Clec2a-binding therapeutics

Abstract

Targeted therapeutics for the treatment of cancers that express CLEC2A are described. The targeted therapeutics include anti-CLEC2A binding domains and engineered formats thereof such as multi-domain binding molecules, antibody conjugates, and recombinant receptors expressed by an immune cell. The targeted therapeutics can be used to treat acute myeloid leukemia (AML), such as KMT2A-r AML cell. Furthermore, the binding domains can be used to isolate cells expressing CLEC2A or to target such cells ex vivo or in vivo for research, diagnostic, or therapeutic purposes.

Description

CLEC2A-BINDING THERAPEUTICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 595,274 filed November 1 , 2023 and U.S. Provisional Patent Application No. 63 / 549,936 filed February 5, 2024, both of which are incorporated herein by reference in their entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3BT8243.XML. The file is 242,406 bytes, was created on October 30, 2024, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] Particular embodiments provide targeted treatments for cancer cells expressing CLEC2A. The targeted therapeutic can include an anti-C-type lectin domain family 2 member A (CLEC2A) binding domain engineered into multi-domain binding molecule, an antibody conjugate, or a recombinant receptor expressed by an immune cell. The binding domains and engineered forms thereof can be used to isolate cells expressing CLEC2A or to target such cells ex vivo or in vivo for research, diagnostic, or therapeutic purposes. In particular embodiments, a CLEC2A- expressing cancer includes KMT2A-rearranged acute myeloid leukemia. The present disclosure also provides anti-CLEC2A binding domains.BACKGROUND OF THE DISCLOSURE

[0004] Acute myeloid leukemia (AML) is a diverse group of diseases classified based on morphology, lineage, and genetics (Rubnitz, Blood 119:5980-8, 2012) and its prognosis depends on several cytogenetic and molecular characteristics. Despite improved survival and remission induction rates, outcomes vary significantly amongst the different biological subtypes of AML (Kim, Blood Res. 55(Suppl): S5-S13, 2020). To better stratify risk and survival outcomes, genomic investigations of AML has led to new genomic classifications and predictive biomarkers (Arber, Semin Hematol. 56: 90-5, 2019; and Arber et a / ., Blood 127: 2391-405, 2016).

[0005] KMT2A-rearranged AML is a high-risk subtype of AML with very poor outcomes and is a highly refractory and fatal disease regardless of age. Current conventional chemotherapy has failed to adequately treat KMT2A-rearranged AML, highlighting the need for novel innovativetreatments to improve their dismal outcomes. There is a lack of appropriate targeted therapies to effectively treat KMT2A-rearranged AML.SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides a method of treating KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML), among other benefits described herein. The method of treating KMT2A- r AML includes targeting C-type lectin domain family 2 member A (CLEC2A) with a CLEC2A binding domain associated therapeutic. The present disclosure also provides anti-CLEC2A binding domains and uses thereof. The binding domains can be used to isolate cells expressing CLEC2A or to target such cells ex vivo or in vivo for research, diagnostic, or therapeutic purposes.

[0007] In particular embodiments, the disclosed anti-CLEC2A binding domains include a binding domain from a 3H10 antibody or a binding domain from a 4H10 antibody. In particular embodiments, the 3H10 binding domain includes a variable heavy domain encoded by the sequence as set forth in SEQ ID NO: 2. In particular embodiments, the 4H10 binding domain includes a variable heavy domain encoded by the sequence as set forth in SEQ ID NO: 4.

[0008] The binding domains disclosed herein can also be engineered into numerous additional formats, such as antibodies and fragments thereof, multi-domain binding molecule, an antibody conjugate, or a recombinant receptor, such as a chimeric antigen receptor (CAR), expressed by an immune cell (e.g., CAR-T cell).

[0009] The current disclosure provides targeted therapies against cancer cells expressing CLEC2A. Treated cancers can include a variety of solid tumor cancers and blood cancers. In particular embodiments, KMT2A-rearranged AML can be treated with targeted therapies against cells expressing CLEC2A.

[0010] In particular embodiments, a targeted therapeutic disclosed herein includes a recombinant receptor (e.g., CAR) expressed by an immune cell, such as a T cell. In certain examples, the recombinant receptor includes a binding domain that binds CLEC2A, a spacer, a CD28 transmembrane domain, and an intracellular component including a 4-1 BB signaling domain and a CD3 signaling domain.

[0011] The present disclosure also provides CLEC2A knock-out and overexpression models for in vivo and in vitro testing as well as a patient derived xenograft animal model.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0012] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves theright to present color images of the drawings in later proceedings.

[0013] FIGs. 1A-1 E. C-type lectin domain family 2 member A (CLEC2A) expression in acute myeloid leukemia (AML). (1A) Waterfall plot of CLEC2A mRNA transcript expression in all pediatric AML with CLEC2A expression in primary fusion subtypes shown in-lay. (1B) Waterfall plot of CLEC2A mRNA transcript expression in specific KMT2A fusions with corresponding pie charts. (1C) CLEC2A mRNA transcript expression in normal tissues. (1 D) Pie chart of primary cytogenetics in CLEC2A-positive AML patients. (1 E) Risk stratification of CLEC2A-positive patients.

[0014] FIGs. 2A-2C. KMT2A fusion oncoprotein binds directly to the CLEC2A promoter. (2A) Antibody graphic demonstrating identification of wild type (WT) KMT2A vs. KMT2A oncoprotein for Cut & Run assay. (2B) Representative binding of KMT2A N-terminal and C-terminal antibody at CLEC2A promoter site on primary patient samples (2C) Correlation of CLEC2A mRNA transcript expression relative to KMT2A oncoprotein binding in representative patient samples.

[0015] FIGs. 3A-3D. CLEC2A is detected on the cell surface in AML. (3A) Flow cytometry histogram plots of cell surface CLEC2A expression on AML cell lines (NOMO-1 , OCI-AML2, and MOLM13) and primary AML patient samples (Patient 1 , Patient 2, and Patient 3). (3B) Binding of top CLEC2A human VH binders against CLEC2A-positive and negative (KO) engineered cell lines. (3C) Schematic of CLEC2A chimeric antigen receptor (CAR) T cell constructs. (3D) Target specific cell killing following co-incubation with CD8+ CLEC2A CAR T vs. unmodified T cells with CLEC2A+ OCI-AML2 or CLEC2A- MOLM13 cells at various effector: target (E:T) cell ratios, done in triplicate with standard error of the mean (SEM) across replicates shown.

[0016] FIGs. 4A-4C. CLEC2A 3H10 intermediate spacer (Int) CAR T cells demonstrate robust and specific in vitro cell killing and cytokine production. (4A) Schematic of CLEC2A 3H 10 Int CAR T construct. (4B) Flow cytometry histogram plots of cell surface CLEC2A expression on AML cell lines with corresponding in vitro cytotoxicity results showing target specific cell killing following coincubation with CD8+ CLEC2A Int CAR T vs. unmodified T cells with CLEC2A+(high) OCI-AML2, CLEC2A+(low) NOMO-1 or CLEC2A- MOLM13 cells at various effectortarget (E:T) cell ratios, done in triplicate with SEM across replicates shown. (4C) Proinflammatory cytokine production (IFNy, IL-2 and TN Fa) from supernatant collected after 24 hours of co-incubation with CLEC2A Int CAR T cells vs. unmodified T cells against AML cell lines, done in triplicate with SEM across replicates shown.

[0017] FIGs. 5A-5F. CLEC2A CAR T cells have in vivo anti-leukemic efficacy against AML cell- derived xenograft model. (5A) Schematic experimental design for in vivo cell-derived xenograft model. (5B) Bioluminescent imaging following treatment with CLEC2A CAR T vs. unmodified Tcells. (5C) Leukemia burden via radiance following treatment with CLEC2A CAR T (triangleleukemia 7 days prior and square-4 days prior) vs. unmodified T (square- leukemia 7 days prior, circle-4 days prior). (5D) Leukemia detected in peripheral blood between CLEC2A CAR T and unmodified T cells. (5E) Overall survival. (5F) Disease-free Survival.

[0018] FIGs. 6A-6C. CLEC2A CAR T cells have expansion and persistence in vivo with ongoing anti-leukemia efficacy. (6A) Percentage of AML and human T cells present in all mice at indicated time points following T cell infusion. (6B) Breakdown of T cell subsets in all mice at indicated time points. (6C) Presence of AML cells and human T cells in the bone marrow at time of harvest for unmodified T cell treated mice and CLEC2A CAR T cell treated mice.

[0019] FIGs. 7A-7C. CLEC2A long (long spacer) CAR T cells eradicate leukemia, but do not improve survival. (7A) Schematic of in vivo experimental design. (7B) Percentage of AML and human T cells present in all mice at indicated time points following T cell infusion. (7C) Presence of AML cells and human T cells in the bone marrow at time of harvest for unmodified T cell treated mice and CLEC2A CAR T cell treated mice.

[0020] FIGs. 8A-8C. CLEC2A long CAR T cells eradicate leukemia, but do not improve survival. (8A) Schematic of flow cytometry assay to assess internalization of cell surface CLEC2A. (8B) Flow cytometry histogram plots (top) of CLEC2A cell surface expression at different time points with mean fluorescence intensity (MFI) graphically displayed in relation to time co-incubated with the CLEC2A antibody (bottom). (8C) Target specific cell killing using the HumZap antibody-toxin conjugate system following 72 hours of target positive or negative cell lines with either CLEC2A antibody (Ab) + toxin or isotype control + toxin control.

[0021] FIGs. 9A-9C. Biophysical characterization of 3H10 VH and 4H10 H. (9A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 3H10 VH and 4H10 VH, indicating the correct size. (9B) Size exclusion chromatography of 3H10 VH and 4H10 VH, indicating a monomeric folding. (9C) Dynamic light scattering of 3H10 VH and 4H10 VH, showing no aggregation along with 7 days incubation at 37°C. 3H 10 exhibited correct size, monomeric folding and large portion of reversible oligomers. 4H10 exhibited the correct size and monomeric folding aggregation resistance.

[0022] FIGs. 10A-10G. 3H10 VH and 4H10 VH binding against human CLEC2A and homologues. (10A) Enzyme-linked immunosorbent assay (ELISA) binding to recombinant CLEC2A for two VH binders, 3H10 VH and 4H10 VH, derived from the phage library. (10B-10G) ELISA binding of 3H10 VH and 4H10 VH to recombinant CLEC2A homologues: (10B) CLEC2B, (10C) CLEC2C, (10D) CLEC2D, (10E) CLEC2L, (10F) CLEC1 B and (10G) CLEC5B. 3H10 VH and 4H10 VH exhibited the specific binding against CLEC2A without non-specific binding against CLEC2Ahomologues.

[0023] FIG. 11. Sequences supporting the disclosure including lgG4 hinge coding sequence (SEQ ID NOs: 172 or 173); lgG4 hinge S10P (SEQ ID NO: 174); Hinge+intermediate spacer (DS) (SEQ ID NO: 175); lgG4-int(DS) coding sequence (SEQ ID NO: 176); lgG4-long coding sequence (SEQ ID NO: 177); CD3 coding sequence (SEQ ID NO: 178); CD3 protein (SEQ ID NOs: 179 or 180); 4-1BB signaling coding sequence (SEQ ID NOs: 181 or 182); 4-1 BB protein (SEQ ID NOs: 183 or 184); CD28TM coding sequence (SEQ ID NOs: 185-187); CD28TM protein (SEQ ID NO: 188 or 189); T2A coding sequence (SEQ ID NO: 190); P2A: (SEQ ID NO: 191); T2A: (SEQ ID NO: 192); E2A: (SEQ ID NO: 193); F2A: (SEQ ID NO: 194); tCD19 coding sequence (SEQ ID NO: 195); Psi (SEQ ID NO: 196); RRE (SEQ ID NO: 197); Flap (SEQ ID NO: 198); Ef1 promoter (SEQ ID NO: 199); CAR 5’ seq primer (SEQ ID NO: 200); CAR 3’ sequencing primer (SEQ ID NO: 201); GM-CSFR signal peptide (SEQ ID NO: 202); WPRE (SEQ ID NO: 204); delU3 (SEQ ID NO: 205); R (SEQ ID NO: 206); U5 (SEQ ID NO: 207); AmpR (SEQ ID NO: 208); CoE1 origin (SEQ ID NO: 209); SV40 (SEQ ID NO: 210); CMV (SEQ ID NO: 211); CLEC2A(3H10)-lgG4sh- CD28TM-41 BB-CD3z-T2A-tCD19 (SEQ ID NO: 212); CLEC2A(3H10)-lgG4sh-CD28TM-41 BB- CD3z-T2A-tCD19 with Signal Peptide (SEQ ID NO: 213); CLEC2A(3H10)-lgG4intDS-CD28TM- 41 BB-CD3z-T2A-tCD19 (SEQ ID NO: 214); CLEC2A(3H10)-lgG4intDS-CD28TM-41BB-CD3z- T2A-tCD19 with Signal Peptide (SEQ ID NO: 215); CLEC2A(3H10)-lgG4long-CD28TM-41 BB- CD3z-T2A-tCD19 (SEQ ID NO: 216); CLEC2A(3H10)-lgG4long-CD28TM-41 BB-CD3z-T2A- tCD19 with Signal Peptide (SEQ ID NO: 217); CLEC2A(4H10)-lgG4sh-CD28TM-41 BB-CD3z- T2A-tCD19 (SEQ ID NO: 218); CLEC2A(4H10)-lgG4sh-CD28TM-41 BB-CD3z-T2A-tCD19 with Signal Peptide (SEQ ID NO: 219); CLEC2A(4H10)-lgG4intDS-CD28TM-41 BB-CD3z-T2A-tCD19 (SEQ ID NO: 220); CLEC2A(4H10)-lgG4intDS-CD28TM-41 BB-CD3z-T2A-tCD19 with Signal Peptide (SEQ ID NO: 221); CLEC2A(4H10)-lgG4long-CD28TM-41 BB-CD3z-T2A-tCD19 (SEQ ID NO: 222); and CLEC2A(4H10)-lgG4long-CD28TM-41 BB-CD3z-T2A-tCD19 with Signal Peptide (SEQ ID NO: 223).DETAILED DESCRIPTION

[0024] For many years, the chosen treatments for cancer were surgery, chemotherapy, and / or radiation therapy. In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular and / or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express particular antigens on their cellular surfaces and these antigens have provided targets for successful antibody- and cell-based therapeutics.

[0025] The use of immunotherapy has revolutionized treatment of B cell malignancies, but this success has not been translated into acute myeloid leukemia (AML). AML is the most common leukemia and leading cause of death from leukemia in the US. Survival for high-risk AML patients remains dismal at 25-30% and thus novel treatments are urgently needed for this group. Molecular heterogeneity of the disease has limited broad utility of directed therapies. KMT2A- rearranged (KMT2A-r AML; aka MLL leukemia) is a particularly high-risk disease with adverse outcome. KMT2A-r AML, results from structural translocations with a resultant transcript fusion involving the KMT2A gene (aka MLL) leading to generation of a fusion oncoprotein that mediates one of the most aggressive subtypes of AML. KMT2A-r is highly prevalent in infants and although they occur less frequently in adults, it is among the most refractory and fatal diseases regardless of age. In addition to high prevalence in de novo AML, prior exposure to chemotherapy for other malignancies puts patients at extraordinary risk of developing secondary KMT2A-r AML. All efforts to develop effective targeted therapies for KMT2A-r AML to date have failed.

[0026] One of the reasons the use of immunotherapy to treat AML remains a challenge is because many immunotherapeutic targets present in AML are also found on normal hematopoietic cells, therefore targeted killing of cells expressing these markers results in unacceptable side effects including myeloablation. Identification of novel AML-restricted targets in high-risk subtypes of AML provides both effective and tolerable immunotherapeutic options that are needed to improve the poor clinical outcomes for these patients.

[0027] The current disclosure is based on transcriptome sequencing in AML further complemented with transcriptome data for TCGA, BeatAML and SWOG RNA sequencing, allowing the identification of KMT2A-r restricted transcripts that are highly expressed in KMT2A-r AML, but substantially absent in normal hematopoiesis. The current disclosure more particularly provides C-type lectin domain family 2 member A (CLEC2A) as a therapeutic target that is highly expressed in KMT2A-r AML, but substantially absent in normal hematopoiesis. The current disclosure also provides research tools to assess therapeutics that bind CLEC2A and resulting therapeutics that bind CLEC2A to treat AML. As disclosed herein, the targeting of CLEC2A results in robust and specific killing of AML leukemic blasts with limited to no hematopoietic toxicity.

[0028] Also presented herein are fully human variable heavy (VH) domains with high affinity and specificity for CLEC2A. These binding domains bind the extracellular domain of CLEC2A with binding EC50 ranging from single to two-digit nanomolar. They also display high specificity for CLEC2A, showing little to no binding with several closely associated, but clinically irrelevant, proteins (CLEC2B, CLEC2C,CLEC2D, CLEC-2 1 B, CLEC2L, CLEC5B, etc.). Further therapeutic advantages include variable VH and Fab formats that can target different epitopes of CLEC2A.The small size of these binding domains is ideal for use in a variety of therapeutic modalities without significant impacts to stability. In addition, these binding domains are fully human to reduce immunogenicity in treatment while many currently available commercial antibodies are animal-derived.

[0029] Additional embodiments provide a CLEC2A knock-out model or a CLEC2A overexpression model. These models can be used for in vitro and in vivo testing alongside their respective parental cell lines. In particular embodiments, a CLEC2A knock-out model can be generated using genetic engineering techniques described herein to knockout CLEC2A expression in CLEC2A-expressing cells. In particular embodiments, genetic engineering techniques include using sgRNA (e.g., CLEC2A sgRNA) and CRISPR / Cas9 technology in cells. In particular embodiments, the CLEC2A-expressing cells include OCI-AML2 cells.

[0030] In particular embodiments, an overexpression model of CLEC2A can be generated by using genetic engineering techniques described herein to clone a CLEC2A vector into non- CLEC2A-expressing cells. In particular embodiments, the CLEC2A vector includes a lentivirus including CLEC2A plasmid. In particular embodiments, the non-CLEC2A-expressing cells include MV4;11 cells. In particular embodiments, non-CLEC2A-expressing cells are transfected with a lentivirus with a CLEC2A plasmid to create a CLEC2A overexpression model.

[0031] These cell-based models can be used for in vitro and in vivo testing with CLEC2A antibodies and immunotherapeutic treatments. To generate an in vivo patient-derived xenograft (PDX) model, CLEC2A+ validated primary patient AML cells can be transduced to express luciferase / GFP to allow for in vivo leukemic monitoring by bioluminescence imaging. Such cells can be injected into an animal model (e.g., NSG mice) to establish a PDX model for therapeutic testing.

[0032] In particular embodiments, the disclosed anti-CLEC2A binding domains include a binding domain from a 3H10 antibody or a binding domain from a 4H10 antibody. In particular embodiments, the 3H10 binding domain includes a variable heavy domain including the sequence as set forth in SEQ ID NO: 1. In particular embodiments, the 3H10 binding domain includes a variable heavy domain encoded by the sequence as set forth in SEQ ID NO: 2. In particular embodiments, the 4H10 binding domain includes a variable heavy domain including the sequence as set forth in SEQ ID NO: 2. In particular embodiments, the 4H10 binding domain includes a variable heavy domain encoded by the sequence as set forth in SEQ ID NO: 4.

[0033] The present disclosure provides anti-CLEC2A binding domains and uses thereof. The binding domains can be used to isolate cells expressing CLEC2A or to target such cells ex vivo or in vivo for research, diagnostic, or therapeutic purposes. The binding domains disclosed hereincan also be engineered into numerous additional formats, such as antibodies and fragments thereof, multi-domain binding molecule, an antibody conjugate (ADC), or a recombinant receptor, such as a chimeric antigen receptor (CAR), expressed by an immune cell (e.g., CAR-T cell).

[0034] CLEC2A antibody binders can be tested by flow cytometry using validated CLEC2A- positive (OCI-AML2) and negative (MV4;11) cell lines and primary patient samples. Selected antibodies can be sequenced to create immunotherapeutics for targeting CLEC2A. For ADC, for example, a selected anti-CLEC2A binding antibody can be conjugated to an appropriate drug (e.g., Calicheamicin). In particular embodiments, a multi-domain binding molecule includes an anti-CLEC2A binding domain and an immune cell activating epitope binding domain.

[0035] In particular embodiments, a multi-domain binding molecule includes an anti-CLEC2A binding domain linked to a second binding domain. In particular embodiments, the second binding domain includes and immune cell activating epitope. In particular embodiments, the anti-CLEC2A binding domain includes an scFv.

[0036] In particular embodiments, an antibody conjugate includes an anti-CLEC2A binding domain linked to a toxin, drug, detectable label, a radioisotope, or a particle. In particular embodiments, the drug is calicheamicin.

[0037] In particular embodiments, a CAR T cell includes an extracellular domain including a binding domain that binds CLEC2A. In particular embodiments, the binding domain that binds CLEC2A is an scFv derived from an anti-CLEC2A binding domain described herein. In particular embodiments, the current disclosure provides CAR having an intermediate spacer region. In particular embodiments, the intermediate spacer region includes the hinge region and the CH3 domain of lgG4. In particular embodiments, the spacer is a short spacer. In particular embodiments, the spacer is a long spacer. In particular embodiments the current disclosure provides CAR having a transmembrane domain including the CD28 transmembrane domain. In particular embodiments, the current disclosure provides CAR having an intracellular effector domains including the 4-1 BB and CD3^ signaling domains.

[0038] Aspects of the current disclosure are now described with additional details and options as follows: (I) Anti-CLEC2A Binding Domains; (II) Multi-Domain Binding Molecules; (III) Expression of Recombinant Proteins; (IV) Antibody Conjugates; (V) Recombinant Receptors; (VI) Genetically Modifying Cell Populations; (VII) Cell Activating Culture Conditions; (VIII) Compositions and Formulations for Administration; (IX) Methods of Use; (X) Kits; (XI) Exemplary Embodiments; (XII) Experimental Example; and (XIII) Closing Paragraphs. These headings are provided for organizational purposes only and should not be construed to limit the teachings or interpretation of the disclosure in any way.

[0039] (I) Anti-CLEC2A Binding Domains. The present disclosure provides binding domains (e.g., antibodies) that bind C-type lectin domain family 2 member A (CLEC2A). In particular embodiments, CLEC2A isoform 1 [Homo sapiens] includes the sequence: MINPELRDGRADGFIHRIVPKLIQNWKIGLMCFLSIIITTVCIIMIATWSKHAKPVACSGDWLGVRD KCFYFSDDTRNWFASKIFCSLQKAELAQIDTQEDMEFLKRYAGTDMHWIGLSRKQGDSWKWT NGTTFNGWFEIIGNGSFAFLSADGVHSSRGFIDIKWICSKPKYFL (SEQ ID NO: 5).

[0040] In particular embodiments, CLEC2A isoform 2 [Homo sapiens] includes the sequence: MINPELRDGRADGFIHRIVPKLIQNWKIGLMCFLSIIITTVCIIMIATWSKHAKPVACSGDWLGVRD KCFYFSDDTRNWTASKIFCSLQKAELAQIDTQEDMEFLKRYAGTDMHWIGLSRKQGDSWKWT NGTTFNGWPSNSKWSCNWSLRQWLLLLGPLR (SEQ ID NO: 6).

[0041] Certain forms of conventional antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.

[0042] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1 ):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0043] Definitive delineation of a CDR and identification of residues including the binding site ofan antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs and bioinformatical tools, such as ABodyBuilder and Paratome can also be used to determine CDR sequences. Additionally, delineation of a CDR can be according to X-ray crystallography.

[0044] In particular embodiments, an anti-CLEC2A binding domain includes a VH domain antibody. In comparison to a conventional antibody, a VH domain antibody does not have a light chain, making them much smaller. In particular embodiments, the anti-CLEC2A binding domain includes a 3H10 VH domain antibody. In particular embodiments, the 3H10 VH domain antibody includes the sequence:EVQLVESGGGLVQPGGSLRLSCAASDFYFADYEMSWVRQAPGKALEWIGEIHHSGTTDYNPS LKSRVTISRDNSKNTLYLQMNSLRAEDTAIYYCATWEAPGYFDYWGQGTLVTVSS (SEQ ID NO: 1).

[0045] In particular embodiments, the 3H10 VH domain antibody is encoded by the sequence: GAAGTACAATTGGTCGAATCTGGTGGGGGCCTGGTCCAGCCTGGAGGGAGCCTTCGATTG AGTTGCGCCGCGTCTGATTTCTACTTCGCTGATTACGAAATGAGTTGGGTTAGGCAGGCTC CCGGAAAGGCACTCGAATGGATTGGCGAAATTCACCATTCCGGCACTACGGACTACAATC CTTCCTTGAAGTCCCGAGTTACAATTAGCAGAGATAACTCTAAGAATACATTGTATTTGCAG ATGAACTCACTGCGGGCAGAGGATACAGCTATTTACTATTGCGCGACTTGGGAGGCTCCT GGGTACTTTGACTATTGGGGCCAAGGGACACTGGTTACTGTCTCTAGT (SEQ ID NO: 2).

[0046] In particular embodiments, the anti-CLEC2A binding domain includes a 4H10 VH domain antibody. In particular embodiments, the 4H10 VH domain antibody includes the sequence: EVQLVESGGGLVQPGGSLRLSCAASDFSFDYYEMSWVRQAPGKGLEWVADISYNGRNTWYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGEDADYWGPGTLVTVSS (SEQ ID NO: 3).

[0047] In particular embodiments, the 4H10 VH domain antibody is encoded by the sequence: GAGGTTCAGCTTGTTGAGTCTGGAGGAGGGTTGGTTCAGCCGGGGGGGTCACTTCGGCTT TCCTGTGCCGCTAGTGACTTTTCCTTTGACTATTATGAGATGAGTTGGGTGAGGCAGGCTC CCGGTAAAGGGCTTGAATGGGTCGCTGATATATCATACAATGGCCGCAACACTTGGTACG CGGACTCAGTAAAAGGTCGGTTTACCATCTCCAGGGATAACTCTAAGAATACTTTGTACCT CCAGATGAATTCTCTGCGCGCTGAGGACACGGCTGTGTATTACTGCGCCAGTGGGGAGGACGCGGACTACTGGGGACCAGGTACATTGGTGACGGTGAGTAGT (SEQ ID NO: 4).

[0048] Referring to the binding domains provided herein, the following CDR sets are provided. A CDR set refers to 3 heavy chain CDRs that together result in binding to CLEC2A.

[0049] Table 1. CDR sequences as defined by IMGT, Kabat, Chothia, North, and Contact CDR definitions.

[0050] The VH domains can be used in a format similar to a tetrameric antibody (or conventional antibody).

[0051] In particular embodiments, an anti-CLEC2A binding domain can be derived from any anti- CLEC2A antibody. In particular embodiments, the anti-CLEC2A antibody includes 3H10, 4H10, or 4G8. Commercially available anti-CLEC2A antibodies include MA5-28532, MAB 72191 , MAB7219, PA5-34409, ABIN2595402, PA5-114260, PA5-111692, or PA5-61762.

[0052] The carboxy-terminal portion of each chain of an antibody defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0053] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. In particular embodiments, a human IgK Fc region includes the sequence: TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33). In particular embodiments, a human IgA Fc region includes the sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 34).

[0054] Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1 , lgG2, lgG3, and lgG4. IgM has subclasses including lgM1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2.

[0055] In particular embodiments, a human lgG1 Fc region includes the sequence: THTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVETVFHQDWENGKEYKCKVSNKAFPVPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35).

[0056] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36)

[0057] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 37).

[0058] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 38).

[0059] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYY MTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKAT TAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFT CFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTL NHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQRE VNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDHGPMK (SEQ ID NO: 39).

[0060] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSG HYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHF PPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTY TCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTW SRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTK GSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 40).

[0061] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0062] As indicated, antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody when in the non-blockedpresence of the antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.

[0063] Unless otherwise indicated, the term “antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains, one full-length heavy chain, or the variable binding domain of a heavy chain as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies can include monoclonal antibodies (mAbs), human or humanized antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of the antibodies disclosed herein.

[0064] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0065] A “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences.

[0066] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin or VHframeworksequences. Generally, the selection of human immunoglobulin V or VHsequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91- 3242, Bethesda Md. (1991), vols. 1-3. In particular embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al. (supra). In particular embodiments, for the VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0067] Binding domains disclosed herein can be utilized to prepare various forms of relevant binding domain molecules. For example, particular embodiments can include other binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.

[0068] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage), as described herein. Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab', Fab'- SH, F(ab')2; diabodies; and linear antibodies.

[0069] Additional examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0070] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VLand V domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VLand VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V and V regions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO 1993 / 16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458.

[0071] Linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv are commonly Gly-Ser linkers, described in more detail elsewhere herein. In particular embodiments, the linker can include a Whitlow linker (GSTGSGSKPGSGEGSTKG; SEQ ID NO: 41).

[0072] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH 1 domains. A F(ab')2fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab')2fragments having increased in vivo half-life, see U.S. Patent 5,869,046. Diabodies include two epitope-binding sites that may be bivalent. See, for example, EP 0404097; WO1993 / 01161 ; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51 , 2011)) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9:129-134, 2003.

[0073] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human I gG1 , 1 gG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described herein.

[0074] In particular embodiments, variants (including Fc variants) have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and / or (6) extended half-life. While the disclosure below describes these modifications in terms of their application to antibodies, when applicable to another particular anti- CLEC2A binding domain format (e.g., bispecific antibodies), the modifications can also be applied to these other formats.

[0075] In particular embodiments, the Fc moiety of an antibody includes a substitution at positions CH2 4, CH2 5, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type IgG 1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH24, CH25, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH2 4, or CH2 5, or both. In particular embodiments, the antibodyincludes both, a CH2 L4A and a CH2 L5A substitution. Such antibodies are referred to herein as a "l_AI_A" variant. Interestingly, a "l_AI_A" mutation in the Fc moiety does not only result in a lack of contribution of the respective antibody in antibody-dependent enhancement (ADE), but also blocks ADE.

[0076] In particular embodiments, an lgG4 Fc region is mutated to form the lgG4_S228P Fc region. lgG4 antibodies can undergo a process called Fab arm exchange which results in functionally monovalent, bispecific antibodies with unknown specificity and thus potentially reduced therapeutic efficacy. Mutating the wildtype lgG4 serine at position 228 within the corehinge region to a proline creates the lgG4_S228P mutant. In particular embodiments, the lgG4_S228P mutant prevents Fab arm exchange.

[0077] In particular embodiments the antibodies can be mutated to increase their affinity for Fc receptors. Exemplary mutations that increase the affinity for Fc receptors include: G236A / S239D / A330L / I332E (GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012. In particular embodiments, an antibody variant includes an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In particular embodiments, alterations are made in the Fc region that result in altered C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.

[0078] In particular embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521 ,541.

[0079] Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e. , between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., W02000 / 61739; WO 2001 / 29246; W02002 / 031140; US2002 / 0164328; W02003 / 085119; W02003 / 084570; US2003 / 0115614; US2003 / 0157108; US2004 / 0093621 ; US2004 / 0110704; US2004 / 0132140; US2004 / 0110282; US2004 / 0109865; W02005 / 035586; W02005 / 035778; W02005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003 / 085107).

[0080] In particular embodiments, modified antibodies include those wherein one or more amino acids have been replaced with a non-amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, an amino acid conjugated to a lipid nanoparticle, an amino acid conjugated to a polymer nanoparticle, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S / T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.

[0081] In particular embodiments, variants include glycosylation variants wherein the number and / or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particular embodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0082] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetyl-phenylalanine residues can be performed.

[0083] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys., 1984, 7:175- 186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers, F. J. et al., Clin. Pharmacol. Then, 49:307-313, 1991). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski etal., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92 / 16221).

[0084] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG- propionaldehyde.

[0085] In particular embodiments, the antibody can be fused or coupled to an Fc polypeptide thatincludes amino acid alterations that extend the in vivo half-life of an antibody that contains the altered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, S254T, T256E, M428L, and / or N434S and can be used together, separately or in any combination. For example, M428L / N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010. Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No. 2010 / 0234575, PCT / US2012 / 070146, and Zwolak, Scientific Reports 7: 15521 , 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251 , 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each of these alterations or combinations of these alterations can be used to extend the half-life of an antibody as described herein.

[0086] In particular embodiments, Fc modifications include hulgG4 ProAlaAla, hulgG2m4, and / or hulgG2sigma mutations. In particular embodiments, one or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter ed a / ., US Patent No. 5,624,821 ; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296.

[0087] (II) Multi-Domain Binding Molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti-CLEC2A binding domain disclosed herein. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least, three, or at least four binding domains that bind an epitope on CLEC2A. In particular embodiments, all of the binding domains of a multi-domain binding molecule bind CLEC2A. In particular embodiments, multi-domain binding molecules include bispecific antibodies, trispecific antibodies, and so on.

[0088] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F(ab')2bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US Pat. No. 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti-ErbB2 / Fc alpha antibody; and US 5,821 ,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody. In particular embodiments, a bispecific antibody can be in the form of a Bispecific T-cell Engaging (BiTE®) antibody.

[0089] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0090] scFv dimers or diabodies may be used, rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker ef al. (Embo Journal, 10, 3655-3659, 1991).

[0091] Bispecific antibodies with extended half-lives are described in, for example, US Patent No. 8,921 ,528 and US Patent Publication No. 2014 / 0308285.

[0092] Methods for making antibodies with two binding domains are known in the art. For example, traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983). Similar procedures are disclosed in, for example, WO 1993 / 008829, Traunecker ef al., EMBO J. 10:3655-3659, 1991 and Holliger & Wnter, Current Opinion Biotechnol. 4, 446-449 (1993).

[0093] In particular embodiments, antibodies with two binding domains can be prepared using chemical linkage. For example, Brennan et al. (Science 229: 81 , 1985) describes a procedure wherein intact antibodies are proteolytical ly cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated then are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives then is reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the antibody having twobinding domains.

[0094] In particular embodiments, bispecific antibodies (or antibodies with two binding domains) can be prepared using knobs-into holes techniques. Knobs-into-holes refers to forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids are replaced by amino acids with short side chains to create a ‘hole’. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a ‘knob’. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (‘knob-hole’) versus homodimer formation (‘hole-hole’ or ‘knob-knob’) is observed (Ridgway, J. B., Protein Eng. 9 (1996) 617-621 ; and WO 96 / 027011).

[0095] In particular embodiments, the ‘knob’ and / or the ‘hole’ may exist in the original polypeptide or may be introduced synthetically (e.g., by altering nucleic acid encoding the polypeptide). To synthetically introduce a knob and / or hole, the nucleic acid encoding the original amino acid residue (or other non-amino acid groups such as, for example carbohydrate groups) in the interface of the polypeptide is replaced with DNA encoding at least one import amino acid residue, wherein the interface refers to amino acid residues in contact between a first heavy chain constant region and one or more amino acid residues (or other non-amino acid groups) in a second heavy chain constant region. The import residues for the formation of a hole are amino acids with smaller side chain volumes than the original amino acid residue such as alanine (A), serine (S), threonine (T), valine (V), or glycine (G). The import residues for the formation of a knob are amino acids with larger side chain volumes than the original amino acid residue such as tyrosine (Y), arginine (R), phenylalanine (F), or tryptophan (W). The percentage of heterodimer can be increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681; Atwell, S.,J. Mol. Biol. 270 (1997) 26-35).

[0096] Two or more binding domains can be linked through a linker to form a multi-domain binding molecule. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0097] Commonly used flexible linkers include a linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 42),(Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 43), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 44), and (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 45). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 46), (Gly4Ser)3(SEQ ID NO: 47), (Gly4Ser)2(SEQ ID NO: 48), (Gly4Ser)i (SEQ ID NO: 49), (Gly3Ser)2(SEQ ID NO: 50), (Gly3Ser)i (SEQ ID NO: 51), (Gly2Ser)2(SEQ ID NO: 52) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 53), GGSGGGSGSG (SEQ ID NO: 54), or GGSGGGSG (SEQ ID NO: 55).

[0098] In particular embodiments, a (Gly4Ser)4linker is encoded by the sequence as set forth in SEQ ID NO: 46.

[0099] In particular embodiments, a linker region is (GGGGS)n(SEQ ID NO: 42) wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the spacer region is (EAAAK)n (SEQ ID NO: 56) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0100] Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357— 1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0101] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51 % proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0102] Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, and disulfide bond cleavage. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0103] T-cell activation can be mediated by two distinct signals: those that initiate antigendependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Immune cell activating multispecific (l-AMS) disclosed herein can target any T-cell activating epitope that upon bindinginduces T-cell activation. Examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3. Binding domains that bind T- cell markers are known in the art. B cell activation can be initiated by binding of an antigen to the B cell receptor (BCR) (e.g., IgM or IgD). Exemplary immune cell activating factors for NK cells include IL-15 and CD137.

[0104] In particular embodiments, a multi-domain binding molecule includes an immune cell engaging molecule. In particular embodiments, an immune cell engaging molecules includes a first binding domain that binds CLEC2A and a second binding domain that binds an immune cell activating epitope. In particular embodiments, the immune cell activating epitope includes an anti- CD3 binding domain, an anti-CD28 binding domain, an anti-4-1 BB binding domain, or an anti- CD8 binding domain.

[0105] In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain (HcFv) including the sequence: EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV SS (SEQ ID NO: 57) and a variable light chain (LcFv) including the sequence: QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPA RFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (SEQ ID NO: 58).

[0106] In particular embodiments, the anti-CD3 binding domain (e.g., scFv) is derived from the OKT3 antibody (the same as the one utilized in blinatumomab). The OKT3 antibody is described in detail in U.S. Patent No. 5,929,212. In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including KASGYTFTRYTMH (SEQ ID NO: 59), a CDRH2 sequence including INPSRGYTNYNQKFKD (SEQ ID NO: 60), and a CDRH3 sequence including YYDDHYCLDY (SEQ ID NO: 61); and a variable light chain including a CDRL1 sequence including SASSSVSYMN (SEQ ID NO: 62), a CDRL2 sequence including RWIYDTSKLAS (SEQ ID NO: 63), and a CDRL3 sequence including QQWSSNPFT (SEQ ID NO: 64). In particular embodiments, the anti-CD3 binding domain is a human or humanized binding domain.

[0107] The following sequence is an scFv derived from OKT3 which retains the capacity to bind CD3:QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGG GSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 65). It may also be used as a CD3 binding domain.

[0108] In particular embodiments, the anti-CD3 binding domain is derived from the 20G6-F3 antibody. In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 66), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 67), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NO: 68); and a variable light chain including a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 69), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 70).

[0109] In particular embodiments, the anti-CD3 binding domain is derived from the 4B4-D7 antibody. In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 71), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 72), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 73); and a variable light chain including a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 74), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 70).

[0110] In particular embodiments, the anti-CD3 binding domain is derived from the 4E7-C9 antibody. In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 71), a CDRH2 sequence including IKDKSNNYAT (SEQ ID NO: 75), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 76); and a variable light chain including a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 77), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 70).

[0111] In particular embodiments, the anti-CD3 binding domain is derived from the 18F5-H10 antibody. In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including a CDRH1 sequence including GFTFTNAW (SEQ ID NO: 78), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 79), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 80); and a variable light chain including a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 81), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 82).

[0112] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WQ2016 / 116626. TR66 may also be used.

[0113] CD28 is a surface glycoprotein present on 80% of peripheral T-cells in humans and is present on both resting and activated T-cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) andis the most potent of the known co-stimulatory molecules (June et al., Immunol. Today 15:321 , 1994; Linsley et al., Ann. Rev. Immunol. 11 :191 , 1993). In particular embodiments, the CD28 binding domain (e.g., scFv) is derived from CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10. Further, 1YJD provides a crystal structure of human CD28 in complex with the Fab fragment of a mitogenic antibody (5.11A1).

[0114] In particular embodiments, the anti-CD28 binding domain is derived from TGN1412. In particular embodiments, the variable heavy chain of TGN1412 includes: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNE KFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS (SEQ ID NO: 83) and the variable light chain of TGN1412 includes: DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 84).

[0115] In particular embodiments, the anti-CD28 binding domain includes a variable heavy chain including a CDRH1 sequence including GYTFTSYYIH (SEQ ID NO: 85), a CDRH2 sequence including CIYPGNVNTNYNEK (SEQ ID NO: 86), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 87); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 88), CDRL2 sequence including KASNLHT (SEQ ID NO: 89), and CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 90).

[0116] In particular embodiments, the anti-CD28 binding domain including a variable heavy chain including a CDRH1 sequence including SYYIH (SEQ ID NO: 91), a CDRH2 sequence including CIYPGNVNTNYNEKFKD (SEQ ID NO: 92), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 87); and a variable light chain including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 88), a CDRL2 sequence including KASNLHT (SEQ ID NO: 89), and a CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 90).

[0117] Activated T-cells express 4-1BB (CD137). In particular embodiments, the anti-4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including YYWS (SEQ ID NO: 97), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 98); and a variable light chain including a CDRL1 sequence including RASQSVS (SEQ ID NO: 99), a CDRL2 sequence including ASNRAT (SEQ ID NO: 100), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 101).

[0118] In particular embodiments, the anti-4-1 BB binding domain includes a variable heavy chain including a CDRH1 sequence including GYSFSTYWIS (SEQ ID NO: 102), a CDRH2 sequence including KIYPGDSYTNYSPS (SEQ ID NO: 103) and a CDRH3 sequence including GYGIFDY(SEQ ID NO: 104); and a variable light chain including a CDRL1 sequence including SGDNIGDQYAH (SEQ ID NO: 105), a CDRL2 sequence including QDKNRPS (SEQ ID NO: 106), and a CDRL3 sequence including ATYTGFGSLAV (SEQ ID NO: 107).

[0119] Particular embodiments disclosed herein including binding domains that bind epitopes on CD8. In particular embodiments, the anti-CD8 binding domain (e.g., scFv) is derived from the OKT8 antibody. For example, in particular embodiments, the anti-CD8 binding domain includes a variable heavy chain including a CDRH1 sequence including GFNIKD (SEQ ID NO: 108), a CDRH2 sequence including RIDPANDNT (SEQ ID NO: 109), and a CDRH3 sequence including GYGYYVFDH (SEQ ID NO: 110); and a variable light chain including a CDRL1 sequence including RTSRSISQYLA (SEQ ID NO: 111), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 112), and a CDRL3 sequence including QQHNENPLT (SEQ ID NO: 113). In particular embodiments, the anti-CD8 binding domain is human or humanized.

[0120] In particular embodiments natural killer cells (also known as NK-cells, K-cells, and killer cells) are targeted for localized activation by l-AMS. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes and can secrete cytokines to recruit other immune cells.

[0121] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0122] Examples of commercially available antibodies that bind to an NK cell receptor and induce and / or enhance activation of NK cells include: 5C6 and 1 D11, which bind and activate NKG2D (available from BioLegend® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend®); P44-8, which binds and activates NKp44 (available from BioLegend®); and SK1 , which binds and activates CD8.

[0123] Particular embodiments utilize binding domains that bind CD16. Commercially available anti-CD16 antibodies include 3G8, A9, NM3E2, ab203883, eBioCB16, EPR22409-124, KD1 , SP175, EPR16784, GRM1 , ASH 1975, B73.1 , MEM -154, DJ130C, 1E12E11 , BLR162J, PD00- 12, and SP189.

[0124] In particular embodiments, an anti-CD16 binding domain is derived from a 3G8 antibody. In particular embodiments, the anti-CD16 binding domain includes a variable heavy chain including the sequence: QVTLKESGPGILQPSQTLSLTCSFSGFSLRTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYN PALKSRLTISKDTSSNQVFLKI ASVDTADTATYYCAQI NPAWFAYWGQGTLVTVSA (SEQ IDNO: 114) and a variable light chain including the sequence:DTVLTQSPASLAVSLGQRATISCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGIPA RFSASGSGTDFTLNIHPVEEEDTATYYCQQSNEDPYTFGGGTKLEIK (SEQ ID NO: 115).

[0125] In particular embodiments, an anti-CD16 antibody is derived from an A9 antibody. In particular embodiments, the anti-CD16 antibody includes a variable heavy chain including the sequence:QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYTNYNE KFKGKATVTADTSSRTAYVQVRSLTSEDSAVYFCARSASWYFDVWGARTTVTVSS (SEQ ID NO: 116) and a variable light chain including the sequence:DIQAVVTQESALTTSPGETVTLTCRSNTGTVTTSNYANWVQEKPDHLFTGLIGHTNNRAPGVP ARFSGSLIGDKAALTITGAQTEDEAIYFCALWYNNHWVFGGGTKLTVL (SEQ ID NO: 117).

[0126] In particular embodiments, an anti-CD16 binding domain is derived from a 3G8 antibody. In particular embodiments, the anti-CD16 binding domain includes a variable heavy chain including a CDRH1 sequence including TSGMGVG (SEQ ID NO: 118), a CDRH2 sequence including HIWWDDDKRYNPALKS (SEQ ID NO: 119), and a CDRH3 sequence including INPAWFAY (SEQ ID NO: 120); and a variable light chain sequence including a CDRL1 sequence including KASQSVDFDGDSFMN (SEQ ID NO: 121), a CDRL2 sequence including TTSNLES (SEQ ID NO: 122), and a CDRL3 sequence including QQSNEDPYT (SEQ ID NO: 123).

[0127] In particular embodiments, an anti-CD16 binding domain is derived from an A9 antibody. In particular embodiments, the anti-CD16 binding domain includes a variable heavy chain including a CDRH1 sequence including NYWLG (SEQ ID NO: 124), a CDRH2 sequence including DIYPGGGYTNYNEKFKG (SEQ ID NO: 125), and a CDRH3 sequence including SASWYFD (SEQ ID NO: 126); and a variable light chain sequence including a CDRL1 sequence including RSNTGTVTTSNYAN (SEQ ID NO: 127), a CDRL2 sequence including HTNNRAP (SEQ ID NO: 128), and a CDRL3 sequence including ALWYNNHWV (SEQ ID NO: 129).

[0128] In particular embodiments, the l-AMS can bind to and block an NK cell inhibitory receptor to enhance NK cell activation. Examples of NK cell inhibitory receptors that can be bound and blocked include KIR2DL1 , KIR2DL2 / 3, KIR3DL1 , NKG2A, and KLRG1. In particular embodiments, a binding domain that binds and blocks the NK cell inhibitory receptors KIR2DL1 and KIR2DL2 / 3 includes a variable heavy chain region of the sequence QVQLVQSGAEVKKPGSSVKVS CKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELS SLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS (SEQ ID NO: 130) and a variable light chain region of the sequenceEIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT (SEQ ID NO: 131). Additional NK cell activating antibodies are described in WO / 2005 / 0003172 and US Patent No. 9,415,104.

[0129] In particular embodiments macrophages are targeted for localized activation by l-AMS. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis.

[0130] The l-AMS can be designed to bind to a protein expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11 b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, 1 L-4Ra, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70, which binds and activates CD11b (available from BioLegend®); KP1 , which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0131] In particular embodiments, l-AMS can target a pathogen recognition receptor (PRR). PRRs are proteins or protein complexes that recognize a danger signal and activate and / or enhance the innate immune response. Examples of PRRs include the TLR4 / MD-2 complex, which recognizes gram negative bacteria; Dectin-1 and Dectin-2, which recognize mannose moieties on fungus and other pathogens; TLR2 / TLR6 or TLR2 / TLR1 heterodimers, which recognize gram positive bacteria; TLR5, which recognizes flagellin; and TLR9 (CD289), which recognizes CpG motifs in DNA. In particular embodiments, l-AMS can bind and activate TLR4 / MD-2, Dectin-1 , Dectin-2, TRL2 / TLR6, TLR2 / TLR1 , TLR5, and / or TLR9.

[0132] In particular embodiments, l-AMS can target the complement system. The complement system refers to an immune pathway that is induced by antigen-bound antibodies and involves signaling of complement proteins, resulting in immune recognition and clearance of the antibody- coated antigens.

[0133] Binding domains of l-AMS and other engineered formats described herein may be joined through a linker. A linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a l-AM. Any appropriate linker may be used.

[0134] Cytolytic properties of l-AMS molecules can be confirmed in comparative in vitro assays. Briefly, for cell line experiments, target cells can be incubated in 96-well round bottom plates at 5-10,000 cells / well containing increasing concentrations of the various l-AMS antibodieswith / without healthy donor T-cells (used at an E:T cell ratio of 1:1 and 3:1). After 48 hours, cell numbers and drug-induced cytotoxicity, using 4',6-diamidi no-2-phenylindole (DAPI) to detect non- viable cells, can be determined by flow cytometry. In experiments where healthy donor T-cells are added, cells can be identified by forward / side scatter properties and negativity for CellVue Burgundy dye. Experiments can include technical duplicates.

[0135] In particular embodiments, T-cell activating epitope binding domains including l-AMS constructs include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, Vp, Ca, or Cp of a known TCR. An insertion, deletion or substitution may be anywhere in a Va, Vp, Ca, or Cp region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain including a modified Va, Vp, Ca, or Cp region can still specifically bind its target with an affinity similar to wild type.

[0136] In particular embodiments, a multi-domain binding molecule includes i) a variable region of an anti-CLEC2A binding domain sequence derived from 5’ RACE (rapid cloning of cDNA ends) cloning and ii) an anti-CD3 binding domain sequence. In particular embodiments, the mutli- domain binding molecules includes a CLEC2A-CD3 bispecific antibody (BsAb). Bispecific molecules can be assembled by synthesizing each scFv as a DNA fragment with overlapping Gibson assembly-compatible ends in the canonical bispecific antibody format. Prototypical intervening regions such as (Gly4Ser)3(SEQ ID NO: 47) linkers can be used between paired variable domains and a short Gly4Ser (SEQ ID NO: 49) linker between the two scFvs.

[0137] Tri-specific antibodies are artificial proteins that simultaneously bind to three different types of antigens, wherein at least one of the antigens is CLEC2A. T ri-specific antibodies are described in, for example, WO2016 / 105450, WO 2010 / 028796; WO 2009 / 007124; WO 2002 / 083738; US 2002 / 0051780; and WO 2000 / 018806.

[0138] In some embodiments, a multi-domain binding molecule includes a basic immunoglobulin structure such as an IgA domain or an IgM domain. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0139] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects,multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0140] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1 , US20180118814A1 , US20180118816A1 , US20190185570A1 , and US20180265596A1 , and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761 , and WO 2019 / 165340.

[0141] Particular embodiments include using IgA and IgM constant region domains to allow the binding portion of molecules provided herein to readily multimerize into dimers, pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0142] Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CA1 and CA2 domains. The main difference between lgA1 and I gA2 resides in the hinge region that lies between the two Fab arms and the Fc region. lgA1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below.

[0143] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the "tailpiece" (tp). The IgA and IgM tp is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tp contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tp differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tp differ at seven amino acid positions.

[0144] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 132). Referring to this SEQ ID NO: 132, the human CA1 domain extends from amino acid 6 to amino acid 98; the human lgA1 hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tp extends from amino acid 331 to amino acid 352.

[0145] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGD LYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLL LGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHG ETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRW LQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQK TIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 133). Referring to this SEQ ID NO: 133, the human CA1 domain extends from amino acid 6 to amino acid 98, the human lgA2 hinge region extends from amino acid 102 to amino acid 111 , the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tp extends from amino acid 318 to amino acid 340.

[0146] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 134, the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. An exemplary precursor secretory component includes the sequence MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGC ITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVS QGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFH CALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKED AGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIK YWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTN GDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALP SQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAA GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSE EQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGA NDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEA QDGPQEA (SEQ ID NO: 135). An exemplary mature secretory component includes KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGR ANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDL GRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQL RLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCR QSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQE GSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLL VDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEP NLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDEN SRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEK VLDSGFREIENKAIQDPR (SEQ ID NO: 136). While not wishing to be bound by theory, and as indicated above, the assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the CA3 and tp domains. See, e.g., Braathen, R., el al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the CA3 and tp domains.

[0147] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CA1 domain or a fragment thereof, and / or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy chain constant region (e.g., SEQ ID NO: 132 or SEQ ID NO: 133), or a variant, derivative, or analog thereof.

[0148] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 132 or amino acids 113 to 340 of SEQ ID NO: 133. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA or IgG hingeregion situated N-terminal to the IgA CA2 domains. For example, the IgA heavy chain constant regions can include amino acids 102 to 353 of SEQ ID NO: 132 or amino acids 102 to 340 of SEQ ID NO: 133. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA CA1 domain situated N-terminal to the IgA hinge region.

[0149] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0150] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0151] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cp4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and / or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment” of an IgM-derived binding molecule.

[0152] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cp4 and tp domains. See, e.g., Braathen, R., et al., J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cp4 and / or tp domains (also referred to herein collectively as Cp4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4-tp domains. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cp1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0153] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J- chain is considered to facilitate polymerization of chains before IgM is secreted from antibodyproducing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroomshaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylationsites: Asn-171, Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.

[0154] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V- Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.

[0155] A “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C- terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cp1), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tp, as indicated above.

[0156] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM Cp4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM Cp3 domain situated N- terminal to the IgM Cp4 and IgM tp domains.

[0157] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cp2 domain situated N-terminal to the IgM Cp3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cp2, Cp3, and Cp4-tp domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAE AKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWN SGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADV FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR VTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 137).

[0158] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM Cp2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM Cp3 domain. An exemplary variant human lgG1 hinge region amino acid sequence in which the cysteine at position 6 is substituted withserine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 138). An exemplary IgM constant region of this type includes the variant human I gG 1 hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and tp domains, and includes the amino acid sequence:VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNG EAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHR PDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPG RYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 139).

[0159] Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” includes the amino acid sequence N-X1-S / T, wherein N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 140 or SEQ ID NO: 141 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0160] The human IgM heavy chain constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 140; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1 , and CAA47714.1). Referring to this SEQ ID NO: 140, the human Cp1 region ranges from amino acid 5 to amino acid 102; the human Cp2 region ranges from amino acid 114 to amino acid 205, the human Cp3 region ranges from amino acid 224 to amino acid 319, the Cp4 regionranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0161] In particular embodiments, an IgM heavy chain constant region includes the sequence: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL GQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDS VTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTI SRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSA PMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLV MSDTAGTCY (SEQ ID NO: 141 ; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 140 by one amino acid at position 191.

[0162] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1 , and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 140 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species, e.g., those shown in FIG. 1 of PCT / US2019 / 020374.

[0163] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 , P313, R344, E345, S401 , E402, and / or E403 of SEQ ID NO: 140. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 140 corresponds to S524 of Kabat; E402 of SEQ ID NO: 140 corresponds to E525 of Kabat; E403 of SEQ ID NO: 140 corresponds to E526 of Kabat; R344 of SEQ ID NO: 140 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 140 corresponds to E468 of Kabat.

[0164] In particular embodiments, “corresponds to’’ means the designated position of SEQ ID NO: 140 and the amino acid in the sequence of the IgM constant region of any species which is homologous to the specified position. See FIG. 1 of PCT / US2019 / 020374.

[0165] In particular embodiments, P311 of SEQ ID NO: 140 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 140 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 140 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 142).

[0166] In certain aspects, S401 of SEQ ID NO: 140 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 140 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVAEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 143).

[0167] In certain aspects, E402 of SEQ ID NO: 140 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 140 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSAEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 144).

[0168] In certain aspects, E403 of SEQ ID NO: 140 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 140 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEAEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 145).

[0169] In certain aspects, R344 of SEQ ID NO: 140 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 140 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 146).

[0170] In certain aspects, E345 of SEQ ID NO: 140 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 140 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 147).

[0171] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody. The precursor form of the human J-chain includes:MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLN NRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKC YTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 148). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 148 and the mature human J-chain extends fromamino acid 23 to amino acid 159 of SEQ ID NO: 148.

[0172] The mature human J-chain includes the amino acid sequenceQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 134).

[0173] The term “J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and / or variant thereof, including a mature human J-chain amino acid sequence provided herein as SEQ ID NO: 134. A functional fragment, derivative, and / or variant of a J-chain has at least 90% sequence identity to the reference J-chain and retains the multimerizing function of the reference J-chain.

[0174] In certain aspects, the J-chain of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid Y102, T103, N49 or S51 of SEQ ID NO: 134.

[0175] By “an amino acid corresponding to” a position of SEQ ID NO: 134 is meant the amino acid in the sequence of the J-chain of any species which is homologous to the referenced residue in the human J-chain. For example, the position corresponding to Y102 in SEQ ID NO: 134 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 134 is conserved in the J-chain amino acid sequences of at least 37 other species. The positions corresponding to N49 and S51 in SEQ ID NO: 134 are conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No. 9,951 ,134 and FIG. 2 of PCT / US2019 / 020374.

[0176] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 134 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 134 can be substituted with alanine (alanine substitution indicated by bold underline): QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 149),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCSTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 150),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCRTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 151).

[0177] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 134 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 134 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD (SEQ ID NO: 152).

[0178] In certain aspects, the variant J-chain or functional fragment thereof of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 134, provided that S51 is not substituted with threonine (T), or wherein the J-chain includes amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 134.

[0179] The amino acids corresponding to N49 and S51 of SEQ ID NO: 134 along with the amino acid corresponding to 150 of SEQ ID NO: 134 include an N-linked glycosylation motif in the J- chain. Accordingly, mutations at N49 and / or S51 (with the exception of a single threonine substitution at S51) can prevent glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 134 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 134 can be substituted with alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect the position corresponding to N49 of SEQ ID NO: 134 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid sequence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 153).

[0180] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 134 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 134 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 134 can be substituted with alanine (A). In a particular aspect the variant J-chain or functional fragment thereof is a variant human J-chain and includes the amino acid sequence: EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDA CYPD (SEQ ID NO: 154).

[0181] Particular embodiments include a heterologous polypeptide (e.g., a single-domain antibody binding domain) fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker including at least 5 amino acids, but no more than 25 amino acids. In certain aspects, the peptide linker includes (GGGGS)n (SEQ ID NO: 42) wherein n is 1-5.

[0182] A single-domain antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible. In certain aspects, the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 134 between cysteine residues 92 and 101 of SEQ ID NO: 134. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 134 at or near a glycosylation site. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 134 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0183] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 134 by chemical or chemo-enzymatic derivatization. In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 134 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In particular embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group including N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), afunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In particular embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post- translational ly attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

[0184] In certain aspects the modified J-chain can include the formula X[Ln]J or J[Ln]X, where J includes a mature native J-chain or functional fragment thereof, X includes a heterologous binding domain, and [Ln] is a linker sequence including n amino acids, where n is a positive integer from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5, 10, 15, or 20.

[0185] J-chains from the following species can also be used in certain embodiments: Pan troglodytes, Pongo abelii, Callithrix jacchus, Macaca mulatta, Papio Anubis, Saimiri boliviensis, Tupaia chinensis, Tursiops truncatus, Orcinus orca, Loxodonta Africana, Leptonychotes weddellii, Ceratotherium si mum, Felis catus, Canis familiaris, Ailuropoda melanoleuca, Mustela furo, Equus caballus, Cavia porcellus, Camelus ferus, Capra hircus, Chinchilla lanigera, Mesocricetus auratus, Ovis aries, Myotis lucifugus, Pantholops hodgsonii, Bos taurus, Mus musculus, Rattus norvegicus, Echinops telfairi, Oryctolagus cuniculus, Monodelphis domestica, Alligator mississippiensis, Chrysemys picta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0186] In particular embodiments, the antibodies can multimerize by optionally including a multimerization domain. A “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0187] In particular embodiments, the multimerization domain is a dimerization domain that allows binding of two complementary monomers to form a dimer. In particular embodiments, a dimerization and docking domain (DDD) can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD). The AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. Additional DDDs and ADs include: the 4-helix bundle type DDD (Newlon, et al. EMBO J. 2001 ; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1)) and HNF-1 (hepatocyte nuclear factor 1) (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). Other AD sequences of potential use may be found in US 2003 / 0232420A1.

[0188] In particular embodiments, complementary binding domains can dimerize. In particular embodiments, the binding domain is a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof can include a part of an FCERI p chain such that said FCERI chains spontaneously dimerize together to form a dimeric antibody (e.g., bispecific antibody). In particular embodiments, an antibody or fragment thereof can include a part of a FCERI a chain and another antibody or fragment thereof part of a FCERI y chain such that saidFCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-domain binding molecule can include a part of FCERI a chain, a part of FCERI |3 chain and a part of FCERI y chain such that said FCERI chains spontaneously tetramerize together to form a tetrameric multi-domain binding molecule.

[0189] Leucine zippers are described in US 5932448; SH2 and SH3 are described in Vidal et al., Biochemistry, 43:7336- 44, 2004); PTB is described in Zhou et al., Nature, 378:584- 592, 1995); WW is described in Sudol Prog Biochys MoL Bio, 65:113-132, 1996; PDZ is described in Kim et al., Nature, 378: 85-88, 1995 and Komau et al., Science, 269:1737-1740, 1995; and WD40 is described in Hu et al., J Biol Chem., 273:33489- 33494, 1998.

[0190] Additional multimerization domains and systems are described in, for example, Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052; Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993; W02012001647A2; US 5821333; GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006), the PTB domain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21 : 5591-5604, 2001), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz etal., Mol Cell Biol, 18:4079-4088, 1998) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014). C4b, dextrameric, and ferritin-based multimerization can be used.

[0191] In particular embodiments, complementary binding domains can be induced using a third molecule or chemical inducer. This method of dimerization requires that one antibody or fragment thereof include a chemical inducer of dimerization binding domain 1 (CBD1) and the second antibody or fragment thereof include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR.

[0192] (III) Expression of Recombinant Proteins. Recombinant proteins disclosed herein can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of proteins, including naturally-associated or heterologous promoter regions. In particular embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the expressed proteins.

[0193] In particular embodiments, mammalian cells are used as a host for expressing nucleotide segments encoding recombinant proteins. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non- antibody-producing myelomas including Sp2 / 0 and NSO. In certain examples, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. In particular embodiments, expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus (see Co et a / ., J. Immunol. 1992, 148:1149).

[0194] Once expressed, recombinant proteins can be purified according to standard procedures of the art, including high-performance liquid chromatography (HPLC) purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982)).

[0195] In particular embodiments, recombinant proteins are formed using the Daedalus expression system as described in Pechman et al. (Am J Physiol 294: R1234-R1239, 2008). The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake et al., Nucleic Acids Res., 39(21) 2011. In some instances, purification by chromatography may not be needed due to the purity of manufacture according to the methods described herein.

[0196] (IV) Antibody Conjugates. Antibody conjugates include an anti-CLEC2A binding domain disclosed herein linked to another molecule, other than an additional binding domain. Examplesof antibody conjugates include antibody immunotoxins, antibody-drug conjugates (ADCs), antibody-detectable label conjugates, antibody radioisotope conjugates, and antibody-particle conjugates.

[0197] Antibody immunotoxins include an anti-CLEC2A binding domain disclosed herein conjugated to one or more cytotoxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1) holotoxins (or class II ribosome inactivating proteins), such as saporin, ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), Bryodin 1 , bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). The toxin may be obtained from essentially any source and can be a synthetic or a natural product.

[0198] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3): 154- 169; Chari, R. V. (2008) AccRes. 41:98-107). See also Kamath & Iyer (Pharm Res. 32(11): 3470- 3479, 2015), which describes considerations for the development of antibody-drug conjugates.The drug moiety (D) of an antibody-drug conjugate may include any compound, moiety or group that has a cytotoxic or cytostatic effect. To prepare antibody-drug conjugates, linker-cytotoxin conjugates can be made by conventional methods analogous to those described by Doronina eta / . (Bioconjugate Chem. 17: 114-124, 2006). Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0199] Antibody-detectable label conjugates include an anti-CLEC2A binding domain disclosed herein linked to a detectable label. Detectable labels can include any suitable label or detectablegroup detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In particular embodiments, detectable labels can include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymatic labels, and affinity tags.

[0200] Fluorescent labels can be particularly useful in cell staining, identification, imaging, and isolation uses. Exemplary fluorescent labels include blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed- Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0201] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0202] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0203] Enzymatic labels can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes can include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0204] Antibody-radioisotope conjugates include a binding domain disclosed herein linked to a radioisotope for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / or treatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to a subject. Therapeutic nuclear medicine is often referred to as radiation therapy or radioimmunotherapy (RIT). Examples of radionuclides that are useful for radiation therapy include225Ac and227Th.225Ac is a radionuclide with the half-life of ten days. As225Ac decays the daughter isotopes221Fr,213Bi, and209Pb are formed.227Th has a half-life of 19 days and forms the daughterisotope223Ra. Additional examples of useful radioisotopes include228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,57Cu,153Dy,157Dy, 159Dy, 1650y 1660y 171E|- 250Es, 254Es, 147E|J157Eu52pe59pe25lpm252Fm, 253Fm, 66Qa, 12Q^146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,150,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, and / or97Zr. Radioisotopes can be used as a type of detectable label called a radiolabel. In particular embodiments, a radioisotope includes131l,90Y, and / or211At. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0205] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, metal particles (e.g., gold, platinum, or silver), liposomes, polymer-based nanoparticles, and / or colored beads, and can be made from organic matter and / or inorganic matter.

[0206] Antibody-particle conjugates can function in the targeted delivery of a payload (e.g., small molecules or genetic engineering components) to a cell ex v / vo or in vivo that expresses the target cell marker. For example, scFv or other binding fragments can be linked to the surface of nparticles to guide delivery to target cells. The linkage can be through, for example, covalent attachment.

[0207] Methods of forming liposomes are described in, for example, US Patent Nos. 4,229,360; 4,224,179; 4,241 ,046; 4,737,323; 4,078,052; 4,235,871 ; 4,501 ,728; and 4,837,028, as well as in Szoka et a / ., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et a / ., Chem. Phys. Lip. 40:89 (1986). For additional information regarding particles, see Yetisgin et a / ., Molecules 2020, 25, 2193.

[0208] Examples of polymers that can be used within particles include polyglutamic acid (PGA); poly(lactic-co-glycolic acid) (PLGA); Polylactic acid (PLA); poly-D-lactic acid (PDLA); PLGA- di methacrylate; polyamines; polyorganic amines (e.g., polyethyleneimine (PEI), polyethyleneimine celluloses); poly(amidoamines) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl-N-alkyl-pyridiumiun), poly(acryloyl-trialkyl ammonium), and Tat proteins.

[0209] In particular embodiments, the particles can include a coating, particularly when used in vivo. A coating can serve to shield the encapsulated cargo and / or reduce or prevent off-target binding. Off-target binding is reduced or prevented by reducing the surface charge of the nanoparticles to neutral or negative. Coatings can include neutral or negatively charged polymer- and / or liposome-based coatings. In particular embodiments, the coating is a dense surface coating of hydrophilic and / or neutrally charged hydrophilic polymer sufficient to prevent the encapsulated cargo from being exposed to the environment before release into a target cell. In particular embodiments, the coating covers at least 80% or at least 90% of the surface of the particle.

[0210] Examples of neutrally charged polymers that can be used as a particle coating include polyethylene glycol (PEG); polypropylene glycol); and polyalkylene oxide copolymers, (PLURONIC®, BASF Corp., Mount Olive, NJ).

[0211] The size of particles can vary over a wide range and can be measured in different ways. In particular embodiments, particles are <130 nm in size. However, particles can also have a minimum dimension of equal to or less than 500 nm, less than 150 nm, less than 140 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, particles are 90 to 130 nm in size.

[0212] Dimensions of the particles can be determined using, e.g., conventional techniques, such as dynamic light scattering and / or electron microscopy.

[0213] In particular embodiments, a binding domain as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the constant region can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags, and transglutaminase recognition sequences. Antibody fragments can also be modified for site-specific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8).

[0214] (V) Recombinant Receptors. Anti-CLEC2A binding domains disclosed herein can be utilized within recombinant receptors such as chimeric antigen receptors (CAR) and / or engineered T cell receptors (eTCR).

[0215] CAR include several distinct subcomponents that allow genetically modified cells (e.g., regulatory T cells) to recognize and kill CLEC2A-expressing cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular componentincludes a binding domain that specifically binds a CLEC2A epitope that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted epitope.

[0216] eTCR disclosed herein include an anti-CLEC2A binding domain disclosed herein linked to the Caand / or Cp chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and |3 chain. Each chain includes a variable region (Vaand Vp) and a constant region (Caand Cp). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes a anti-CLEC2A binding domain as the variable region of the a and p chain. In particular embodiments, eTCR include a Caand / or Cp chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Caor Cp.

[0217] In particular embodiments, the extracellular component of a recombinant receptor includes a binding domain that binds CLEC2A. Particular embodiments of binding domains include an anti- CLEC2A binding domain and / or the CDRs thereof as disclosed herein.

[0218] Recombinant receptors can additionally include intracellular effector domains, transmembrane domains, spacer regions, linkers (described elsewhere herein), and control features.

[0219] (V-a) Intracellular Component. In particular embodiments, the intracellular component includes an effector domain. Intracellular effector domains activate the expressing cell when the binding domain binds the antigen. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0220] An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effectordomain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co-stimulatory domains.

[0221] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.

[0222] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11, CD3y, CD35, CD3c, CD3 , CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRp (FccRIb), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lek, LRP, NKG2D, NOTCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7- H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, CD8 , IL2R|3, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0223] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3c, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcRp (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0224] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a co- stimulatory domain, or any combination thereof.

[0225] Additional examples of intracellular signaling components include the cytoplasmicsequences of the CD3 chain, and / or co- receptors that act in concert to initiate signal transduction following binding domain engagement.

[0226] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and anti-cancer activity in v / vo (Song etal. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8|3, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1 , CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0227] In particular embodiments, the intracellular signaling components include CD3 (encoded by, for example, SEQ ID NO: 178) and / or a variant of 4-1 BB (encoded by, for example, SEQ ID NOs: 181 or 182). In particular embodiments, the intracellular signaling components include a variant of CD3 (SEQ ID NOs: 179 or 180) and / or a portion of 4-1 BB (SEQ ID NOs: 183 or 184).

[0228] In particular embodiments, the intracellular signaling component includes (i) all or a portion of the signaling domain of CD3 , (ii) all or a portion of the signaling domain of 4-1 BB, or (iii) all or a portion of the signaling domain of CD3 and 4-1 BB.

[0229] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-likeorphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1 R, IL2R, IL7R, or IL15R).

[0230] (V-b) Transmembrane Domain. As indicated, transmembrane domains within a recombinant receptor serve to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.

[0231] The transmembrane domain can be derived either from a natural and / or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In particular embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2RP, IL2Ry, IL7R a, ITGA1 , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI Ic, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9(CD229), PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.

[0232] In particular embodiments, a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof.

[0233] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15amino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the recombinant receptor is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In particular embodiments, the transmembrane domain is encoded by the nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NOs: 185-187). In particular embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 188 or 189).

[0234] (V-c) Spacer Regions. Spacer regions are used to create appropriate distances and / or flexibility from other recombinant receptor sub-components. In particular embodiments, the length of a spacer region is customized for binding targeted cells and mediating destruction. In particular embodiments, a spacer region length can be selected based upon the location of a cellular marker epitope, affinity of a binding domain for the epitope, and / or the ability of the targeting agent to mediate cell destruction following target binding.

[0235] Spacer regions typically include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.

[0236] In particular embodiments, a spacer region is 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, or 75 amino acids. These lengths qualify as short spacer regions.

[0237] In particular embodiments, a spacer region is 76 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, or 179 amino acids. These lengths qualify as intermediate spacer regions.

[0238] In particular embodiments, a spacer region is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids, 220 amino acids, 230 amino acids, 240 amino acids, or 250 amino acids. These lengths qualify as long spacer regions.

[0239] Exemplary spacer regions include all or a portion of an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is a human immunoglobulin hinge region. As used herein, a “wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.

[0240] An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an IgG 1 , 1 gG2, 1 gG3, or I gG4 hinge region. Sequences from IgG 1 , lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.

[0241] In particular embodiments, the spacer is a short spacer including an I gG4 hinge region. In particular embodiments the short spacer is encoded by either of SEQ ID NOs: 172 or 174. In particular embodiments, the spacer is an lgG4 hinge S10P. In particular embodiments, the lgG4 hinge S10P is encoded by SEQ ID NO: 174. In particular embodiments, the spacer is an intermediate spacer including an lgG4 hinge region and an lgG4 hinge CH3 region. In particular embodiments the intermediate spacer is encoded by SEQ ID NO: 175. In particular embodiments, the spacer is a hinge and intermediate spacer (DS). In particular embodiments, the hinge and intermediate spacer (DS) is encoded by SEQ ID NO: 176. In particular embodiments, the spacer is a long spacer including an lgG4 hinge region, an lgG4 CH3 region, and an lgG4 CH2 region. In particular embodiments the long spacer is encoded by SEQ ID NO: 177.

[0242] Other examples of hinge regions that can be used in recombinant receptors described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.

[0243] In particular embodiments, a spacer region includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. A “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the ECD of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington etal., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11 , 2002). These type II C-lectin or CD molecules may also have junction amino acids(described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an ECD ranging from amino acids 94-233. The CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-116, which may be flanked by additional junction amino acids. Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1 ; NP 001773.1; AAL65234.1 ; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively).

[0244] (V-d) Control Features Including Tag Cassettes, Transduction Markers, and / or Suicide Switches. In particular embodiments, a recombinant receptor construct can include one or more control features. In particular embodiments, a control feature can include a tag cassette, a transduction marker, or a suicide switch. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate genetically modified cells in vitro, in vivo and / or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a recombinant receptor, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the recombinant receptor.

[0245] In particular embodiments, a recombinant receptor includes a T2A ribosomal skip element that separates the expressed recombinant receptor from a truncated CD19 (tCD19) transduction marker. In particular embodiments, the T2A ribosomal skip element is encoded by SEQ ID NO: 190.

[0246] Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 155), Flag tag (DYKDDDDK; SEQ ID NO: 156), Xpress tag (DLYDDDDK; SEQ ID NO: 157), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 158), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 159), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 160), Myc tag (EQKLISEEDL; SEQ ID NO: 161), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 162), STREP® tag II (WSHPQFEK SEQ ID NO: 163 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 164), Softag 3 (TQDPSRVG; SEQ ID NO: 165), and V5 tag(GKPIPNPLLGLDST; SEQ ID NO: 166).

[0247] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma- Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0248] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et a / ., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wang et a / ., Blood 118: 1255, 2011); an ECD of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1( 5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278).

[0249] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into a recombinant receptor construct as a suicide switch.

[0250] Control features may be present in multiple copies in a recombinant receptor or can be expressed as distinct molecules with the use of a skipping element (SEQ ID NOs: 191-194). For example, a recombinant receptor can have one, two, three, four or five tag cassettes and / or one, two, three, four, or five transduction markers could also be expressed. For example, embodiments can include a recombinant receptor construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13 / 463,247.

[0251] One advantage of including at least one control feature in a recombinant receptor is that cells expressing the recombinant receptor administered to a subject can be increased or depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing a recombinant receptor by using an antibody specific for the tag cassette, using a cognate binding molecule specific for the control feature, or by using a second modified cell expressing a recombinant receptor and having specificity for the control feature. Elimination of modified cells may be accomplished usingdepletion agents specific for a control feature. For example, if tEGFR is used, then an anti-tEGFR binding domain (e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal) may be used, or an anti-tEGFR / anti-CD3 bispecific scFv, or an anti-tEGFR CAR T cell may be used.

[0252] In certain embodiments, modified cells expressing a recombinant receptor or other chimeric molecule may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, iron-oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI-scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0253] Thus, modified cells expressing at least one control feature within a recombinant receptor can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and / or eliminated as compared to a modified cell without a tag cassette.

[0254] (VI) Genetically Modifying Cell Populations to Express Recombinant Proteins. Cell populations can be genetically modified.

[0255] In particular embodiments, an immune cell is genetically modified to express a recombinant receptor. In particular embodiments, the immune cell is a T cell. In particular embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) having an anti- CLEC2A binding domain.

[0256] In particular embodiments, a cell is genetically modified to knockout CLEC2A expression. In particular embodiments, the cell is a CLEC2A-expressing cell. In particular embodiments, the CLEC2A-expressing cell includes an OCI-AML2 cell.

[0257] In particular embodiments, a cell is genetically modified to overexpress CLEC2A. In particular embodiments, the cell is a non-CLEC2A expressing cell. In particular embodiments, the non-CLEC2A expressing cell includes an MV4;11 cell.

[0258] Cell types, sample collection, and genetic engineering techniques are further described in subheadings of this section.

[0259] (Vl-a) Cells. The present disclosure describes cells genetically modified to express a recombinant receptor or genetically modified to knockout or overexpress CLEC2A. Cells genetically modified to express a recombinant receptor can include T-cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e. , HSPC). In particular embodiments, genetically modified cells include T-cells. Cells genetically modified to knockout oroverexpress CLEC2A can include any cell useful for research purposes. For research on AML, leukemic cell lines, such as AML cell lines are of particular use. AML cell lines include 0CI-AML2, MV4;11 , M0LM16, CTS, UoC-M1, KG-1 , K-562, Kasumi-1 , Kasumi-6, SKNO-1 , HL-60, PLB-985, NB4, PL-21 , UF-1 , HT93, AP-1060, 0CI-AML3, MUTZ-11 , MUTZ-8, MUTZ-3, ME-1 , THP-1 , U- 937, MOLM-13, MV4-11 , HEL, 0CI-M1, 0CI-M2, F-36P, TF-1 , AS-E2, CMK, ELF-153, UT-7, M- 07, MEG-01 , or M EGAL.

[0260] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCR[3) genes and are called a- and |3-TCR chains.

[0261] y5 T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In y8 T-cells, the TCR is made up of one y-chain and one 5-chain. This group of T-cells is much less common (2% of total T-cells) than the cc|3 T-cells.

[0262] CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.

[0263] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0264] Cytotoxic T-cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0265] "Central memory" T-cells (or "TCM") as used herein refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.

[0266] "Effector memory" T-cell (or "TEM") as used herein refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.

[0267] "Naive" T-cells as used herein refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.

[0268] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells express CD8, CD16 and CD56 but do not express CD3.

[0269] NK cells include NK-T cells. NK-T cells are a specialized population of T cells that express a semi invariant T cell receptor (TCR ab) and surface antigens typically associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-related immunosurveillance or immunosuppression. Like natural killer cells, NK-T cells can also induce perforin-, Fas-, and TNF-related cytotoxicity. Activated NK-T cells are capable of producing IFN-y and IL-4. In particular embodiments, NK-T cells are CD3+ / CD56+.

[0270] Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components. Monocytes / macrophages express CD11 b, F4 / 80; CD68; CD11c; IL-4Ra; and / or CD163.

[0271] Immature dendritic cells (i.e. , pre-activation) engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells. Dendritic cells express CD1 a, CD1 b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.

[0272] Hematopoietic Stem / Progenitor Cells or HSPC refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0273] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to all other hematopoietic cell types.

[0274] A hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetaltissue that is capable of further differentiation into mature cell types. In certain embodiments, hematopoietic progenitor cells are CD24l0Lin- CD117+hematopoietic progenitor cells. HPC can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.

[0275] HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof. Also, the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. In particular embodiments, the HSPC are CD34+ cells.

[0276] OCI-AML2 cells are acute myeloid leukemia cells established from the peripheral blood of a 65-year-old man with acute myeloid leukemia (AML FAB M4) in 1986 at diagnosis. OCI-AML2 cells are described to express the retinoic acid receptor gene and carry the DNMT3A R635W mutation. This cell line expresses CLEC2A.

[0277] MV4; 11 are macrophages that were isolated from the blast cells of a 10-year-old male with biphenotypic B-myelomonocytic leukemia. This cell line does not express CLEC2A.

[0278] A statement that a cell or population of cells is "positive" for or expressing a particular marker refers to the detectable presence on or in the cell of the particular marker. When referring to a surface marker, the term can refer to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype- matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0279] A statement that a cell or population of cells is "negative" for a particular marker or lacks expression of a marker refers to the absence of substantial detectable presence on or in the cell of a particular marker. When referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a levelsubstantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0280] Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or allogeneic when appropriate and can also be in vivo or ex vivo. In particular embodiments, cells to be genetically modified include CD4+ or CD8+ T cells. In particular embodiments, cells to be genetically modified include OCI-AML2 cells or MV4;11 cells.

[0281] (Vl-b) Cell Sample Collection and Cell Enrichment. Methods of sample collection and enrichment are known by those skilled in the art. In some embodiments, cells are derived from cell lines. In particular embodiments, cells are derived from humans.

[0282] In some embodiments, cells are derived or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. In particular embodiments, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in particular embodiments, contain lymphocytes, including T cells, monocytes, macrophages, granulocytes, B cells, other nucleated white blood cells, HSC, HPC, HSPC, red blood cells, cancer cells (e.g., AML cells) and / or platelets, and in some aspects, contains cells other than red blood cells and platelets and further processing is necessary.

[0283] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In particular embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations.

[0284] Isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and / or simultaneously. In particular embodiments, the isolation, culture, and / or engineering of the different populations is carried out from the same starting material, such as from the same sample.

[0285] In particular embodiments, a sample can be enriched for cells by using density-based cellseparation methods and related methods. For example, white blood cells can be separated from other cell types in the peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.

[0286] In particular embodiments, a bulk cell population can be used that has not been enriched for a particular cell type. In particular embodiments, a selected cell type can be enriched for and / or isolated based on cell-marker based positive and / or negative selection.

[0287] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells but need not result in a complete removal of all such cells.

[0288] In some embodiments, an antibody or binding domain for a cellular marker is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ); see also US 4,452,773; US 4,795,698; US 5,200,084; and EP 452342.

[0289] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto.

[0290] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry.

[0291] Cell-markers for different cell subpopulations are described above. In particular embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0292] CD3+, CD28+ T cells can be positively selected for and expanded using anti-CD3 / anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0293] In particular embodiments, a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD8+ and CD4+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to arelatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0294] In some embodiments, enrichment for central memory T (TCM) cells is carried out. In particular embodiments, memory T cells are present in both CD62L subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L, CDS and / or CD62L+CD8+ fractions, such as by using anti-CD8 and anti-CD62L antibodies.

[0295] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CCR7, CD45RO, and / or CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L.

[0296] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or RORI, and positive selection based on a marker characteristic of central memory T cells, such as CCR7, CD45RO, and / or CD62L, where the positive and negative selections are carried out in either order.

[0297] In particular embodiments, PBMCs are isolated over Lymphoprep (StemCell Technologies, Cat# 07851). In particular embodiments CD4+ and / or CD8+ T cells are isolated from PBMCs using negative magnetic selection. In particular embodiments, negative magnetic selection includes using Easy Sep Human CD4+ T cell Isolation Kit II (StemCell Technologies, Cat # 17952) and Easy Sep Human CD8+ T cell Isolation Kit II (StemCell Technologies, Cat # 17953).

[0298] Other cell types can be enriched based on known marker profiles and techniques. For example, CD34+ HSC, HSP, and HSPC can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0299] (Vl-c) Genetic Engineering Techniques. Desired genes (e.g., recombinant receptors, antibodies, CLEC2A overexpression, or CLEC2A knockout) disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection,I i pofecti on, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, heritable and expressible by its cell progeny.

[0300] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that encodes a molecule of interest. This definition includes various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not substantially affect the function of the encoded molecule of interest. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the molecule of interest. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type (e.g., mammalian cells). Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.

[0301] Gene sequences encoding a molecule of interest can be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence.

[0302] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids.

[0303] Polynucleotide gene sequences encoding more than one portion of an expressed molecule can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acidsequence resulting in expression of the latter. A promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0304] In any of the embodiments described herein, a polynucleotide can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the CAR construct and a polynucleotide encoding a transduction marker (e.g., tCD19 or tEGFR). Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim ef a / . (PLOS One 6:e18556 (2011).

[0305] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids, cosmids, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0306] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 , and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0307] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0308] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses,adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes(Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch andKlotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med.75:267-282).

[0309] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including CAR transgenes are described in, e.g., US 8,119,772; Walchli, et al., 2011, PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Then 14:1155; Frecha, etal., 2010, Mol. Then 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0310] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839,US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661 , WO2014 / 093694, WO2014 / 093701 , WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726,WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351 ,WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462,WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, W02016205711, WO2017 / 106657, WO2017 / 127807 and applications related thereto. As an example, the CRISPR / Cas system can be used to knock-out expression of CLEC2A within a cell. A method of knocking out expression of CLEC2A could include introducing a Cas protein (e.g., Cas9 protein) and anti-CLEC2A sgRNA to the cell. The sgRNA is designed to be complementary to a target sequence within the coding region of the gene to be knocked out, in this case CLEC2A. Example sgRNA that can be used in the methods described herein include ATTAATCCAGAGCTGCGGGA (SEQ ID NO: 167),CAGTCCCCTGAACATGCCAC (SEQ ID NO: 168), TAAACCTGTGGCATGTTCAG (SEQ ID NO: 169), GCTCTGCCATCCCGCAGCTC (SEQ ID NO: 170), or ACGCAGGAACTGATATGCAC (SEQ ID NO: 171). Alternatively, a CLEC2A sgRNA CRISPR / Cas9 lentivector, such as Cat. No.16305111 from Applied Biological Materials, can be used.

[0311] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. For information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0312] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. For information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0313] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0314] Nanoparticles that result in selective in vivo genetic modification of targeted cell types have been described and can be used within the teachings of the current disclosure. In particular embodiments, the nanoparticles can be those described in WQ2014153114, WQ2017181110, and WQ201822672.

[0315] In particular embodiments, T cells are transduced with a lentivirus encoding recombinant receptor (e.g., CAR).

[0316] In particular embodiments, CRISPR / Cas9 and sgRNA are used to knockout CLEC2A expression in OCI-AML2 cells.

[0317] In particular embodiments, MV4;11 cells are transfected with a lentivirus with a CLEC2A plasmid.

[0318] (VII) Cell Activating Culture Conditions. Cell populations can be incubated in a cultureinitiating media to expand genetically modified cell populations. The incubation can be carried out in a culture vessel, such as a bag, cell culture plate, flask, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica- coated glass plate, tube, tubing set, well, vial, or other container for culture or cultivating cells.

[0319] Culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0320] In some aspects, incubation is carried out in accordance with techniques such as those described in US 6,040,1 77, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1 :72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0321] Exemplary culture media for culturing T cells include (i) RPMI supplemented with non- essential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) RPMI with HEPES, 5- 15% human serum, 1-3% L-Glutamine, 0.5-1.5% penicillin / streptomycin, and 0.25x10-4 - 0.75x10-4 M p-MercaptoEthanol; (iii) RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES, 100 U / ml penicillin and 100 m / mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 10mM HEPES, 100 U / ml penicillin and 100 m / mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax (Gibco), and 2% N-acetyl cysteine (Sigma-Aldrich, St. Louis, MO). T cell culture media are also commercially available from Hyclone (Logan, UT). Additional T cell activating components that can be added to such culture media are described in more detail below.

[0322] In some embodiments, the T cells are expanded by adding to the culture-initiating media feeder cells (e.g., such that the resulting population of cells contains at least 5, 10, 20, or 40 or more feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the numbers of T cells). In some aspects, the nondividing feeder cells can include gamma-irradiated feeder cells. In particular embodiments, a time sufficient to expand the numbers of T cells includes 24 hours. In particular embodiments, the ratioof T cells to feeder cells is 1 :1 , 2:1 , or 1 :2. In particular embodiments, the feeder cells include cells expressing CLEC2A. In particular embodiments, the feeder cells include cancer cells. In particular embodiments, the feeder cells include AML feeder cells.

[0323] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least 25°C, at least 30°C, or 37°C.

[0324] The activating culture conditions for T cells include conditions whereby T cells of the culture-initiating media proliferate or expand. T cell activating conditions can include one or more cytokines, for example, interleukin (IL)-2, IL-7, IL-15 and / or IL-21. IL-2 can be included at a range of 10 - 100 ng / ml (e.g., 40, 50, or 60 ng / ml). IL-7, IL-15, and / or IL-21 can be individually included at a range of 0.1 - 50 ng / ml (e.g., 5, 10, or 15 ng / ml).

[0325] In particular embodiments, T cell activating culture condition conditions can include T cell stimulating epitopes. T cell stimulating epitopes include CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1 BB (CD 137), B7-H3, CTLA-4, Frizzled- 1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1 R, LAT, LFA-1 , LIGHT, MHCI, MHCII, NKG2D, 0X40, ROR2 and RTK.

[0326] CD3 is a primary signal transduction element of T cell receptors. In particular embodiments, the CD3 stimulating molecule (i.e., CD3 binding domain) can be derived from the OKT3 antibody (see US 5,929,212; US 4,361 ,549; ATCC® CRL-8001 ™; and Arakawa et al., J. Biochem. 120, 657-662 (1996)), the 20G6-F3 antibody, the 4B4-D7 antibody, the 4E7-C9, or the 18F5-H10 antibody.

[0327] In particular embodiments, CD3 stimulating molecules can be included within culture media at a concentration of at least 0.25 or 0.5 ng / ml or at a concentration of 2.5 - 10 pg / ml. Particular embodiments utilize a CD3 stimulating molecule (e.g., OKT3) at 5 pg / ml.

[0328] An exemplary binding domain for CD28 can include or be derived from TGN1412, CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, and CD28.3 (deposited as a synthetic single chain Fv construct under GenBank Accession No. AF451974.1 ; see also Vanhove et al., BLOOD, 15 Jul. 2003, Vol. 102, No. 2, pages 564-570).

[0329] 4-1BB binding domains can be derived from LOB12, lgG2a, LOB12.3, or lgG1 as described in Taraban et al. Eur J Immunol. 2002 December; 32(12):3617-27. In particular embodiments a 4-1 BB binding domain is derived from a 4-1 BB binding domain described in US 9,382,328; US 6,569,997; US 6,303,121 ; and Mittler et al. Immunol Res. 2004; 29(1 -3): 197-208.

[0330] 0X40 (CD134) and / or ICOS activation may also be used. 0X40 binding domains are described in US20100196359, US 20150307617, WO 2015 / 153513, W02013 / 038191 andMelero et al. Clin Cancer Res. 2013 Mar. 1 ; 19(5): 1044-53. Exemplary binding domains that can bind and activate ICOS are described in e.g., US20080279851 and Deng et al. Hybrid Hybridomics. 2004 June; 23(3):176-82.

[0331] When in soluble form, T-cell activating agents can be coupled with another molecule, such as polyethylene glycol (PEG) molecule.

[0332] In particular embodiments, a solid phase can be added to a culture media. Such solid phases can include, for example, beads, hollow fibers, resins, membranes, and polymers.

[0333] Exemplary beads include magnetic beads, polymeric beads, and resin beads (e.g., Strep- Tactin® Sepharose, Strep-Tactin® Superflow, and Strep-Tactin® MacroPrep I BA GmbH, Gottingen)). Anti-CD3 / anti-CD28 beads are commercially available reagents for T cell expansion (Invitrogen).

[0334] In particular embodiments, T cells are activated with anti-CD3 / CD28 beads (3:1 beads: cell, Gibco, 11131 D) on Retronectin-coated plates. In particular embodiments, CAR T cells are sorted with CD19 microbeads 8 to 10 days post activation. In particular embodiments, sorted cells are further expanded in CTL (+50 U / mL IL-2) media.

[0335] Culture conditions for HSC / HSP can include expansion with a Notch agonist (see, e.g., US 7,399,633; US 5,780,300; US 5,648,464; US 5,849,869; and US 5,856,441 and growth factors present in the culture condition as follows: 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6 and 10 ng / ml IL-3. In more specific embodiments, 50, 100, or 200 ng / ml SCF; 50, 100, or 200 ng / ml of Flt-3L; 50 or 100 ng / ml TPO; 50 or 100 ng / ml IL-6; and 10 ng / ml IL-3 can be used.

[0336] (VIII) Compositions and Formulations for Administration. Any of the binding domains described herein (e.g., antibodies, multi-domain binding molecules, or antibody conjugates) in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the antibodies can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within nanoparticles (e.g., liposomes or polymer-based nanoparticles) and / or as part of a vector delivery system (e.g., a viral vector or plasmid). Binding domains (e.g., antibodies, multi-domain binding molecules, antibody conjugates) and / or nucleic acids encoding antibodies are collectively referred to herein as “active ingredients”. Certain examples may include formulations. Formulations include cells genetically modified to express a binding domain disclosed herein (e.g., as in a CAR) within a pharmaceutically-acceptable carrier.

[0337] Salts and / or pro-drugs of the active ingredients can also be used.

[0338] A pharmaceutically-acceptable salt includes any salt that retains the activity of the activeingredient and is acceptable for pharmaceutical use. A pharmaceutically-acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0339] Suitable pharmaceutically-acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0340] Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine, arginine and procaine.

[0341] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.

[0342] Exemplary generally used pharmaceutically-acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0343] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0344] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0345] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0346] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0347] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0348] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0349] The compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The formulations and / or compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration.

[0350] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0351] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredient and a suitable powder base such as lactose or starch.

[0352] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to over 100 days. In particularembodiments, the compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0353] In certain examples, cells are genetically modified to express a binding domain (e.g., antibody) disclosed herein, for example, as part of an antibody or fragment thereof (e.g., as part of a CAR or eTCR). In these embodiments, genetically modified cells can be prepared as formulations for delivery in buffers such as Hanks' solution, Ringer's solution, or physiological saline. Cells can be genetically modified using methods known in the art. Exemplary targeted genetic engineering approaches include the use of CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), and / or transcription activator like effector nucleases (TALENs). In particular embodiments, the cells are B cells genetically modified to express an antibody. Methods to genetically modify a B cell to express an antibody are described in PCT / US2018 / 056789.

[0354] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107 cells, greater than 108 cells, greater than 109 cells, greater than 1010 cells, or greater than 1011 cells.

[0355] In particular embodiments, cells are in a formulation volume of a liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Hence, the density of administered cells is typically greater than 104 cells / ml, 105 cells / ml, 106 cells / ml, 107 cells / ml, or 108 cells / ml.

[0356] In certain examples, compositions include a secondary treatment. Examples of secondary treatments are described elsewhere herein.

[0357] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically-acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically-acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions and formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0358] (IX) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens)and research animals (e.g., monkeys, rats, mice, fish) with compositions and / or formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0359] An “effective amount” is the amount of a composition or formulation necessary to result in a desired physiological change in a subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model, clinical study, or clinical study relevant to the assessment of a condition’s development (e.g., cancer’s development), progression, and / or resolution, as well as the effects of the condition. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0360] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer to be treated or displays only early signs or symptoms of the cancer to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the cancer. Thus, a prophylactic treatment functions as a preventative treatment against a cancer. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a cancer.

[0361] "Immune response" refers to a response of the immune system to produce to neutralize and / or destroy. In particular embodiments, an immune response can be an innate and / or adaptive response.

[0362] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a cancer and is administered to the subject for the purpose of reducing the severity or progression of the cancer. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer and / or reduce, control, or eliminate side effects of the cancer.

[0363] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0364] In particular embodiments, therapeutically effective amounts provide anti-cancer effects. Anti-cancer effects include a decrease in the number of cancer cells, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited cancer cell proliferation, prolonged subject life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0365] In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against a cancer cell.

[0366] In particular embodiments, the cancer includes a solid cancer or a blood cancer. In particular embodiments, the cancer includes any cancer with CLEC2A expression. In particular embodiments, the cancer includes leukemia. In particular embodiments, the leukemia is AML. In particular embodiments, the AML is KMT2A-r AML. In particular embodiments, the cancer cell is a KMT2A-r AML cell, expressing CLEC2A.

[0367] Formulations and / or compositions disclosed herein can also be used to treat a complication or disease related to KMT2A-r AML. For example, complications relating to AML may include a preceding myelodysplastic syndrome (MDS, formerly known as “preleukemia”), secondary leukemia, in particular secondary AML, high white blood cell count, and absence of Auer rods. Among others, leukostasis and involvement of the central nervous system (CNS), hyperleukocytosis, residual disease, are also considered complications or diseases related to AML.

[0368] In particular embodiments, a method of treating KMT2A-r AML in a subject includes administering a therapeutically effective amount of a CLEC2A targeted therapeutic to the subject, thereby treating the subject. In particular embodiments, the CLEC2A targeted therapeutic includes an antibody, a multi-domain binding molecule, a conjugate (i.e. , antibody conjugate), or a recombinant receptor expressed by a cell. In particular embodiments, the CLEC2A targeted therapeutic includes an anti-CLEC2A binding domain. In particular embodiments, the anti- CLEC2A binding domain is derived from an anti-CLEC2A antibody or includes the binding domains disclosed herein.

[0369] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC50 as determined in cell culture against a particular target. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0370] Useful doses of active ingredients within compositions range from, for example, 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg,0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0371] Exemplary doses of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0372] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0373] The compositions and formulations described herein can be administered by, for example, injection, infusion, perfusion, or lavage. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intrathecal, intratumoral, intramuscular, subcutaneous, and / or sublingual administration.

[0374] The compositions and / or formulations described herein can be administered on top of the current standard of care for AML patients, or in combination or alternation with any other compound or therapy that the healthcare provider deems beneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative. In particular embodiments, the compositions and / or formulations described herein can be administered with a secondary treatment. In particular embodiments, the compositions and / or formulations described herein can be administered at the same time or at a different time as a secondary treatment.

[0375] In certain embodiments, formulations and / or compositions are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities (e.g., secondary treatments). In particular embodiments, secondary treatments include chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.

[0376] Additional embodiments provide a CLEC2A knock-out model or a CLEC2A overexpression model. These models can be used for in vitro and in vivo testing alongside their respective parental cell lines. In particular embodiments, a CLEC2A knock-out model can be generated using genetic engineering techniques described herein to knockout CLEC2A expression in cells. In particular embodiments, genetic engineering techniques include using sgRNA and CRISPR / Cas9 technology in cells. In particular embodiments, the cells are CLEC2A- expressing cells. In particular embodiments, the CLEC2A-expressing cells include OCI-AML2 cells.

[0377] In particular embodiments, an overexpression model of CLEC2A can be generated by using genetic engineering techniques described herein to clone a CLEC2A vector into cells. In particular embodiments, the CLEC2A vector includes a lentivirus including CLEC2A plasmid. In particular embodiments, the cells include non-CLEC2A-expressing cells. In particular embodiments, the non-CLEC2A-expressing cells include MV4;11 cells. In particular embodiments, non-CLEC2A-expressing cells are transfected with a lentivirus with a CLEC2A plasmid to create a CLEC2A overexpression model.

[0378] These cell-based models can be used for in vitro and in vivo testing with CLEC2A antibodies and immunotherapeutic treatments. To generate an in vivo patient-derived xenograft (PDX) model, CLEC2A+ AML cells can be genetically modified to express a genetic construct and injected into an animal model, thereby creating a PDX model. As an example, a PDX model can be manufactured by obtaining CLEC2A+ validated primary patient AML cells, transducing the cells to express luciferase / GFP to allow for in vivo leukemic monitoring by bioluminescence imaging, and injecting the cells into an animal model (e.g., NSG mice) to establish a PDX model for therapeutic testing.

[0379] (X) Kits. The current disclosure also includes kits. Kits can include various components to practice methods disclosed herein. For example, depending on the aspect of the methods practiced, kits could include one or more of binding domains, multi-domain binding molecules, antibody conjugates, recombinant receptors, or nucleic acids encoding binding domains, multidomain binding molecules, antibody conjugates, or recombinant receptors (e.g., CAR); CLEC2A targeted therapeutics; nucleic acids encoding a self-cleaving peptide; lentiviral genetic construct; a nucleic acid encoding an scFv; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding EGFRt; a nucleic acid encoding CD19t; cells (e.g., immune cells, T-cells, CD4 T cells, CD8 T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC), untransduced T cells,anti-CLEC2A CAR T cells, CLEC2A ko cells, cells overexpressing CLEC2A); cell lines (e.g., OCI- AML2 cells, MV4;11 cells, CLEC2A-expressing cells, non-CLEC2A-expressing cells); engineered cells (e.g., CLEC2A knockout cells, CLEC2A overexpression cells); tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom); genetic expression components (e.g., genes for expression provided by vectors (e.g., lentiviral vector, retroviral vector), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or nanoparticles); cell formulation or activation components (e.g., saline, buffered saline, phosphate buffered saline (PBS); biocompatible buffers such as, Ca++ / Mg++ free PBS; physiological saline, water, Hanks' solution, Ringer's solution, T cell stimulating epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads; OKT3, TGN1412), culture-initiating compositions, RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, non-dividing EBV-transformed lymphoblastoid cells (LCL), IL-21 , human serum albumin (HSA) or other human serum components orfetai bovine serum, dextrose, stabilizers, preservatives); combination therapy components (e.g., local anesthetics, chemotherapeutic agents, immunosuppressive agents, anti-inflammatory agents); PCR amplification sequences; cytokines (e.g., IL-2, IL-7, IL-15, IL-21); culture vessels; reference levels; animal models (CLEC2A knockout animal model, CLEC2A overexpression model, PDX animal model); primer pairs; GAPDH; IFN-y enzyme-linked immunosorbent assay (ELISA); culture plates; etc.

[0380] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0381] (XI) Exemplary Embodiments.1. A binding domain that binds CLEC2A, the binding domain including a heavy chain variable domain including a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3, wherein: the CDRH1 includes a sequence as set forth in SEQ ID NO: 7, the CDRH2 includes a sequence as set forth in SEQ ID NO: 8, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 9, according to North; the CDRH1 includes a sequence as set forth in SEQ ID NO: 10, the CDRH2 includes a sequence as set forth in SEQ ID NO: 11 , and the CDRH3 includes a sequence as set forthin SEQ ID NO: 12, according to Kabat; the CDRH1 includes a sequence as set forth in SEQ ID NO: 13, the CDRH2 includes a sequence as set forth in SEQ ID NO: 14, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 9, according to IMGT; the CDRH1 includes a sequence as set forth in SEQ ID NO: 15, the CDRH2 includes a sequence as set forth in SEQ ID NO: 16, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 12, according to Chothia; the CDRH1 includes a sequence as set forth in SEQ ID NO: 17, the CDRH2 includes a sequence as set forth in SEQ ID NO: 18, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 19, according to Contact; the CDRH1 includes a sequence as set forth in SEQ ID NO: 20, the CDRH2 includes a sequence as set forth in SEQ ID NO: 21 , and the CDRH3 includes a sequence as set forth in SEQ ID NO: 22, according to North; the CDRH1 includes a sequence as set forth in SEQ ID NO: 23, the CDRH2 includes a sequence as set forth in SEQ ID NO: 24, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 25, according to Kabat; the CDRH1 includes a sequence as set forth in SEQ ID NO: 26, the CDRH2 includes a sequence as set forth in SEQ ID NO: 27, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 22, according to IMGT; the CDRH1 includes a sequence as set forth in SEQ ID NO: 28, the CDRH2 includes a sequence as set forth in SEQ ID NO: 29, and the CDRH3 includes a sequence as set forth in SEQ ID NO: 25, according to Chothia; or the CDRH1 includes a sequence as set forth in SEQ ID NO: 30, the CDRH2 includes a sequence as set forth in SEQ ID NO: 31 , and the CDRH3 includes a sequence as set forth in SEQ ID NO: 32, according to Contact. The binding domain of embodiment 1 , wherein the heavy chain variable domain has at least 90% sequence identity to a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3. The binding domain of embodiments 1 or 2, wherein the heavy chain variable domain has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3. The binding domain of any of embodiments 1-3, wherein the heavy chain variable domain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4. The binding domain of any of embodiments 1-4, wherein the heavy chain variable domain is encoded by a sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.The binding domain of any of embodiments 1-5, wherein the binding domain is a VH domain antibody. A nucleic acid encoding the binding domain of any of embodiments 1-6. A multi-domain binding molecule including at least two binding domains, wherein at least one binding domain of the at least two binding domains includes the binding domain of any of embodiments 1-6. The multi-domain binding molecule of embodiment 8, wherein the multi-domain binding molecule includes an immune cell engaging molecule. The multi-domain binding molecule of embodiment 9, wherein the immune cell engaging molecule activates an immune cell. The multi-domain binding molecule of embodiment 10, wherein the immune cell includes B cell, T cell, natural killer (NK) cell, or macrophage. The multi-domain binding molecule of embodiment 11 , wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell. The multi-domain binding molecule of any of embodiments 9-12, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Tolllike receptors 1-9, IL-4Ra, or MARCO. The multi-domain binding molecule of any of embodiments 9-13, wherein a binding domain of the immune cell engaging molecule binds CD3 or CD28. The multi-domain binding molecule of any of embodiments 9-13, wherein a binding domain of the immune cell engaging molecule binds CD16. The multi-domain binding molecule of any of embodiments 8-15, wherein the at least two binding domains include at least two copies of the binding domain of any of embodiments 1- 6. The multi-domain binding molecule of embodiment 16, wherein the at least two copies are joined by a protein linker. The multi-domain binding molecule of embodiment 17, wherein the protein linker is a Gly-Ser linker. The multi-domain binding molecule of embodiment 18, wherein the Gly-Ser linker is (GlyxSery)nwherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.The multi-domain binding molecule of any of embodiments 8-19, including 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the binding domain of any of embodiments 1-6. The multi-domain binding molecule of any of embodiments 8-20, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer. The multi-domain binding molecule of any of embodiments 16-21 , wherein the at least two copies are linked to an Fc region of an antibody. A single-chain variable fragment (scFv) including the binding domain of any of embodiments 1-6. The scFv of embodiment 23, further including a linker. The scFv of embodiment 24, wherein the linker includes a Gly-Ser linker. The scFv of embodiment 25, wherein the Gly-Ser linker includes SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. The scFv of embodiment 24, wherein the linker includes a Whitlow linker. A conjugate including the binding domain of embodiment 1-6 linked to a toxin, a drug, a detectable label, a radioisotope, or a particle. The conjugate of embodiment 28, wherein the binding domain includes an scFv. The conjugate of embodiments 28 or 29, wherein the toxin includes a plant toxin or bacterial toxin. The conjugate of embodiment 30, wherein the plant toxin includes saporin, ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, Bryodin 1, bouganin, or gelonin. The conjugate of embodiment 30, wherein the bacterial toxin includes diphtheria toxin or Pseudomonas exotoxin. The conjugate of any of embodiments 28-32, wherein the toxin includes a holotoxin or a hemitoxin. The conjugate of embodiment 28, wherein the drug includes calicheamicin, actinomycin D, anthracycline, auristatin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1- dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, or stereoisomers, isosteres, analogs, or derivatives thereof.The conjugate of embodiments 28 or 34, wherein the drug includes calicheamicin. The conjugate of embodiment 28, wherein the detectable label includes a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, or an affinity tag. The conjugate of embodiment 28, wherein the radioisotope includes228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,57Cu, 153Dy, 157Dy 1590y 1650y 1660y 171Er250Es254Es, 147Eu157Eu52pe59Ee, 251 pm252F m, 253pm ,66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174ml_u,175mLu,257Md,250Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr. The conjugate of embodiments 28 or 37, wherein the radioisotope does not emit daughter radionuclides. The conjugate of embodiment 28, wherein the particle includes a metal nanoparticle, a liposome, or a polymer nanoparticle. A recombinant receptor that, when expressed by a cell, includes an extracellular component including the binding domain of any of embodiments 1-6 or the scFv of any of embodiments 23-27. The recombinant receptor of embodiment 40, wherein the recombinant receptor includes a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR). The recombinant receptor of embodiment 41 , wherein the CAR includes an intracellular component linked to the extracellular component by a transmembrane domain. The recombinant receptor of embodiment 42, wherein the intracellular component includes an effector domain including: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH 1 , 0X40, ROR2, SLAMF1 , TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof. The recombinant receptor of embodiment 43, wherein the effector domain includes all or a portion of a signaling domain of CD3 and all or a portion of a signaling domain of 4-1 BB. The recombinant receptor of embodiment 44, wherein the CD3 signaling domain is encoded by a sequence as set forth in SEQ ID NO: 178 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 178. The recombinant receptor of embodiments 44 or 45, wherein the CD3£ signaling domain includes a sequence as set forth in SEQ ID NO: 179 or SEQ ID NO: 180 or a sequence havingat least 90% sequence identity to the sequence as set forth in SEQ ID NO: 179 or SEQ ID NO: 180. The recombinant receptor of any of embodiments 44-46, wherein the 4-1 BB signaling domain is encoded by a sequence as set forth in SEQ ID NO: 181 or SEQ ID NO: 182 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 181 or SEQ ID NO: 182. The recombinant receptor of any of embodiments 44-47, wherein the 4-1 BB signaling domain includes a sequence as set forth in SEQ ID NO: 183 or SEQ ID NO: 184 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 183 or SEQ ID NO: 184. The recombinant receptor of any of embodiments 42-48, wherein the transmembrane domain includes a transmembrane region of: an a, p or £ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof. The recombinant receptor of any of embodiments 42-49, wherein the transmembrane domain includes a CD28 transmembrane domain. The recombinant receptor of embodiment 50, wherein the CD28 transmembrane domain is encoded by SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187. The recombinant receptor of embodiments 50 or 51 , wherein the CD28 transmembrane domain includes a sequence as set forth in SEQ I D NO: 188 or SEQ ID NO: 189 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 188 or SEQ ID NO: 189. The recombinant receptor of any of embodiments 41-52, wherein the CAR further includes a spacer region. The recombinant receptor of embodiment 53, wherein the spacer region includes an intermediate spacer region, long spacer region, or short spacer region. The recombinant receptor of embodiment 54, wherein the intermediate spacer region is 135 amino acids or less. The recombinant receptor of embodiments 54 or 55, wherein the intermediate spacer region is 131 amino acids or less and includes a hinge region and a CH3 domain of lgG4. The recombinant receptor of any of embodiments 54-56, wherein the intermediate spacer region is encoded by a sequence as set forth in SEQ ID NO: 175 or a sequence having atleast 90% sequence identity to the sequence as set forth in SEQ ID NO: 175. The recombinant receptor of any of embodiments 54-56, wherein the intermediate spacer region is encoded by a sequence as set forth in SEQ ID NO: 176 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 176. The recombinant receptor of embodiment 54, wherein the long spacer region is greater than 200 amino acids and includes an lgG4 hinge, an lgG4 CH3 region, and an lgG4 CH2 region. The recombinant receptor of embodiments 54 or 59, wherein the long spacer region is encoded by a sequence as set forth in SEQ ID NO: 177 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 177. The recombinant receptor of embodiment 54, wherein the short spacer region is less than 50 amino acids and includes an I gG4 hinge. The recombinant receptor of embodiments 54 or 61 , wherein the short spacer region is encoded by a sequence as set forth in SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174. The recombinant receptor of any of embodiments 40-62, further including a control feature selected from a tag cassette, a transduction marker, and a suicide switch. The recombinant receptor of embodiment 63, wherein the transduction marker includes a truncated CD 19. The recombinant receptor of embodiment 64, wherein the truncated CD19 is encoded by SEQ ID NO: 195 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 195. The recombinant receptor of any of embodiments 40-65, further including a ribosomal skip element. The recombinant receptor of embodiment 66, wherein the ribosomal skip element includes T2A, P2A, E2A, or F2A. The recombinant receptor of embodiments 66 or 67, wherein the ribosomal skip element includes T2A. The recombinant receptor of any of embodiments 67-68, wherein T2A is encoded by SEQ ID NO: 190 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190. The recombinant receptor of embodiment 41 , wherein the eTCR includes a constant alpha domain (Ca) and a constant beta domain (Cp). The recombinant receptor of embodiment 70, wherein the scFv of any of embodiments 23-27is linked to the Cadomain and / or the Cp domain. A genetic construct encoding the binding domain of any of embodiments 1-6 or the recombinant receptor of any of embodiments 40-71. The genetic construct of embodiment 72, including a sequence as set forth in SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, or SEQ ID NO: 223 or has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, or SEQ ID NO: 223. A nanoparticle encapsulating the genetic construct of embodiments 72 or 73. A cell genetically modified to express the binding domain of any of embodiments 1-6 or the recombinant receptor of any of embodiments 40-71. The cell of embodiment 75, wherein the cell is an immune cell. The cell of embodiment 76, wherein the immune cell is a T cell, B cell, natural killer (NK) cell, NK-T cell, monocyte / macrophage, hematopoietic stem cells (HSC), or a hematopoietic progenitor cell (HPC). The cell of any of embodiments 75-77, wherein the cell is a T cell selected from a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell. The cell of any of embodiments 75-78, wherein the cell is a CD8+ T cell and / or a CD4+ T cell. The cell of any of embodiments 75-79, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject. The cell of any of embodiments 75-80, wherein the cell is in vivo or ex vivo. A composition including the binding domain of any of embodiments 1-6, the multi-domain binding molecule of any of embodiments 8-22, the scFv of any of embodiments 23-27, the conjugate of any of embodiments 28-39, the genetic construct of any of embodiments 72-73, or the nanoparticle of embodiment 74; and a pharmaceutically-acceptable carrier. A formulation including the cell of any of embodiments 75-81 and a pharmaceutically- acceptable carrier. The formulation of embodiment 83, wherein the cells are T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, hematopoietic stem cells (HSC), or hematopoietic progenitor cells. The formulation of embodiment 84, wherein the T cells are selected from CD3+ T cells, CD4+T cells, CD8+ T cells, central memory T cells, effector memory T cells, and / or naive T cells. The formulation of embodiments 84 or 85, wherein the T cells are CD8+ T cells and / or CD4+ T cells. A method of treating a subject in need thereof including administering a therapeutically effective amount of the composition of embodiment 82 or the formulation of any of embodiments 83-86 to the subject thereby treating the subject in need thereof. The method of embodiment 87, wherein the subject in need thereof has cancer. The method of embodiment 88, wherein the cancer includes cancer cells expressing CLEC2A. The method of embodiments 88 or 89, wherein the cancer includes leukemia. The method of embodiment 90, wherein the leukemia is acute myeloid leukemia (AML). The method of embodiment 91 , wherein the AML includes KMT2A-r AML. The method of any of embodiments 87-92, wherein the administering includes intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intrarectal, intrathecal, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration. A method of treating KMT2A-r AML in a subject including administering a therapeutically effective amount of a CLEC2A-targeted therapeutic, wherein the CLEC2A-targeted therapeutic includes an anti-CLEC2A binding domain, thereby treating the subject. The method of embodiment 94, wherein the anti-CLEC2A binding domain includes the binding domain of any of embodiments 1-6. The method of embodiments 94 or 95, wherein the anti-CLEC2A binding domain is derived from an anti-CLEC2A antibody. The method of embodiment 96, wherein the anti-CLEC2A antibody includes MA5-28532 or MAB 72191. The method of any of embodiments 94-97, wherein the CLEC2A-targeted therapeutic includes a multi-domain binding molecule, an antibody conjugate, or a recombinant receptorexpressing immune cell. The method of embodiment 98, wherein the multi-domain binding molecule includes an anti- CLEC2A binding domain linked to a binding domain that binds an immune cell activating epitope. . The method of embodiment 99, wherein the immune cell activating epitope includes CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.. The method of embodiments 99 or 100, wherein the immune cell activating epitopeincludes CD3. . The method of embodiments 99 or 100, wherein the immune cell activating epitope includes CD28. . The method of embodiments 99 or 100, wherein the immune cell activating epitope includes CD16. . The method of any of embodiments 98-103, wherein the multi-domain binding molecule includes the multi-domain binding molecule of any of embodiments 8-22. . The method of embodiment 98, wherein the antibody conjugate includes an anti-CLEC2A binding domain linked to a toxin, a drug, or a radioisotope. . The method of embodiment 105, wherein the toxin includes a holotoxin or a hemitoxin.. The method of embodiment 105, wherein the drug includes actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1- dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, or stereoisomers, isosteres, analogs, or derivatives thereof. . The method of embodiment 105, wherein the drug includes calicheamicin. . The method of embodiment 105, wherein the radioisotope includes228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,2S2Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn, "Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr. . The method of any of embodiments 98-109, wherein the antibody conjugate includes the conjugate of any of embodiments 28-39. . The method of embodiment 98, wherein the recombinant receptor-expressing immune cell includes a recombinant receptor that, when expressed by an immune cell, includes an extracellular component including the anti-CLEC2A binding domain. . The method of embodiment 1 11 , wherein the recombinant receptor includes a CAR.. The method of embodiment 112, wherein the CAR includes an intracellular component linked to the extracellular component by a transmembrane domain. . The method of embodiment 113, wherein the intracellular component includes an effector domain including: 4-1BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, l_AG3, NKG2D, NOTCH1 , 0X40, ROR2, SLAMF1 , TCRa, TCR0, TRIM, Wnt, Zap70, or a combination thereof. . The method of embodiment 114, wherein the effector domain includes all or a portion of a signaling domain of CD3£ and all or a portion of a signaling domain of 4-1 BB. . The method of any of embodiments 113-115, wherein the transmembrane domain includes a transmembrane region of: an a, [3 or £ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof. . The method of any of embodiments 113-116, wherein the transmembrane domain includes a CD28 transmembrane domain. . The method of any of embodiments 111-117, wherein the recombinant receptor includes the recombinant receptor of any of embodiments 40-71. . The method of any of embodiments 111-118, wherein the immune cell includes a T cell.. The method of embodiment 119, wherein the T cell includes a CD8+ T cell or a CD4+ T cell. . A method of manufacturing a CLEC2A knock-out model including introducing a CLEC2A sgRNA and a Cas9 protein into a cell. . The method of embodiment 121 , wherein the cell includes a CLEC2A-expressing cell.. The method of embodiment 122, wherein the CLEC2A-expressing cell includes an OCI- AML2 cell. . The method of any of embodiments 121-123, wherein the introducing includes electroporating. . The method of any of embodiments 121-124, wherein the CLEC2A sgRNA is complementary to a sequence within a coding region of CLEC2A. . A CLEC2A knock-out model includes a cell genetically modified to lack CLEC2A expression. . The CLEC2A knock-out model of embodiment 126, manufactured according to the method of any of embodiments 121-125. . A method of manufacturing a CLEC2A overexpression model including introducing aCLEC2A vector into a cell.. The method of embodiment 128, wherein the cell includes a non-CLEC2A-expressing cell.. The method of embodiment 129, wherein the non-CLEC2A-expressing cell includes an MV4;11 cell. . The method of any of embodiments 128-130, wherein the CLEC2A vector includes a lentivirus including a CLEC2A plasmid. . A CLEC2A overexpression model includes a cell genetically modified to have increased CLEC2A expression compared to a same type of cell found in nature. . The CLEC2A overexpression model of embodiment 132, manufactured according to the method of any of embodiments 128-131. . A method of manufacturing a patient-derived xenograft (PDX) model including: obtaining CLEC2A+ AML cells; and injecting CLEC2A+ A L cells into an animal. . The method of embodiment 134, wherein the CLEC2A+ AML cells are patient derived.. The method of embodiments 134 or 135, further including transducing CLEC2A+ AML cells with a genetic construct. . The method of embodiment 136, wherein the genetic construct expresses a fluorescent label. . The method of embodiment 137, wherein the fluorescent label includes luciferase and / or GFP. . The method of any of embodiments 134-138, wherein the animal includes a mouse.. The method of embodiment 139, wherein the mouse includes an NSG mouse. . An patient-derived xenograft (PDX) model includes an animal injected with CLEC2A+ AML cells. . The PDX model of embodiment 141 , wherein the animal includes a mouse. . The PDX model of embodiment 142, wherein the mouse includes an NSG mouse. . The PDX model of any of embodiments 141-143, wherein the CLEC2A+ AML cells are genetically modified to express a genetic construct. . The PDX model of embodiment 144, wherein the genetic construct includes a fluorescent label. . The PDX model of embodiment 145, wherein the fluorescent label includes luciferase and / or GFP. . A kit including the binding domain of any of embodiments 1-6, the multi-domain binding molecule of any of embodiments 8-22, the scFv of any of embodiments 23-27, the conjugate of any of embodiments 28-39, the recombinant receptor of any of embodiments 40-71 , thegenetic construct of embodiments 72 or 73, the nanoparticle of embodiment 74, the cell of any of embodiments 75-81 , the composition of embodiment 82, the formulation of any of embodiments 83-86, the CLEC2A knock-out model of embodiments 126 or 127, the CLEC2A overexpression model of embodiments 132 or 133, or the PDX model of any of embodiments 141-146.

[0382] (XII) Experimental Example. KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. This study interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. The study describes a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. The study evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity.

[0383] Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from recent COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary next generation sequencing (NGS) to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) >1. Although, CLEC2A was detected in only 18% of AML patients (252 / 1486; 17% pediatric and 90 / 378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, FIG. 3A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), FIG. 3B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL.

[0384] The study further evaluated a potential causal link between KMT2A fusions and CLEC2Aexpression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. AutoCUT&RUN profiling of genome-wide occupancy of KMT2A oncoproteins was used to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). The study found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression.

[0385] The study further evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells and target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI- AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate.

[0386] Clinical outcomes for patients with and without CLEC2A expression was evaluated Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p<0.0001 and OS 38% vs. 63%, p<0.0001 for patients with and without CLEC2A expression respectively). Given high rates of co-occurrence with KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), clinical outcomes within the KMT2A-r cohort were evaluated as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039).

[0387] This example describes CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

[0388] (XIII) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1 , 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0389] To the extent not explicitly provided herein, coding sequences for proteins disclosed herein and protein sequences for coding sequences disclosed herein can be readily derived from one of ordinary skill in the art.

[0390] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0391] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0392] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0393] It is known in the art that certain amino acids may be substituted by other amino acidshaving a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0394] As detailed in US 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5+1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0395] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistical ly-significant degree.

[0396] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0397] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer(Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, H I- 20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0398] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 g / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0399] "Binds" refers to an association of a binding domain (of, for example, an anti-CLEC2A binding domain) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds” is also referred to as “binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107M-1, at least 108M-1, at least 109M’1, at least 1010M-1, at least 1011M-1, at least 1012M’1, or at least 1013M’1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107M’1, up to 106M’1, up to 105M’1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10’5M to 10’13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and US 5,283,173, US 5,468,614, or the equivalent).

[0400] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0401] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient orcomponent. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in binding between a disclosed binding domain and its epitope or a statistically significant reduction in the ability to treat cancer, as described herein.

[0402] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0403] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0404] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0405] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0406] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0407] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0408] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0409] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0410] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMSWhat is claimed is:

1. A binding domain that binds CLEC2A, the binding domain comprising a heavy chain variable domain comprising a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3, wherein: the CDRH1 comprises a sequence as set forth in SEQ ID NO: 7, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 8, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 9, according to North; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 10, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 11 , and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 12, according to Kabat; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 13, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 14, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 9, according to IMGT; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 15, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 16, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 12, according to Chothia; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 17, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 18, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 19, according to Contact; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 20, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 21 , and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 22, according to North; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 23, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 24, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 25, according to Kabat; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 26, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 27, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 22, according to IMGT; the CDRH1 comprises a sequence as set forth in SEQ ID NO: 28, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 29, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 25, according to Chothia; or the CDRH1 comprises a sequence as set forth in SEQ ID NO: 30, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 31 , and the CDRH3 comprises a sequenceas set forth in SEQ ID NO: 32, according to Contact.

2. The binding domain of claim 1 , wherein the heavy chain variable domain has at least 90% sequence identity to a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

3. The binding domain of claim 1 , wherein the heavy chain variable domain has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

4. The binding domain of claim 1 , wherein the heavy chain variable domain is encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

5. The binding domain of claim 1 , wherein the heavy chain variable domain is encoded by a sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

6. The binding domain of claim 1, wherein the binding domain is a VH domain antibody.

7. A nucleic acid encoding the binding domain of claim 1 .

8. A multi-domain binding molecule comprising at least two binding domains, wherein at least one binding domain of the at least two binding domains comprises the binding domain of claim 1.

9. The multi-domain binding molecule of claim 8, wherein the multi-domain binding molecule comprises an immune cell engaging molecule.

10. The multi-domain binding molecule of claim 9, wherein the immune cell engaging molecule activates an immune cell.11 . The multi-domain binding molecule of claim 10, wherein the immune cell comprises B cell, T cell, natural killer (NK) cell, or macrophage.

12. The multi-domain binding molecule of claim 11 , wherein the T cell is a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.

13. The multi-domain binding molecule of claim 9, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.

14. The multi-domain binding molecule of claim 9, wherein a binding domain of the immune cell engaging molecule binds CD3 or CD28.

15. The multi-domain binding molecule of claim 9, wherein a binding domain of the immune cell engaging molecule binds CD16.

16. The multi-domain binding molecule of claim 8, wherein the at least two binding domains comprise at least two copies of the binding domain of claim 1.

17. The multi-domain binding molecule of claim 16, wherein the at least two copies are joined by a protein linker.

18. The multi-domain binding molecule of claim 17, wherein the protein linker is a Gly-Ser linker.

19. The multi-domain binding molecule of claim 18, wherein the Gly-Ser linker is (GlyxSery)n wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

20. The multi-domain binding molecule of claim 8, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the binding domain of claim 1 .

21. The multi-domain binding molecule of claim 8, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.

22. The multi-domain binding molecule of claim 16, wherein the at least two copies are linked to an Fc region of an antibody.

23. A single-chain variable fragment (scFv) comprising the binding domain of claim 1.

24. The scFv of claim 23, further comprising a linker.

25. The scFv of claim 24, wherein the linker comprises a Gly-Ser linker.

26. The scFv of claim 25, wherein the Gly-Ser linker comprises SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55.

27. The scFv of claim 24, wherein the linker comprises a Whitlow linker.

28. A conjugate comprising the binding domain of claim 1 linked to a toxin, a drug, a detectable label, a radioisotope, or a particle.

29. The conjugate of claim 28, wherein the binding domain comprises an scFv.

30. The conjugate of claim 28, wherein the toxin comprises a plant toxin or bacterial toxin.

31. The conjugate of claim 30, wherein the plant toxin comprises saporin, ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, Bryodin 1 , bouganin, or gelonin.

32. The conjugate of claim 30, wherein the bacterial toxin comprises diphtheria toxin or Pseudomonas exotoxin.

33. The conjugate of claim 28, wherein the toxin comprises a holotoxin or a hemitoxin.

34. The conjugate of claim 28, wherein the drug comprises calicheamicin, actinomycin D, anthracycline, auristatin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1- dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine,propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, or stereoisomers, isosteres, analogs, or derivatives thereof.

35. The conjugate of claim 28, wherein the drug comprises calicheamicin.

36. The conjugate of claim 28, wherein the detectable label comprises a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, or an affinity tag.

37. The conjugate of claim 28, wherein the radioisotope comprises228Ac,111Ag,124Am,74As,211At, 209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy, 157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga, 72Ga,146Gd,153Gd,58Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K, 43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na, 95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr,191Pt,243Pu, 225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te, 234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe, 85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.

38. The conjugate of claim 28, wherein the radioisotope does not emit daughter radionuclides.

39. The conjugate of claim 28, wherein the particle comprises a metal nanoparticle, a liposome, or a polymer nanoparticle.

40. A recombinant receptor that, when expressed by a cell, comprises an extracellular component comprising the binding domain of claim 1 or the scFv of claim 23.41 . The recombinant receptor of claim 40, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR).

42. The recombinant receptor of claim 41 , wherein the CAR comprises an intracellular component linked to the extracellular component by a transmembrane domain.

43. The recombinant receptor of claim 42, wherein the intracellular component comprises an effector domain comprising: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH 1 , 0X40, ROR2, SLAMF1 , TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

44. The recombinant receptor of claim 43, wherein the effector domain comprises all or a portion of a signaling domain of CD3 and all or a portion of a signaling domain of 4-1 BB.

45. The recombinant receptor of claim 44, wherein the CD3£ signaling domain is encoded by a sequence as set forth in SEQ ID NO: 178 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 178.

46. The recombinant receptor of claim 44, wherein the CD3£ signaling domain comprises asequence as set forth in SEQ ID NO: 179 or SEQ ID NO: 180 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 179 or SEQ ID NO: 180.

47. The recombinant receptor of claim 44, wherein the 4-1 BB signaling domain is encoded by a sequence as set forth in SEQ ID NO: 181 or SEQ ID NO: 182 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 181 or SEQ ID NO: 182.

48. The recombinant receptor of claim 44, wherein the 4-1 BB signaling domain comprises a sequence as set forth in SEQ ID NO: 183 or SEQ ID NO: 184 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 183 or SEQ ID NO: 184.

49. The recombinant receptor of claim 42, wherein the transmembrane domain comprises a transmembrane region of: an a, [3 or £ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

50. The recombinant receptor of claim 42, wherein the transmembrane domain comprises a CD28 transmembrane domain.51 . The recombinant receptor of claim 50, wherein the CD28 transmembrane domain is encoded by SEQ I D NO: 185, SEQ I D NO: 186, or SEQ I D NO: 187 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187.

52. The recombinant receptor of claim 49, wherein the CD28 transmembrane domain comprises a sequence as set forth in SEQ ID NO: 188 or SEQ ID NO: 189 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 188 or SEQ ID NO: 189.

53. The recombinant receptor of claim 41 , wherein the CAR further comprises a spacer region.

54. The recombinant receptor of claim 53, wherein the spacer region comprises an intermediate spacer region, long spacer region, or short spacer region.

55. The recombinant receptor of claim 54, wherein the intermediate spacer region is 135 amino acids or less.

56. The recombinant receptor of claim 54, wherein the intermediate spacer region is 131 amino acids or less and comprises a hinge region and a CH3 domain of lgG4.

57. The recombinant receptor of claim 54, wherein the intermediate spacer region is encoded by a sequence as set forth in SEQ ID NO: 175 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 175.

58. The recombinant receptor of claim 54, wherein the intermediate spacer region is encoded by a sequence as set forth in SEQ ID NO: 176 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 176.

59. The recombinant receptor of claim 54, wherein the long spacer region is greater than 200 amino acids and comprises an lgG4 hinge, an I gG4 CH3 region, and an lgG4 CH2 region.

60. The recombinant receptor of claim 54, wherein the long spacer region is encoded by a sequence as set forth in SEQ ID NO: 177 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 177.61 . The recombinant receptor of claim 54, wherein the short spacer region is less than 50 amino acids and comprises an lgG4 hinge.

62. The recombinant receptor of claim 54, wherein the short spacer region is encoded by a sequence as set forth in SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174.

63. The recombinant receptor of claim 40, further comprising a control feature selected from a tag cassette, a transduction marker, and a suicide switch.

64. The recombinant receptor of claim 63, wherein the transduction marker comprises a truncated CD19.

65. The recombinant receptor of claim 64, wherein the truncated CD19 is encoded by SEQ ID NO: 195 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 195.

66. The recombinant receptor of claim 40, further comprising a ribosomal skip element.

67. The recombinant receptor of claim 66, wherein the ribosomal skip element comprises T2A, P2A, E2A, or F2A.

68. The recombinant receptor of claim 66, wherein the ribosomal skip element comprises T2A.

69. The recombinant receptor of claim 67, wherein T2A is encoded by SEQ ID NO: 190 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 190.

70. The recombinant receptor of claim 41 , wherein the eTCR comprises a constant alpha domain (Ca) and a constant beta domain (Cp).71 . The recombinant receptor of claim 70, wherein the scFv of claim 23 is linked to the Cadomain and / or the Cp domain.

72. A genetic construct encoding the binding domain of claim 1 or the recombinant receptor of claim 40.

73. The genetic construct of claim 72, comprising a sequence as set forth in SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, orSEQ ID NO: 223 or has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 , SEQ ID NO: 222, or SEQ ID NO: 223.

74. A nanoparticle encapsulating the genetic construct of claim 72.

75. A cell genetically modified to express the binding domain of claim 1 or the recombinant receptor of claim 40.

76. The cell of claim 75, wherein the cell is an immune cell.

77. The cell of claim 76, wherein the immune cell is a T cell, B cell, natural killer (NK) cell, NK-T cell, monocyte / macrophage, hematopoietic stem cells (HSC), or a hematopoietic progenitor cell (HPC).

78. The cell of claim 75, wherein the cell is a T cell selected from a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.

79. The cell of claim 75, wherein the cell is a CD8+ T cell and / or a CD4+ T cell.

80. The cell of claim 75, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject.81 . The cell of claim 75, wherein the cell is in vivo or ex vivo.

82. A composition comprising the binding domain of claim 1 , the multi-domain binding molecule of claim 8, the scFv of claim 23, the conjugate of claim 28, the genetic construct of claim 72, or the nanoparticle of claim 74; and a pharmaceutical ly-acceptable carrier.

83. A formulation comprising the cell of claim 75 and a pharmaceutically-acceptable carrier.

84. The formulation of claim 83, wherein the cells are T cells, B cells, natural killer (NK) cells, NK- T cells, monocytes / macrophages, hematopoietic stem cells (HSC), or hematopoietic progenitor cells.

85. The formulation of claim 84, wherein the T cells are selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells, and / or naive T cells.

86. The formulation of claim 84, wherein the T cells are CD8+ T cells and / or CD4+ T cells.

87. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the composition of claim 82 or the formulation of claim 83 to the subject thereby treating the subject in need thereof.

88. The method of claim 87, wherein the subject in need thereof has cancer.

89. The method of claim 88, wherein the cancer comprises cancer cells expressing CLEC2A.

90. The method of claim 88, wherein the cancer comprises leukemia.91 . The method of claim 90, wherein the leukemia is acute myeloid leukemia (AML).

92. The method of claim 91 , wherein the AML comprises KMT2A-r AML.

93. The method of claim 87, wherein the administering comprises intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intrarectal, intrathecal, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration.

94. A method of treating KMT2A-r AML in a subject comprising administering a therapeutically effective amount of a CLEC2A-targeted therapeutic, wherein the CLEC2A-targeted therapeutic comprises an anti-CLEC2A binding domain, thereby treating the subject.

95. The method of claim 94, wherein the anti-CLEC2A binding domain comprises the binding domain of claim 1.

96. The method of claim 94, wherein the anti-CLEC2A binding domain is derived from an anti- CLEC2A antibody.

97. The method of claim 96, wherein the anti-CLEC2A antibody comprises MA5-28532 or MAB 72191.

98. The method of claim 94, wherein the CLEC2A-targeted therapeutic comprises a multi-domain binding molecule, an antibody conjugate, or a recombinant receptor-expressing immune cell.

99. The method of claim 98, wherein the multi-domain binding molecule comprises an anti- CLEC2A binding domain linked to a binding domain that binds an immune cell activating epitope.

100. The method of claim 99, wherein the immune cell activating epitope comprises CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 , CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors 1-9, IL-4Ra, or MARCO.

101. The method of claim 99, wherein the immune cell activating epitope comprises CD3.

102. The method of claim 99, wherein the immune cell activating epitope comprises CD28.

103. The method of claim 99, wherein the immune cell activating epitope comprises CD16.

104. The method of claim 98, wherein the multi-domain binding molecule comprises the multidomain binding molecule of claim 8.

105. The method of claim 98, wherein the antibody conjugate comprises an anti-CLEC2A binding domain linked to a toxin, a drug, or a radioisotope.

106. The method of claim 105, wherein the toxin comprises a holotoxin or a hemitoxin.

107. The method of claim 105, wherein the drug comprises actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1- dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine,maytansinoid, mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine, taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, or stereoisomers, isosteres, analogs, or derivatives thereof.

108. The method of claim 105, wherein the drug comprises calicheamicin.

109. The method of claim 105, wherein the radioisotope comprises228Ac,111Ag,124Am,74As, 211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,57Cu, 153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm, 66Ga,72Ga,146Gd,153Gd,68Ge,3H,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln, 185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174ml_u,175mLu,257Md,250Md,28Mg,52Mn, 90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,191Os,32P,201Pb,101Pd,143Pr, 191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta, 154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe, 133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.

110. The method of claim 98, wherein the antibody conjugate comprises the conjugate of claim 28.11 1. The method of claim 98, wherein the recombinant receptor-expressing immune cell comprises a recombinant receptor that, when expressed by an immune cell, comprises an extracellular component comprising the anti-CLEC2A binding domain.

112. The method of claim 11 1 , wherein the recombinant receptor comprises a CAR.

113. The method of claim 112, wherein the CAR comprises an intracellular component linked to the extracellular component by a transmembrane domain.

114. The method of claim 113, wherein the intracellular component comprises an effector domain comprising: 4-1 BB (CD137), CD3y, CD36, CD3E, CD3 , CD27, CD28, DAP10, ICOS, l_AG3, NKG2D, NOTCH1 , 0X40, ROR2, SLAMF1 , TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

115. The method of claim 1 14, wherein the effector domain comprises all or a portion of a signaling domain of CD3 and all or a portion of a signaling domain of 4-1 BB.

116. The method of claim 113, wherein the transmembrane domain comprises a transmembrane region of: an a, p or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

117. The method of claim 1 13, wherein the transmembrane domain comprises a CD28 transmembrane domain.

118. The method of claim 111 , wherein the recombinant receptor comprises the recombinant receptor of claim 40.

119. The method of claim 111 , wherein the immune cell comprises a T cell.

120. The method of claim 119, wherein the T cell comprises a CD8+ T cell or a CD4+ T cell.

121. A method of manufacturing a CLEC2A knock-out model comprising introducing a CLEC2A sgRNA and a Cas9 protein into a cell.

122. The method of claim 121 , wherein the cell comprises a CLEC2A-expressing cell.

123. The method of claim 122, wherein the CLEC2A-expressing cell comprises an OCI-AML2 cell.

124. The method of claim 121 , wherein the introducing comprises electroporating.

125. The method of claim 121 , wherein the CLEC2A sgRNA is complementary to a sequence within a coding region of CLEC2A.

126. A CLEC2A knock-out model comprises a cell genetically modified to lack CLEC2A expression.

127. The CLEC2A knock-out model of claim 126, manufactured according to the method of claim 121.

128. A method of manufacturing a CLEC2A overexpression model comprising introducing a CLEC2A vector into a cell.

129. The method of claim 128, wherein the cell comprises a non-CLEC2A-expressing cell.

130. The method of claim 129, wherein the non-CLEC2A-expressing cell comprises an MV4;11 cell.

131. The method of claim 128, wherein the CLEC2A vector comprises a lentivirus comprising a CLEC2A plasmid.

132. A CLEC2A overexpression model comprises a cell genetically modified to have increased CLEC2A expression compared to a same type of cell found in nature.

133. The CLEC2A overexpression model of claim 132, manufactured according to the method of claim 128.

134. A method of manufacturing a patient-derived xenograft (PDX) model comprising: obtaining CLEC2A+ AML cells; and injecting CLEC2A+ A L cells into an animal.

135. The method of claim 134, wherein the CLEC2A+ AML cells are patient derived.

136. The method of claim 134, further comprising transducing CLEC2A+ AML cells with a genetic construct.

137. The method of claim 136, wherein the genetic construct expresses a fluorescent label.

138. The method of claim 137, wherein the fluorescent label comprises luciferase and / or GFP.

139. The method of claim 134, wherein the animal comprises a mouse.

140. The method of claim 139, wherein the mouse comprises an NSG mouse.

141. An patient-derived xenograft (PDX) model comprises an animal injected with CLEC2A+ AML cells.

142. The PDX model of claim 141 , wherein the animal comprises a mouse.

143. The PDX model of claim 142, wherein the mouse comprises an NSG mouse.

144. The PDX model of claim 141 , wherein the CLEC2A+ AML cells are genetically modified to express a genetic construct.

145. The PDX model of claim 144, wherein the genetic construct comprises a fluorescent label.

146. The PDX model of claim 145, wherein the fluorescent label comprises luciferase and / or GFP.

147. A kit comprising the binding domain of claim 1 , the multi-domain binding molecule of claim 8, the scFv of claim 23, the conjugate of claim 28, the recombinant receptor of claim 40, the genetic construct of claim 72, the nanoparticle of claim 74, the cell of claim 75, the composition of claim 82, the formulation of claim 83, the CLEC2A knock-out model of claim 126, the CLEC2A overexpression model of claim 132, or the PDX model of claim 141 .

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