comprises a first, second, and third peptide chain:wherein the first peptide chain comprises an amino acid sequence having 100% similarity to the amino acid sequence set forth in SEQ ID NO: 139.

47. The TCRm antibody or fragment of any one of claims 44-46, wherein the second peptide chain comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 140.

48. The TCRm of any one of claims 44-46, wherein the second peptide chain comprises an amino acid sequence having at least 95% similarity to SEQ ID NO: 140.

49. The TCRm of any one of claims 44-46, wherein the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 140.

50. The TCRm antibody or fragment thereof of any one of claims 44-49, wherein the third peptide chain comprises an amino acid sequence having at least 90% similarity to SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143.

51. The TCRm antibody or fragment thereof of any one of claims 44-50, wherein the third peptide chain comprises an amino acid sequence having at least 95% similarity to SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143.

52. The TCRm antibody or fragment thereof of any one of claims 44-50, wherein the third peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143.

53. The TCRm antibody or fragment thereof of any one of claims 1-52, wherein the TCRm comprises a first, second, and third peptide chain:wherein the first peptide chain comprises an amino acid as set forth in SEQ ID NO: 139.wherein the second peptide chain comprises an amino acid as set forth in SEQ ID NO: 140, wherein the third peptide chain comprises an amino acid as set forth in SEQ ID NO: 141.

54. The TCRm antibody or fragment thereof of any one of claims 1-52, wherein the TCRm comprises a first, second, and third peptide chain: wherein the first peptide chain comprises an amino acid as set forth in SEQ ID NO: 139.wherein the second peptide chain comprises an amino acid as set forth in SEQ ID NO:

140. wherein the third peptide chain comprises an amino acid as set forth in SEQ ID NO: 142.

55. The TCRm antibody or fragment thereof of any one of claims 1-52, wherein the TCRm comprises a first, second, and third peptide chain:wherein the first peptide chain comprises an amino acid as set forth in SEQ ID NO:

139. wherein the second peptide chain comprises an amino acid as set forth in SEQ ID NO:

140. wherein the third peptide chain comprises an amino acid as set forth in SEQ ID NO: 143.

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