Multi-specific immune cell engaging molecules to treat CD19 and CD33 co-expressing cancers

Abstract

Multi-specific immune cell engaging molecules are provided herein. These immune cell engaging molecules bind cancer cells co-expressing CD19 and CD33 while also binding immune cells, allowing the immune cells to recognize and destroy the CD19 and CD33-expressing cancer cells.

Description

MULTI-SPECIFIC IMMUNE CELL ENGAGING MOLECULESTO TREAT CD19 AND CD33 CO-EXPRESSING CANCERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 716,136 filed on November 4, 2024, the entire contents of which are incorporated by reference herein.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides multi-specific immune cell engaging molecules and uses thereof. The multispecific immune cell engaging molecule bind CD19, CD33, and an immune activating epitope and can be used to treat CD19 and CD33 co-expressing cancers, such as B cell malignancies including chronic phase leukemia, accelerated phase leukemia, blast phase leukemia, mixed phenotype acute leukemia (MPAL), and subsets of B-cell acute lymphoblastic leukemia (B-ALL).BACKGROUND OF THE DISCLOSURE

[0003] Blood cells are continually generated from self-renewing progenitors in the bone marrow called hematopoietic stem cells (HSCs). Those stem cells have the capacity to self-renew and they have the potential to differentiate in all types of blood cells. They give rise to progenitors that are more restricted in their differentiation potential, and finally to functionally mature cells.

[0004] Common lymphoid progenitors (CLPs) give rise to T lymphocytes, B lymphocytes, and natural killer (NK) cells while common myeloid progenitors (CMPs) give rise to granulocyte-monocyte progenitors (GMPs), which then differentiate into monocytes macrophages and granulocytes, and to megakaryotic erythroid progenitors (MEP), which produce megakaryocytes platelets and erythrocytes. Both CMPs and CLPs can give rise to dendritic cells. All of these stem and progenitor cell populations are separable as pure populations by using cell surface markers.

[0005] Leukemia consists of abnormal proliferation of CLPs, CMPs, MEPs, GMPs, progenitor dendritic cells (DC), progenitor T cells, progenitor NK cells, and progenitor B cells when one of these cell types gains self-renewal and loses the ability to differentiate. Depending on what kind of progenitor undergoes the transformation, different types of leukemia can occur, namely acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). ALL can be further divided into B cell ALL (B-ALL) or T cell ALL (T-ALL).

[0006] Mixed phenotype acute leukemia (MPAL) is small subtype of leukemia that does not fall into either AML or ALL categories because it co-expresses markers of both lineages. Depending on the type of cell markers expressed, MPAL can be further classified into subtypes: B / Myeloid being the most common, T / Myeloid, NOS (not otherwise specified) and Acute undifferentiated leukemia when cells fail to express any markers of differentiation (usually only expressing CD34 and HLA DR). MPAL remains a diagnostic challenge as aberrant expression of markers of the other lineage is a common event (i.e., ALL with aberrant CD33 expression or AML with aberrant CD7 or CD10 expression.

[0007] While MPAL is rare (3-5% of all leukemia), it affects both children and adults with a bi-modal distribution.Outcomes are worse than ALL and either worse than AML or similar to AML depending on the cohorts and whether or not children are included.SUMMARY OF THE DISCLOSURE

[0008] The present disclosure provides multi-specific immune cell engaging molecules including an immune cell activating epitope binding domain, an anti-CD 19 binding domain, and an anti-CD33 binding domain. The multi-specific immune cell engaging molecule targets both a lymphoid marker and a myeloid marker such that it can be used to treat CD19 and CD33 co-expressing cancers, such as B cell malignancies including chronic phase leukemia, accelerated phase leukemia, blast phase leukemia, mixed phenotype acute leukemia (MPAL), and subsets of B-cell acute lymphoblastic leukemia (B-ALL). In particular embodiments, the multi-specific immune cell engaging molecules have strong avidity and binding to cancer cells expressing both CD19 and CD33 but have low affinity and binding for cells expressing only CD19 or CD33. In particular embodiments, the multi-specific immune cell engaging molecules are affinity-tuned to decrease binding affinity of one or more binding domains.BRIEF DESCRIPTION OF THE FIGURES

[0009] 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 the right to present color images of the drawings in later proceedings.

[0010] FIGs. 1A, 1B. CD19 and CD33 were identified as appropriate targets for B-myeloid mixed phenotype acute leukemia (MPAL) after analyzing aggregated transcriptome data for 31 patients. (1A) RNAseq data of 31 B / myeloid MPAL patient samples. (1B) Expression of CD19 and CD33 by flow cytometry in a patient sample diagnosed with B / myeloid MPAL.

[0011] FIG. 2. Exemplary binding molecule formats.

[0012] FIGs. 3A, 3B. Exemplary multi -specific T cell engaging (MTE) molecules disclosed herein. (3A) These embodiments may include asymmetric "knob into holes" human IgG 1 scaffold incorporating LALAPG effector silencing mutations. (3B) These embodiments do not include Fc regions. The MTE depicted: in the top left can be referred to as #SCRI-002879 or MTE_hsCD33_Gem_Y74A_Fab-hsCD19_FMC63-hsCD3_MM194_Var1, top right #SCRI-002879 or MTE_hsCD33_Gem_Y74A_Fab-hsCD19_FMC63-hsCD3_MM194_Var2, bottom left SSCRI-002881 or MTE_hsCD33_Gem_Y74A_Fab-hsCD19_FMC63-hsCD3_MM194_Var3, and bottom right #SCRI-002881 or MTE_hsCD33_Gem_Y74A_Fab-hsCD19_FMC63-hsCD3_MM194_Var4. These MTE designs that do not contain an Fc domain are smaller than Fc-based designs, which can provide a shorter serum half-life and more tissue penetration. These MTEs also have favorable manufacturability characteristics in terms of expression level and stability, providing final yields in the range of 1.0 - 1.7 mg and concentrations in the range of 0.42 - 0.67 mg / ml..

[0013] FIG. 4. Table of example commercially available binding domains for combinatorial assembling of exemplary CD19: CD33: CD3 MTEs.

[0014] FIG. 5. To evaluate the activity of candidate MTEs on CD 19 and CD33 single antigen vs. dual antigen positive cells, MOLM-13 cell line variants were engineered to obtain a defined set of MOLM-13 lines that express neither antigen, each antigen individually, or both antigens together.

[0015] FIGs. 6A, 6B. Site density of CD 19 and CD33 on native JIH-5 and engineered MOLM-13 cell lines. (6A) Levels of CD19 and CD33 expression in engineered MOLM-13 cell lines. (6B) Comparison of CD19 expression level between engineered MOLM-13 cell line and WT JIH-5 MPAL cells. Engineered MOLM-13 CD19 single and CD19 / CD33 dualversus single positive (SP) CD19+ or CD33+ MOLM-13 cells. The binding of the MTE is assessed using an anti-Fc (Allophycocyanin, APC). The difference in Median Fluorescent Intensity (MFI) for the different cell types reveals the enhanced specificity of MTEs for DP cells. (7B) Heat map of the AMFI between CD19+ / CD33+DP cells vs. CD19+SP cells.

[0017] FIGs. 8A, 8B. Testing candidates for specific cytotoxicity of CD19-CD33+ tumor cells. (8A) Cytotoxicity ofCD 19FMC63ScFv / CD33GEMTuzuMAB Fab / CD3mICrometi94(SEQ |D NOs: 226, 240, and 248) MTEs for MOLM-13 cells in T-cell killing assays, demonstrating a therapeutic window between DP cells versus SP cells. With the parental design (no affinity variants employed), MTE directed cytotoxicity against DP MOLM-13 cells with an IC50 of 0.2 pM versus an IC50 of 1.3 pM for SP CD19+MOLM-13 cells and 35.4 pM for the SP CD33 MOLM-13 cells. This difference in IC50 demonstrates a potential "therapeutic window” where DP cells are specifically targeted for lysis while SP cells are spared (8B) SEQ ID NOs: 334 and 337 (top) and SEQ ID NOs: 338 and 337 (bottom), both lacking Fc, each demonstrated a therapeutic window comparable to that observed for the reference Fc-based design of FIG. 8A.

[0018] FIG. 9. MTE disclosed herein, CD19FMC63 scFV / CD33GEMTUZUMAB Fab / CD3micromet194(SEQ ID NOs: 342, 344, and

[0021] FIG. 12. Comparison of wild type (WT, SEQ ID NOs: 226, 240, and 248) vs 3 CD19 affinity mutants (Y261A SEQ ID NOs: 231, 240, and 248, Y70A SEQ ID NOs: 232, 240, and 248, Y260A SEQ ID NOs: 230, 240, and 248) in CD 19+CD33+ cells, CD19+ only cells, and CD33+only cells. There was no difference in cytotoxicity for WT or 3 CD19 affinity mutants in DP CD19+ CD33+ cells, revealing that the avidity for target cells expressing both targets ismaintained. When comparing different CD19 binding domains integrated into 4 different MTEs, decreased affinity was observed for SP CD19+ cells (IC50 going from 1.3 pM to 62.9 pM) to no observable cytotoxicity. Notably, no effect on cytotoxicity for CD33 only cells was observed, further revealing the modularity of the affinity-tuning approach. The targeting of CD33 SP cells has hence become the limiting factor for the potential therapeutic window.

[0022] FIG. 13. Examplary affinity-tuned mutant MTEs.

[0023] FIG. 14. Screening for the optimal binding domain combination. Top, difference in IC50 observed for MTECD 1 9FMC63 SCFV / CD33GEMTUZUM B Fab Y74A / CD3micrometi94(SEQ |D Nos:342]345,and 248). Bottom, each combination of binding domains shows a distinct difference in ICso-

[0024] FIGs. 15A-15C. In vivo dose determination study with 1, 10, 100 pig / dose / mouse. (15A) Timeline of dose cycle. (15B) Table of dose and number of activated T cells. (15C) IVIS imaging of mice at day 7 engrafted with JIH-5 P3 YFP-fflux.and 248) at the bottom).

[0028] FIGs. 19A, 19B. Therapeutic window cytokine profile. In in vitro T cell-mediated killing assays, a MSD V-plex custom assay was used to measure the levels of T cell activation cytokines, specifically IL-2, TNF-a, and IFN-y, in the supernatant. Within the therapeutic window (indicated by the black box in 19A), the T cells showed significantly higher activation, as evidenced by elevated levels of all three cytokines (19B), when co-cultured with target cells expressing342, 345, and 248) was confirmed by comparing it to the standard of care, Blinatumomab in NSG-SGM3 mice engrafted with human B / myeloid leukemia cells expressing both targets CD19 and CD33, called JIH-5. Both therapies demonstrated excellent reduction of disease burden, as evidenced by a significant decrease in total flux in IVIS imaging (20A), when compared to the control group receiving only T cells and no therapeutic agents. While a residual signalwas detected in some mice treated with MTE and Blinatumomab (20B, lower panel), these results indicate that the MTE is equally as efficacious as Blinatumomab in this xenograft model. I VIS imaging was conducted at day 35.

[0030] FIG. 21. For safety studies, humanized mice obtained from Taconic Biosciences were used. These off-the-shelf models are created by engrafting immunodeficient mice with human hematopoietic stem cells (HSC). The engraftment leads to the development of a functional human immune system, including key lineages such as B cells, T cells, and CD33+ myeloid cells. By using this model, it can specifically be tested whether treatments have an on-target, off-tumor effect by sparing normal B cells and myeloid cells and thus a robust safety profile can be established (data from Taconic Biosciences Inc ). 10 week timepoint: n=99 mice from 20 human donors (4-6 mice per donor); 22and 248) at 3pg led to off-target toxicity, specifically characterized by the significant depletion of normal B cells (22A, for both MTEs tested). This confirmed that the 3pg dose was too high in this model and necessitated further dosefinding studies to establish the true therapeutic window (see 22C and 22D, showing toxicity for cells with one antigen outside the therapeutic window in 22C and 22D). Importantly, by incorporating a tuned CD33 binder variant (SEQ ID NOs: 342, 345, and 248), the desired outcome of sparing normal CD33+ cells (22B) was achieved.

[0032] FIGs. 23A-23C. In a subsequent humanized mouse experiment, a safe dosing regimen for the MTE (SEQ ID NOs: 342, 345, and 248) was identified, showing that B cell depletion is avoided at doses of 0.3pg and 0.03pg (23A). This establishes a promising therapeutic window. CD33+ cells were spared at all doses. The MTE used in this experiment was FMC63WT: GemtuzumabY74A: MT194 (SEQ ID NOs: 342, 345, and 248).

[0033] FIGs. 24A-24C. Mice were euthanized at week 19 and it was confirmed that B cell depletion in the spleen and BM was observed only at the highest dose of 3ug (24A). CD33+ cells were spared at all doses in BM and spleen (24B). An increase in the amount of CD8+PD-1 + T cells was observed in the spleen of animals treated with the highest dose, suggesting more activated CD8+ T cells at 3ug compared to lower doses (24C).

[0034] FIGs. 25A-25G. (25A) Anti-CD19 scFv with linkers underlined. (25B) Anti-CD33 binding domains. (250) Anti-003 binding domains with linkers underlined, and signal peptides in bold. (25D) Scaffolds including multimerization domains with signal peptide, location to insert binding domain, and the multimerization domain (linkers underlined). (25E) Example sequences including binding domains within scaffolds including multimerization domains. (25F) Overexpression constructs for CD 19 and CC33.

[0035] FIG. 26. Example immune cell engaging molecule architectures.DETAILED DESCRIPTION

[0036] The present disclosure provides affinity-tuned multi-specific immune cell engaging molecules (ICEm) including an immune cell activating epitope (ICAE) binding domain, an anti-CD19 binding domain, and an anti-CD33 bindingdomain. The ICEm target both a lymphoid marker and a myeloid marker such that it can be used to treat CD19 and CD33 co-expressing cancers, such as B cell malignancies including chronic phase leukemia, accelerated phase leukemia, blast phase leukemia, mixed phenotype acute leukemia (MPAL), and subsets of B-cell acute lymphoblastic leukemia (B-ALL). In particular embodiments, the multi-specific immune cell engaging molecules have strong avidity and binding to cancer cells that express CD19 and CD33 and low affinity and binding for cells expressing only CD19 or CD33.

[0037] In particular embodiments, an ICEm includes a first polypeptide sequence including a first multimerization domain and a second polypeptide sequence including a second multimerization domain. In particular embodiments, an ICEm includes a first polypeptide sequence including a knob and a second polypeptide sequence including a hole. In particular embodiments, an ICEm includes an IgM Fc region and a joining (J)-chain. In particular embodiments, an ICEm lacks an Fc region of an antibody. Exemplary scaffolds for multimerization are shown in FIG. 25D.

[0038] In particular embodiments, the ICAE binding domain binds an epitope on a T cell, an NK cell, or a macrophage. In particular embodiments, the ICAE binding domain binds CD3. In particular embodiments, an anti-CD3 binding domain is derived from Micromet194, ADI-26906, F2B, or ABLYNX. In particular embodiments, an anti-CD19 binding domain is derived from FMC63, FMC63 Y70A, FMC63 Y260A, FMC63 Y261A, Blinatumomab, TNB-334354, or TNB-341620. In particular embodiments, an anti-CD33 binding domain is derived from Lintuzumab, Gemtuzumab, Nb7, Nb12, Nb16, or Nb87. In particular embodiments, one or more of the binding domains are selected from FDA approved antibodies, antibodies that have gone through human clinical trials, or have been extensively studied. This approach lowers the risk of safety and immunogenicity concerns.

[0039] FMC63, for example is an scFv employed in several FDA approved CD19 targeting CAR T cells including Kymriah (Novartis, Switzerland), Yescarta (Kite Pharma, Inc., Santa Monica, CA), Tecartus (Kite Pharma, Inc.), and Breyanzi (Juno Therapeutics, Inc., Seattle, WA). In addition to testing the wildtype FMC63 scFv, a set of affinity tuned variants (Y70A, Y260A, Y261A) were used that reduce FMC63 CAR T cell activity. Blinatumomab (Blincyto, Amgen Inc., Thousand Oaks, CA) is the first FDA approved bispecific antibody targeting CD19 and CD3. Additionally, the Teneobio VHH advanced into clinical trials with the CD19: CD3 bispecific TNB-486. The sequence of this VHH (clone TNB-334354) as well as a lower affinity VHH (clone TNB-341620) can be found in WO Publication No. 2020018922. For targeting CD33, scFv and Fab format binding domains derived from lintuzumab and gemtuzumab sequences were used. For targeting CD3 a set of CD3 binding domains were selected with a range of affinities and / or activation potentials. CD3 clone Micromet194 is a high affinity binder derived from the parental CD3 clone SP34. ADI26906 is a medium affinity anti-CD3 clone from Adimab (Lebanon, NH), and the Ablynx (Ablynx N. V., Belgium) VHH is a lower affinity anti-CD3 VHH.

[0040] In particular embodiments, the anti-CD19 binding domain and the anti-CD33 binding domain are modified to have reduced binding affinity. Binding affinity can be reduced by replacing one or more tyrosines within a complementarity determining region (CDR) of the binding domain with a less hydrophobic amino acid, such as alanine.Reducing the binding affinity is beneficial as it increases the necessity of a cell co-expressing CD19 and CD33 in order for the ICEm to bind

[0041] In particular embodiments, an ICEm includes an anti-CD19 binding domain described in FIG. 25A, an anti-CD33 binding domain described in FIG. 25B, and an anti-CD3 binding domain described in FIG. 25C within a scaffold for multimerization described in FIG. 25D. In particular embodiments, the ICEm includes a sequence described in FIG.25E.

[0042] In particular embodiments, an ICEm includes a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes a first multimerization domain and the second polypeptide chain includes a second multimerization domain. In particular embodiments, the first polypeptide chain includes a first binding domain and an lgG1 Fc hole and the second polypeptide chain includes a second binding domain and an lgG1 Fc knob. In particular embodiments, a third binding domain is on the first polypeptide chain or the second polypeptide chain. In particular embodiments, the first polypeptide chain includes an anti-CD 19 binding domain and an lgG1 Fc hole and the second polypeptide chain includes an anti-CD33 binding domain and an lgG1 Fc knob. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 226-232, 342, or 343. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 233-238 or 344-347. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 344 or 345. In particular embodiments, either the first polypeptide chain or the second polypeptide chain further includes an ICAE binding domain. In particular embodiments, the ICAE binding domain includes an anti-CD3 binding domain. In particular embodiments, the anti-CD3 binding domain includes a sequence of SEQ ID NO: 211, 212, 217, or 218. In particular embodiments, the anti-CD3 binding domain includes a sequence of SEQ ID NO: 248

[0043] In particular embodiments, an ICEm includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain includes a first multimerization domain and the second polypeptide chain includes a second multimerization domain. In particular embodiments, the first polypeptide chain includes a first binding domain and an lgG1 Fc hole, the second polypeptide chain includes a heavy chain of a second binding domain and an lgG1 Fc knob, and the third polypeptide chain includes a light chain of the second binding domain. In particular embodiments, a third binding domain is on the first polypeptide chain or the second polypeptide chain. In particular embodiments, the first polypeptide chain includes an anti-CD19 binding domain and an lgG1 Fc hole, the second polypeptide chain includes a heavy chain of an anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide chain includes a light chain of the anti-CD33 binding domain. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 226-232, 342, or 343. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 342. or In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 343. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 239 and the third polypeptide chain includes a sequence of SEQ ID NO: 241. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 240 and the third polypeptide chain includes asequence of SEQ ID NO: 242. In particular embodiments, either the first polypeptide chain or the second polypeptide chain further includes an ICAE binding domain. In particular embodiments, the ICAE binding domain includes an anti-003 binding domain. In particular embodiments, the anti-CD3 binding domain includes a sequence of SEQ ID NO: 211, 212, 217, or 218.

[0044] In particular embodiments, an ICEm includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain includes a first multimerization domain and the second polypeptide chain includes a second multimerization domain. In particular embodiments, the first polypeptide chain includes a first binding domain and an lgG1 Fc hole, the second polypeptide chain includes a heavy chain of a second binding domain and an lgG1 Fc knob, and the third polypeptide chain includes a light chain of the second binding domain and a third binding domain. In particular embodiments, the first polypeptide chain includes an anti-CD19 binding domain and an lgG1 Fc hole, the second polypeptide chain includes a heavy chain of an anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide chain includes a light chain of the anti-CD33 binding domain and an anti-CD3 binding domain. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 226-232, 342, or 343. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 342. or In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 343. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 239 and the third polypeptide chain includes a sequence of SEQ ID NO: 243, 245, or 247. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 240 and the third polypeptide chain includes a sequence of SEQ ID NO: 244, 246, or 248. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 344, 345, 246, or 347 and the third polypeptide chain includes a sequence of SEQ ID NO: 248.

[0045] In particular embodiments, an ICEm includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain includes a first multimerization domain and the second polypeptide chain includes a second multimerization domain. In particular embodiments, i) the first polypeptide chain includes a first binding domain, a second binding domain, and an lgG1 Fc hole; ii) the second polypeptide chain includes a heavy chain of a third binding domain and an lgG1 Fc knob; and iii) the third polypeptide chain includes a light chain of the third binding domain. In particular embodiments, the first polypeptide chain includes an anti-CD 19 binding domain, an anti-CD33 binding domain, and an lgG1 Fchole. In particular embodiments, the second polypeptide chain includes a heavy chain of an anti-CD3 binding domain and an IgG 1 Fc knob. In particular embodiments, the third polypeptide chain includes a light chain of the anti-CD3 binding domain. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 249-256, 334, 336, 338, 340, 348, or 349. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 214 and the third polypeptide chain includes the sequence of SEQ ID NO: 213.

[0046] In particular embodiments, an ICEm includes (i) an IgM Fc including an a first binding domain and a second binding domain; and (ii) a joining (J)-chain including an ICAE binding domain. In particular embodiments, an ICEmincludes (i) an IgM Fc including an anti-CD19 binding domain and an anti-CD33 binding domain; and (ii) a joining (J)-chain including an ICAE binding domain. In particular embodiments, the ICAE binding domain includes an anti-CD3 binding domain. In particular embodiments, the J-chain including an anti-CD3 binding domain includes a sequence of SEQ ID NO: 257-259. In particular embodiments, the IgM Fc including the anti-CD 19 binding domain and the anti-0033 binding domain includes a sequence of SEQ ID NO: 326-333.

[0047] In particular embodiments, an ICEm includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain includes a first multimerization domain and the second polypeptide chain includes a second multimerization domain. In particular embodiments, i) the first polypeptide chain includes a first binding domain and an lgG1 Fc hole; the second polypeptide chain includes a heavy chain of a second binding domain and an lgG1 Fc knob; and the third polypeptide chain includes a light chain of the second binding domain and a third binding domain. In particular embodiments, the first polypeptide chain includes an anti-CD19 binding domain and an lgG1 Fc hole. In particular embodiments, the second polypeptide chain includes a heavy chain of an anti-CD3 binding domain and an lgG1 Fc knob. In particular embodiments, the third polypeptide chain includes a light chain of the anti-CD3 binding domain and an anti-CD33 binding domain. In particular embodiments, the first polypeptide chain includes a sequence of SEQ ID NO: 228 or 229. In particular embodiments, the second polypeptide chain includes a sequence of SEQ ID NO: 214. In particular embodiments, the third polypeptide chain includes the sequence of SEQ ID NO: 260-271.

[0048] In particular embodiments, the first polypeptide chain includes linkers, a signal peptide, and / or a tag. In particular embodiments, the second polypeptide chain includes linkers, a signal peptide, and / or a tag.

[0049] In particular embodiments, an ICEm lacks an Fc region of an antibody ICEm designs that lack an Fc region of an antibody are depicted in FIG. 3B. Examples include SEQ ID NOs: 334, 335, 337, 338, and 339.

[0050] Aspects of the disclosure are now described in additional detail and with additional options to practice the disclosure as follows: (I) Binding Domains; (II) Cancer Markers and Binding Domains Thereof; (III) Immune Cell Activating Epitopes and Binding Domains Thereof; (IV) Multimerization Domains and Linkers; (V) Variants & Nucleic Acid Sequences; (VI) Expression of Proteins; (VI I) Modifications to Provide Administration Benefits; (VIII) Antibody Conjugates; (IX) Compositions for Administration; (X) Kits; (XI) Methods of Use; (XII) Exemplary Embodiments; (XIII) Experimental Example; (XIV) Prophetic Example; and (XV) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0051] (I) Binding Domains. In certain examples, an ICEm includes three or more binding domains, wherein each binding domain binds a target antigen. Binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. In particular embodiments, a binding domain includes any molecule that can bind a marker or antigen. In particular embodiments, the binding domain includes a protein. In particular embodiments, the protein includes an antibody or a peptide (e.g., miniprotein). In particular embodiments, the binding domain includes anantibody, a peptide (e.g., miniprotein, peptide aptamer), a receptor ligand, a receptor (e.g., T cell receptor), a nucleic acid aptamer, or a fragment thereof

[0052] As used herein, the term "antibody" refers to a monomeric or multimeric protein comprising one or more polypeptide chains that comprise antigen-binding sites. An antibody binds specifically to an antigen and may be able to modulate the biological activity of the antigen. As used herein, the term "antibody" can include "full length antibody" and "antibody fragments." The terms "binding site" or "antigen-binding site" as used herein denotes the region(s) of an antibody molecule to which a ligand actually binds. The term "antigen-binding site" includes an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or in the case of heavy chain only antibodies, an antibody heavy chain variable region.

[0053] As is understood by those of ordinary skill in the art, a conventional antibody includes two heavy chains and two light chains. Each heavy chain includes a variable region and a first, second, and third constant region, while each light chain includes a variable region and a constant region. Mammalian heavy chains are classified as a, 5, E, y, and p, and mammalian light chains are classified as A or K. Immunoglobulins including the a, 5, E, y, and p heavy chains are classified as immunoglobulin (lg)A, IgD, IgE, IgG, and IgM The complete antibody forms a “Y” shape. The stem of the Y includes the second and third constant regions (and for IgE and IgM, the fourth constant region) of two heavy chains bound together and disulfide bonds (inter-chain) are formed in the hinge. Heavy chains y, a and 5 have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains p and E have a constant region composed of four immunoglobulin domains. The second and third constant regions are referred to as “CH2 domain” and “CH3 domain”, respectively. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0054] Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”.

[0055] CDR sets can be based on, for example, 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. (2008) Mol Immunol. 45:3832-3839 (“Martin” numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand et al. (1998) Protein Eng. 11:1015-1025; Gelfand et al. (1996) Proc Natl Acad Sci USA. 93:3675-3678; Gelfand et al. (1998) J Comput Biol. 5:467-477 (“Gelfand” numbering scheme)); Contact (MacCallum et al. (1996) J. Mol. Biol. 262:732-745 (Contact numbering scheme)); IMGT (Lefranc et al. (2003) Dev Comp Immunol 27(1):55-77 (“IMGT” numbering scheme)); AHo (Honegger and Pliickthun (2001) J Mol Biol 309(3):657-670 (“AHo” numbering scheme)); North (North et al. (2011) J Mol Biol. 406 (2): 228-256 (“North” numbering scheme)); or other numbering schemes. Software programs and bioinformatical tools, such as ABodyBuilder (Leem et al. (2016) MAbs 8(7): 1259-1268), PIGSPro (Lepore et al. (2017) Nucleic Acids Res 45(W1): W17-W23), Kotai AntibodyBuilder (Yamashita et al. (2014) Bioinformatics 30(22):3279— 3280), Rosetta Antibody (Weitzner et al. (2017) Nature Protocols 12:401-416), Paratome (Kunik et al. (2012) Nucleic Acids Res 40: W521-W524), Antibody i-Patch (Krawczyk etal. (2013) Protein Eng Des Sei 26(10):621 -629), and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107— 5108 can also be used to determine CDR sequences.

[0056] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the heavy chain of the antibody are referred to as CDRH1, CDRH2, and CDRH3, whereas the CDRs located in the variable domain of the light chain of the antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0057] References to "VH” or “VH” refer to the variable region of an immunoglobulin heavy chain. References to "VL" or “VL” refer to the variable region of an immunoglobulin light chain.

[0058] Antibodies that specifically bind an antigen can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target antigen. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and / or determined. Many relevant antibodies are also publicly known and commercially available.

[0059] In particular embodiments, antibodies specifically bind to a surface molecule on a cell (e.g., cancer cell or immune cell) and do not cross react with nonspecific components such as bovine serum albumin or other unrelated antigens.

[0060] “Antibody fragment” refers to at least one portion of an antibody that retains the ability to specifically bind an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, single chain variable (scFv) antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment including VH and constant CH1 domains, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid variable heavy only (VHH) domains, an immunoglobulin new antigen receptor (Ig N AR), or a picobody, multi-specific antibodies formed from antibody fragments such as a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N. Y.; Houston et al., 1988, Proc Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). An antigen binding fragment can also be incorporated into a single-domain antibody, maxibody, minibody, nanobody, picobody, intrabody, diabody, triabody, tetrabody, v-NAR, IgNAR, and bis-scFv (see, e.g., Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0061] “scFv” refers to an engineered fusion protein including the VH and VL of an antibody linked via a linker and capable of being expressed as a single chain polypeptide. The scFv retains the specificity of the intact antibody from which it is derived. In particular embodiments, a linker connecting the variable regions can include glycine-serine linkers, including, for example, those shown as SEQ ID NOs: 148-162 or described elsewhere herein. In particular embodiments, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may include VL-linker-VH or may include VH-linker-VL.

[0062] "Single-domain antibody (sdAb)", or also referred to as "VHH antibody", refers to an antibody molecule having an antigen binding ability, including a heavy chain variable region without a light chain. From a structural point of view, a single-domain antibody can also be considered an antigen-binding fragment of an antibody molecule. In various embodiments, the antibody includes a VHH. Camelids (camels, dromedary, and llamas) contain, in addition to conventional heavy and light chain antibodies, two-chain antibodies (containing only variant heavy chains). The dimeric antibodies are coded for by a distinct set of VH segments referred to as VHH genes. The VH and VHH are interspersed in the genome (i.e., they appear mixed in between each other). The identification of an identical D segment in a VH and VHH cDNA suggests the common use of the D segment for VH and VHH. Natural VHH-containing antibodies are missing the entire CH1 domain of the constant region of the heavy chain. The exon coding for the CH1 domain is present in the genome but is spliced out due to the loss of a functional splice acceptor sequence at the 5' side of the CH1 exon. As a result the VDJ region is spliced onto the CH2 exon. When a VHH is recombined onto such constant regions (CH2, CH3), an antibody is produced in which the half-antibody is a single chain instead of a light chain / heavy chain pair (i.e., an antibody of two heavy chains without a light chain interaction). Binding of an antigen is different from that seen with a conventional antibody, but high affinity is achieved the same way, i.e., through hypermutation of the variable region and selection of the cells expressing such high affinity antibodies. In particular embodiments, the sdAb is derived from a camelid, shark, or a cow. In particular embodiments, the sdAb is humanized. In particular embodiments, the sdAb is recombinantly produced.

[0063] In particular embodiments, VHH are produced by immunizing a transgenic mouse in which endogenous murine antibody expression has been eliminated and camelid transgenes have been introduced. VHH mice are disclosed in US8,883,150, US8,921,524, US8,921,522, US8,507,748, US8,502,014, US 2014 / 0356908, US2014 / 0033335, US2014 / 0037616, US2014 / 0356908, US2013 / 0344057, US2013 / 0323235, US2011 / 01 18444, and US2009 / 0307787. The VHH mice are immunized and the resulting primed spleen cells fused with a murine myeloma cells to form hybridomas. In other embodiments, VHH are produced by immunizing llamas with a desired antigen, andisolating sequences encoding the VHH regions of resulting antigen-binding antibodies. In alternative embodiments, VHH are isolated using a phage display library See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204.

[0064] The Immunoglobulin New Antigen Receptors (IgNARs) are an unconventional subset of antibodies identified in fish. In domain structure, IgNAR proteins are reportedly similar to other immune effector molecules, being disulphide-bonded homodimers of two polypeptide chains having five constant domains (CNARs) and one variable domain (VNAR). However, unlike conventional antibodies, there are no associated light chains and the individual variable domains are independent in solution and do not appear to associate across a hydrophobic interface (as seen for conventional VH / VL type antibodies).

[0065] IgNARs have minimally variable loop regions analogous to conventional CDR1 and CDR2 loops, with diversity being concentrated in an elongated loop region analogous to a conventional CDR3 loop. The elongated loop region can reportedly vary in length from 5 to 23 residues in length, though the modal classes are more in the order of 15 to 17 residues. This is significantly larger than for conventional murine and human antibodies, but approximate to the extended CDR3 loops found in camelid single VH antibodies.

[0066] Cattle possess exceptional antibodies with ultra-long complementarity-determining regions (ulCDRs) that can include 40-70 amino acids. The bovine ulCDR is folded into a stalk and a disulfide-rich knob domain. The binding to the antigen is via the 3-6 kDa knob. There exists an immense sequence and structural diversity in the knob that enables binding to different antigens. Isolated bovine knobs, also referred to as picobodies are small antigen-binding domains derived from a conventional antibody. In particular embodiments, binding domains disclosed herein include picobodies.

[0067] In addition to antibodies, binding domains disclosed herein can include peptides. A peptide is a compound including two or more amino acids linked in a chain and can include miniproteins or peptide aptamers.

[0068] Miniproteins refer to a diverse group of proteins characterized by small (1-10 kDa) size, stability, and versatility in drug-like roles. Miniproteins may retain the potency and specificity advantages of antibodies while avoiding some of their liabilities. Miniproteins may be cysteine reinforced and include Avimers, Kunits, and cysteine-dense peptide (CDPs). Miniproteins may have a hydrophobic core and include Affibodies, Adnectin + Centyrins, Nanofittins + Affitins, and Fynomers. Miniproteins may be chemically stabilized and include p-hairpins, stapled peptides, and bicycles. For additional information about the characterization of miniproteins, see e.g, Crook et al., Trends Biochem Sci. 45(4):332-346, 2020; Crook et al, Sci. Transl. Med. 14(645) 2022; Bryan et al. Proc Natl Acad Sci USA, 18(29):e2102164118, 2021.

[0069] A “peptide aptamer1’ refers to a polypeptide, generally between 2-20 amino acid residues in length, capable of binding target proteins and interfering with their function in living cells and organisms. They include conformationally-constrained random sequence peptide loops (called 'variable regions') displayed by a scaffold protein. They bind their cognate targets with a strong affinity and, usually, a high specificity, which allows them to discriminate between closely related members within a protein family, or even between different allelic variants of a given protein. So far, peptide aptamers have mostly been selected through yeast two-hybrid screening experiments, for their ability to bind a giventarget protein.

[0070] In particular embodiments, the binding domains disclosed herein can include receptor ligands, receptors, or fragments thereof. Receptors are proteins expressed by a cell that transmit signals upon binding to their cognate receptor ligand. Several receptor ligand and receptor pairs are known in the art and include CD33 and alpha-2, 6-linked sialic acid, T cell receptor (TCR) and major histocompatibility complex (MHC), CD28 and B7 (CD80 and CD86), Fc receptors (FcyR) and immunoglobin, toll-like receptor (TLR) and pathogen associated molecular patterns (PAMPs), and PD-L1 and PD-1.

[0071] A nucleic acid aptamer is a single stranded nucleic acid molecule that structurally bind to targets with high affinity and specificity. The unique properties, including high specific affinity, structural stability and ease of synthesis, make aptamers promising therapeutic and diagnostic agents. In addition, nucleic acid aptamers have less batch-to-batch variation (than antibodies) because they are manufactured by chemical synthesis. Nucleic acid aptamers are often usually generated by a process called “systematic evolution of ligands by exponential enrichment” (SELEX). The targets of nucleic acid aptamers range from small molecules, for example cocaine, proteins like VEGF (vascular endothelial growth factor), and even whole cells.

[0072] (II) Cancer Markers and Binding Domains Thereof. Cancer markers are markers preferentially expressed by cancer cells. In particular embodiments, a cancer marker includes CD19. In particular embodiments, a cancer marker includes CD33. In particular embodiments, an ICEm includes a binding domain that binds CD 19 (i.e., anti-CD19 binding domain) and a binding domain that binds CD33 (i.e., anti-CD33 binding domain).

[0073] CD 19 is a type-l transmembrane glycoprotein of 95 kDa that belongs to the immunoglobulin superfamily and is widely expressed on B cells throughout most stages of B-cell differentiation, though its expression is down-regulated during their terminal differentiation to plasma cells. In particular embodiments, CD19 includes the sequence:MPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPL ASWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLM SPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSL ELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCL CSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGN PSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSF SNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSY ENMDNPDGPDPAWGGGGRMGTWSTR (SEQ ID NO: 1).

[0074] CD33 is a myeloid cell surface antigen that is not expressed on blood stem cells or within the hematopoietic system, but it can be expressed on the surface of natural B-lymphocytes, activated T-lymphocytes, and natural killer (NK) cells. In particular embodiments, CD33 (full length) includes the sequence:MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKL DQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTY VPQNPTTGIFPGDGSGKQETRAGWHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQ KKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 2).

[0075] Splice variants of CD33 can result in truncated forms of CD33. For example, lack of exon 2 (AE2) can result in a splice variant. In particular embodiments, CD33 (AE2 variant) includes the sequence:MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHG TNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGWHGAIGGAGVTALLALCLCLIFFIVKTHR RKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVR TQ (SEQ ID NO: 3).

[0076] In particular embodiments, CD33 (with C-terminal truncation) includes the sequence:MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKL DQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEP GHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTY VPQNPTTGIFPGDGSGKQETRAGWHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPVR(SEQ ID NO: 4).

[0077] As will be understood by one of ordinary skill in the art, targeted cancer markers can lack signal peptides, such as the underlined segments of sequences underlined in SEQ ID NOs: 2-4 (representative CD33 antigens).

[0078] In particular embodiments, the cancer marker binding domain binds CD19. In particular embodiments, the anti-CD 19 binding domain is derived from an antibody such as FMC63 scFv, FMC63 scFv Y70A, FMC63 scFv Y260A, FMC63 scFv Y261 A, Blinatumomab scFv, TNB-334354 VHH, or TNB-341620 VHH.

[0079] In particular embodiments, the anti-CD19 binding domain is derived from an FMC63 scFv and includes a variable heavy chain including the sequence:EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 5) and a variable light chain including the sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 6).

[0080] In particular embodiments, the anti-CD19 binding domain is derived from an FMC63 Y70AscFv and includes a variable heavy chain including the sequence:EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 7) and a variable light chain including the sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKALNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 8).

[0081] In particular embodiments, the anti-CD 19 binding domain is derived from an FMC63 Y260AscFv and includes a variable heavy chain including the sequence:EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYAYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 9) and a variable light chain including the sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 10).

[0082] In particular embodiments, the anti-CD19 binding domain is derived from an FMC63 Y261AscFv and includes a variable heavy chain including the sequence:EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYYAGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 11) and a variable light chain including the sequence:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 12).

[0083] In particular embodiments, the anti-CD19 binding domain is derived from an Blinatumomab scFv and includes a variable heavy chain including the sequence:QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADES SSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 13) and a variable light chain including the sequence:DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNI HPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 14).

[0084] In particular embodiments, the anti-CD 19 binding domain is derived from a VHH including the sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGQEWVATINQDGSDKDYVDSVKGRFTISRDNA KKSLYLQMNSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 15);EVQLVESGGGLAQPGGSLRLSCAASGFTFSSFWMSWVRQAPGKGLEWVATMNQDGSEKDYVDSVKGRFTISRDN AKNSLYLQMNSLTAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 16);EVQLVESGGGMVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVATINQDGSEKDYVDSVKGRFTISRDNA KKSLLLQMNSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 17);EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFWMSWVRQAPGKGLEWVATISQAGSEKDYVDSVKGRFTISRDNA KKSLYLQMNSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 18);EVQLVESGGGLVKPGGSLRLSCEASGFTFSSYWMSWVRQAPGKGLEWVAHIKQDGSEKYYVDSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 19);EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYWMSWVRQAPGKGLEWVANINKAGSEEFYVDSVKGRFTISRDNA KNSLYLQMDSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 20);EVQLVESGGGLVQPGGSLTLSCVASGFTFSDYYMSWIRQAPGKGLEWVANIKQDGSEKFYVDSVKGRFTISRDNPK NSLYLQMDSLRVEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 21); or EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKEYVDSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCASGVYSFDYRGQGTLVTVSS (SEQ ID NO: 22).

[0085] Additional examples of CD 19 binding domains (i.e., binding domains that bind CD19) include: a sequence having a CDR1 including the sequence GNINSRNCMG (SEQ ID NO: 23) or RNCMG (SEQ ID NO: 24), a CDR2 including the sequence AIGQVTGRSYYVDSVKG (SEQ ID NO: 25), and a CDR3 including the sequence APGCLLSALRSADYRN (SEQ ID NO: 26); a sequence having a CDR1 including the sequence GNINSRNC (SEQ ID NO: 27), a CDR2 including the sequence IGQVTGRS (SEQ ID NO: 28), and a CDR3 including the sequence AAAPGCLLSALRSADYRN (SEQ ID NO: 29); or a sequence having a CDR1 including the sequence GDTLSNKWMG (SEQ ID NO: 30) or NKWMG (SEQ ID NO: 31), a CDR2 including the sequence TIRTDHAGTYADSVKG (SEQ ID NO: 32), and a CDR3 including the sequence SYSGATTFRY (SEQ ID NO: 33).

[0086] In particular embodiments, a mouse anti-CD19 binding domain includes a variable heavy chain sequence including the sequence:EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSN TAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVIWTVSS (SEQ ID NO: 34) and a variable light chain sequence including the sequence:DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSM QTEDEGVYFCQQGLTYPRTFGGGTKLELK (SEQ ID NO: 35).

[0087] In particular embodiments, anti-CD 19 binding domains can be found in FIG. 25A.

[0088] In particular embodiments, CDRs for anti-CD19 binding domains are provided in Table 1. CDRs are provided according to Kabat but CDRs according to other CDR definitions could be determined using methods well known to those of skill in the art.

[0089] Table 1. Anti-CD19 binding domain CDRs according to Kabat.

[0090] In particular embodiments, the cancer marker binding domain binds CD33. In particular embodiments, the anti-CD33 binding domain is derived from an antibody such as Lintuzumab (huM195) scFv, a Lintuzumab Fab, a Gemtuzumab scFv, a Gemtuzumab Fab, a Nb7 VHH, a Nb12 VHH, a Nb16 VHH, or a Nb87 VHH.

[0091] In particular embodiments, the anti-CD33 binding domain is derived from HuM195 and includes a variable heavy chain including the sequence:QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADEST NTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS (SEQ ID NO: 36) and a variable light chain including the sequence:DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTL TISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 37).

[0092] In particular embodiments, the anti-CD33 binding domain is derived from Gemtuzumab and includes a variableheavy chain including the sequence:EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTN TAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 38) and a variable light chain including the sequence:DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLT ISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK (SEQ ID NO: 39).

[0093] In particular embodiments, the anti-CD33 binding domain is derived from an Nb12 VHH including the sequence:QVQLQESGGGLVQPGGSLRLSCAASGFTFGSYDMAWVRQAPGKGPEWVSSINSSGGSTEYASSVKGRFTVSRDN AKNMLYLQMDRLKLEDTAVYYCADEGNPSTTWYYEDQGTQVTVSS (SEQ ID NO: 40).

[0094] In particular embodiments, the anti-CD33 binding domain is derived from an Nb16 including the sequence: QVQLQESGGGLVQAGGSLRLSCASSGSILSMNVMGWWRQAPGKQREMVAQITRIGDTNYSSSMKGRFTISRDNAD NTLYLQMNRLEPEDTAVWFCANNNRSTYYYYWGQGTQVTVSS (SEQ ID NO: 41).

[0095] In particular embodiments, the anti-CD33 binding domain is derived from an Nb7 including the sequence: QVQLQESGGQLVQAGGSLRLSCTAFRSVGVIDVMGWWRQAPGKQRELVATVTSGSSTTWADSVKGRFAISRDNAK TVSLQMNSLKPEDTAVYYCADDDEGKLLNRRSVWYYWSQGTQVTVSS (SEQ ID NO: 42).

[0096] In particular embodiments, the anti-CD33 binding domain is derived from an Nb87 including the sequence: QVQLQESGGGLVQAGGSLRLSCAASGSIGSINFIFWYRQAPGKQREFVARISRSGRSDYVESVKGRFTISRDNAKNT VYLQMNNLKSEDTAVYYCALGNNWGQGTQVTVSS (SEQ ID NO: 43).

[0097] In particular embodiments, anti-CD33 binding domains can be found in FIG. 25B.

[0098] Additional examples of CD33 binding domains (i.e., binding domains that bind CD33) include a sequence having a CDR1 including the sequence RSSGIDVMG (SEQ ID NO: 44), a CDR2 including the sequence EISGVGDTN (SEQ ID NO: 45), and a CDR3 including the sequence of HSFLDLVGA (SEQ ID NO: 46); a CDR1 including the sequence GSINSINVME (SEQ ID NO: 47), a CDR2 including the sequence GITSDGDTN (SEQ ID NO: 48), and a CDR3 including the sequence RDWGSLTDY (SEQ ID NO: 49); a CDR1 including the sequence GRTISDYWG (SEQ ID NO: 50), a CDR2 including the sequence AISRYGTTY (SEQ ID NO: 51), and a CDR3 including the sequence LQNDVRNNHSPTSYDY (SEQ ID NO: 52).

[0099] In particular embodiments, CDRs for anti-CD33 binding domains are provided in Table 2. CDRs are provided according to Kabat but CDRs according to other CDR definitions could be determined using methods well known to those of skill in the art.

[0100] Table 2. Anti-CD33 binding domain CDRs according to Kabat.

[0101] ICEm disclosed herein bind at least two cancer markers, CD19 and CD33. Targeting both CD19 and CD33 increases the specificity of the ICEm for cancerous cells and decreases its binding to non-cancerous cells. In particular embodiments, ICEm can include more than two cancer marker binding domains. In particular embodiments, ICEm include an anti-CD 19 binding domain, an anti-CD33 binding domain, and a third cancer marker binding domain. In particular embodiments, an ICEm targets cancer markers that are co-expressed in cancerous tissue.

[0102] (III) Immune Cell Activating Epitopes and Binding Domains Thereof. In particular embodiments, the ICEm include a binding domain that binds an ICAE. An “immune cell activating epitope binding domain" or “ICAE binding domain” refers to the binding domain of an ICEm that binds an ICAE. Immune cells that can be targeted for localized activation by ICEm of the current disclosure include, for example, T cells, natural killer (NK) cells, and macrophages. In particular embodiments, if the ICEm targets T cells, it can be considered a multi-specific T cell engager (MTE). In particular embodiments, the binding domain that targets an immune cell for localized activation is derived from an antibody that binds a protein that activates the immune cell. In particular embodiments, an ICAE includes a T cell activating epitope, NK cell activating epitope, or macrophage activating epitope.

[0103] T -cell activation, for example can be mediated by two distinct signals: those that initiate antigen-dependent 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). MTE disclosed herein can target any T cell activating epitope that upon binding induce 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. T cell suppressive receptors that can be blocked include 4-1 BB, PD-1, LAG3, TIM-3, BTLA, CTLA-4, and CD200. Binding domains that bind T-cell activating epitopes are known in the art.

[0104] CD3 is a primary signal transduction element of T cell receptors. CD3 is composed of a group of invariant proteins called gamma (y), delta (A), epsilon (2), zeta (Z) and eta (H) chains. The y, A, and 2 chains are structurally-related, each containing an Ig-like extracellular constant domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The Z and H chains have a distinctly different structure: both have a very short extracellular region of only 9 amino acids, a transmembrane region and a long cytoplasmic tail including 113 and 115 amino acids in the Z and H chains, respectively. The invariant protein chains in the CD3 complex associate to form noncovalent heterodimers of the Z chain with a y chain (Zy) or with a A chain (ZA) or of the Z and H chain (ZH), or a disulfide-linked homodimer of two Z chains (ZZ). 90% of the CD3 complex incorporate the ZZ homodimer.

[0105] The cytoplasmic regions of the CD3 chains include a motif designated the immunoreceptor tyrosine-based activation motif (ITAM). This motif is found in a number of other receptors including the Ig-o / lg-P heterodimer of the B-cell receptor complex and Fc receptors for IgE and IgG. The ITAM sites associate with cytoplasmic tyrosine kinases and participate in signal transduction following TCR-mediated triggering. In CD3, the y, A and Z chains each contain a single copy of ITAM, whereas the Z and H chains harbor three ITAMs in their long cytoplasmic regions. Indeed, the Z and H chains have been ascribed a major role in T cell activation signal transduction pathways.

[0106] CD3 is expressed on all mature T cells. In particular embodiments, an anti-CD3 binding domain is derived from Micromet194 scFv, ADI-26906 scFv, F2B, or ABLYNXVHH.

[0107] In particular embodiments, the anti-CD3 binding domain is derived from Micromet194 and includes a variable heavy chain including the sequence:EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 53) and a variable light chain including the sequence:QTWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 54).

[0108] In particular embodiments, the anti-CD3 binding domain is derived from ADI26906 and includes a variable heavy chain including the sequence:QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQCLEWMGWIDLENANTIYDAKFQGRVTITRDTSA STAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 55) and a variable light chain including the sequence:DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSLQAEDVAVYYCKQSYSRRTFGCGTKVEIK (SEQ ID NO: 56).

[0109] In particular embodiments, the anti-CD3 binding domain is derived from F2B and includes a variable heavy chain including the sequence:EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNA KNSLYLQMNSLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSS (SEQ ID NO: 57) and a variable light chain including the sequence:EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK (SEQ ID NO: 58).

[0110] In particular embodiments, the anti-CD3 binding domain includes a variable heavy chain including the sequence:EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTN TAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 59) and a variable light chain including the sequence:EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSL QSEDFAVYYCQQYNNWPWTFGQGTKVEIK (SEQ ID NO: 60).

[0111] In particular embodiments, the anti-CD3 binding domain includes a VHH. In particular embodiments the anti-003 binding domain is derived from Ablynx and includes the sequence:EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNA KNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS (SEQ ID NO: 61).

[0112] In particular embodiments, the anti-CD3 binding domain includes the sequence:EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGSGGGSGGGSGGGSQTWTQEPS LTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL (SEQ ID NO: 62);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 63);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 64);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 65);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 66);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 67);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 68);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 69);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 70);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGRFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 71);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 72);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 73);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 74);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 75);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 76);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 77);MAESGGGSVQTGGSLRLSCAYTASSVCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 78);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGRFAISQDYAKKTLYLQ MSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 79);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTKTGYADSVKGRFAISQDYAKKTLYLQ MSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 80);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 81);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 82);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 83);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 84);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 85);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTREGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 86);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 87);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTKTGYADSVKGRFAISQDYAKKTLYLQMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 88);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 89);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLTQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 90);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 91);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPKTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 92);MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYNYWGQGTQVTV (SEQ ID NO: 93); or MAESGGGSVQTGGSLRLSCAYTASSLCMAWFRQAPGKEREGVAVTRDGLPQTGYADSVKGRFAISQDYAKKTLYL QMSSLKPEDTARYYCAARPTSPCTVDGELLASTYDYWGQGTQVTV (SEQ ID NO: 94).

[0113] In particular embodiments, the anti-CD3 binding domain is derived from a CD3 antibody such as the 0KT3 antibody (the same as the one utilized in blinatumomab). The 0KT3 antibody is described in detail in U. S. Patent No.5,929,212. It includes a variable light chain including a CDRL1 sequence including SASSSVSYMN (SEQ ID NO: 95), a CDRL2 sequence including RWIYDTSKLAS (SEQ ID NO: 96), and a CDRL3 sequence including QQWSSNPFT (SEQ ID NO: 97). In particular embodiments, the anti-CD3 binding domain (i.e., binding domain that binds the ICAE CD3) is derived from an antibody including a variable heavy chain including a CDRH1 sequence including KASGYTFTRYTMH (SEQ ID NO: 98), a CDRH2 sequence including INPSRGYTNYNQKFKD (SEQ ID NO: 99), and a CDRH3 sequence including YYDDHYCLDY (SEQ ID NO: 100).

[0114] In particular embodiments, the anti-CD3 binding domain can include or be derived from an scFv derived from OKT3 which retains the capacity to bind CD3. This scFv includes the sequence:QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKS SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASP GEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQW SSNPFTFGSGTKLEINR (SEQ ID NO: 101).

[0115] In particular embodiments, the anti-CD3 binding domain is derived from an anti-CD3 binding domain including a variable light chain including a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 102), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 103). In particular embodiments, the anti-CD3 binding domain is derived from a 20G6-F3 antibody including a variable heavy chain including a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 104), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 105), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NO: 106).

[0116] In particular embodiments, the anti-CD3 binding domain is derived from an anti-CD3 binding domain includinga variable light chain including a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 107), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 108). In particular embodiments, the anti-CD3 binding domain is derived from a 4B4-D7 antibody including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 109), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 110), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 111).

[0117] In particular embodiments, the anti-CD3 binding domain is derived from a 4E7-C9 antibody including a variable light chain including a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 112), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 113). In particular embodiments, the anti-CD3 binding domain is derived from a 4E7-C9 antibody including a variable heavy chain including a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 114), a CDRH2 sequence including IKDKSNNYAT (SEQ ID NO: 115), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 116).

[0118] In particular embodiments, the anti-CD3 binding domain is derived from a 18F5-H10 antibody including a variable light chain including a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 117), a CDRL2 sequence including KVS, and a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 118). In particular embodiments, the anti-CD3 binding domain is derived from a 18F5-H10 antibody including a variable heavy chain including a CDRH1 sequence including GFTFTNAW (SEQ ID NO: 119), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 120), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 121). T

[0119] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WO2016 / 116626. TR66 may also be used. In particular embodiments, additional anti-CD3 binding domains can be found in FIG. 25C.

[0120] In particular embodiments, CDRs for anti-CD3 binding domains are provided in Table 3. CDRs are provided according to Kabat but CDRs according to other CDR definitions could be determined using methods well known to those of skill in the art.

[0121] Table 3. Anti-CD3 binding domain CDRs according to Kabat.

[0122] 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) and is 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 anti-CD28 binding domain is derived from a CD28 antibody. CD28 antibodies include CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0123] Activated T-cells express 4-1 BB (CD137). 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.

[0124] Particular embodiments can include activating CD4+ T cells by binding CD3, TLR2 or CD28 and / or by blocking the suppression of CD4 T cells by binding 4-1 BB, PD-1, LAG3, TIM-3, BTLA, CTLA-4, CD200, and / or VISTA. In particular embodiments, the ICAE includes 4-1BB, PD-1, LAG3, TIM-3, BTLA, CTLA-4, CD200, or VISTA. In particular embodiments, the ICAE binding domain includes a binding domain that binds 4-1 BB, PD-1, LAG3, TIM-3, BTLA, CTLA-4, CD200, or VISTA.

[0125] TLR2 (UniProt ID No. 060603) is involved in the innate immune response to bacterial lipoproteins and other microbial cell wall components. In particular embodiments, the anti-TLR2 binding domain is derived from an anti-TLR2 antibody. Commercially available anti-TLR2 antibodies include anti-hTLR2-lgA and mAb-hTLR2 (both available from Invivogen).

[0126] In particular embodiments, ICEm bind an epitope of co-stimulatory receptor 4-1 BB. 4-1 BB, also called CD 137 or TNFSF9 (UniProt ID No. Q07011) is a T-cell co-stimulatory receptor. In particular embodiments, an anti-4-1 BB binding domain is derived from an anti-4-1 BB antibody including a variable light chain including a CDRL1 sequenceincluding RASQSVS (SEQ ID NO: 122), a CDRL2 sequence including ASNRAT (SEQ ID NO: 123), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 124) and a variable heavy chain including a CDRH1 sequence including YYWS (SEQ ID NO: 125), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 126).

[0127] Cytotoxic T-cells destroy tumor cells. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein at 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. Particular embodiments can include activating CD8 T cells by binding CD3, CD28, or 4-1 BB and / or by blocking the suppression of CD8 T cells by binding PD-1, LAG3, TIM-3, or VISTA.

[0128] Particular embodiments disclosed herein including binding domains that bind epitopes on CD8. In particular embodiments, the anti-CD8 binding domain is derived from the anti-0KT8 antibody.

[0129] In particular embodiments, natural killer cells (also known as NK cells, K cells, and killer cells) are targeted for localized activation. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes, and can secrete cytokines to recruit other immune cells

[0130] 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.

[0131] Examples of commercially available antibodies that bind to an NK cell receptor and induce and / or enhance activation of NK cells include: 5C6 and 1D11, 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®); SK1, which binds and activates CD8; and 3G8 which binds and activates CD16.

[0132] In particular embodiments macrophages are targeted for localized activation. 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.

[0133] The ICEm can be designed to include a binding domain that binds to a molecule expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and MARCO. Commercially available antibodies that bind to molecules 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®).

[0134] In particular embodiments, the ICEm includes a binding domain that binds an epitope of CD40. CD40 (or Tumor necrosis factor receptor superfamily member 5, UniProt ID No. P25942) is a receptor that can transduceactivating signals in macrophages.

[0135] In particular embodiments, examples of inhibitory proteins expressed by macrophages (and their precursors, monocytes) include programmed cell death ligands 1 and 2 (PD-L1 and PD-L2) and galectin 9 (Gal-9).

[0136] In particular embodiments, the ICEm includes a binding domain that binds an epitope of Gal-9 (UniProt ID No.000182) In particular embodiments, an anti-Gal-9 binding domain can be derived from an anti-Gal-9 antibody that blocks binding to TIM-3. An example of a commercially available anti-Gal-9 antibody that blocks TIM-3 binding is 9M1-3 (available from Biolegend).

[0137] (IV) Multimerization Domains and Linkers. An ICEm includes three or more binding domains. Binding domains can multimerize to form an ICEm using a multimerization domain or a linker.

[0138] A “multimerization domain” is a domain that causes two or more monomers (e.g., proteins) to interact with each other through covalent and / or non-covalent association (s). Multimerization domains are highly conserved protein sequences that can include different types of sequence motifs such as leucine zipper, helix loop-helix, ankyrin and PAS (Feuerstein et al, Proc. Natl. Acad. Sci. USA, 91:10655-10659, 1994). Multimerization domains present in proteins can bind to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer, and / or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type (US Patent No. 10030065).

[0139] Dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface" with an engineered "knob" and / or "protruberance" (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336-44, 2004), PTB (Zhou et al., Nature, 378:584-592, 1995), WW (Sudol Prog Biochys MoL Bio, 65: 113-132, 1996), PDZ (Kim et al., Nature, 378: 85-88, 1995; Komau et al., Science, 269:1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489-33494, 1998). Additional examples of molecules that contain dimerization domains / motifs are receptor dimer pairs such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-l and GPIIIb / llla, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993) and variants of some of these domains with modified affinities (PCT Publication No. WO 2012 / 001647).

[0140] In particular embodiments, the ICEm can be prepared using knobs-into holes techniques. Knobs-into-holes refers to forcing the pairing of two different binding domains (e.g., antibody heavy chains) by introducing mutations into the structure (e.g., in the CH3 domains) to modify the contact interface. For example, 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 co-expressing these two 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).

[0141] 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 preferred 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 preferred 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., et al., J. Mol. Biol. 270 (1997) 26-35).

[0142] In particular embodiments, the ICEm includes (i) a first polypeptide chain including a mutated human lgG1 Fc such that it has a knob mutation (referred to as lgG1 Fc Knob) and (ii) a second polypeptide chain including a mutated human lgG1 Fc such that it has a hole mutation (referred to as lgG1 Fc Hole).

[0143] In particular embodiments, the lgG1 Fc Knob includes the sequence: GSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKGS (SEQ ID NO: 127) with the mutations underlined and the lgG1 Fc Hole includes the sequence: GSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKG (SEQ ID NO: 128) with the mutations (e.g., hinge mutant (C to S), knob / hole mutation, and N297A mutation) underlined.

[0144] In particular embodiments, the lgG1 Fc Knob includes the sequence: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 129) with the mutations underlined and the lgG1 Fc Hole includes the sequence: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 130) with the mutations underlined.

[0145] In particular embodiments, the lgG1 Fc Knob with the LALAPG Fc mutation includes the sequence: EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 131) and the lgG1 Fc Hole with the LALAPG Fc mutation includes the sequence: EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVL LHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 132).

[0146] In particular embodiments, the sequence corresponding to a dimerization motif / domain includes the leucine zipper domain of Jun (US5932448; RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN (SEQ ID NO: 133)), the dimerization domain of Fos (US 5932448; LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA (SEQ ID NO: 134)), a consensus sequence for a WW motif (PCT Publication No. WO 1997 / 037223), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621, 2005;WREFCESHARAAALDFARRFRLYLASHPQYAGPGAEAAFSRRFAELFLQHFEAEVARAS (SEQ ID NO: 135)), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen et al., EMBO J., 25: 785-797, 2006; THRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIE (SEQ ID NO: 136)), 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;LGEVHRFHVTVAPGTKLESGPATLHLCNDVLVLARDIPPAVTGQWKLSDLRRYGAVPSGFIFEGGTRCGYWAGVFFL SSAEGEQISFLFDCIVRGISPTKG (SEQ ID NO: 137)), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21: 5591-5604, 2001;DCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDATVADMLQDVYHWTL KIQLHSCPKLEDLPPEQWSHTTVRNALKDLLKDMNQSS (SEQ ID NO: 138)), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463;CAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQ LSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVTCLYCNSFSKQS KQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCLSESTNSTERNVMWGGCGSQLFSYAAFSDSNIIT VWDTALYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKETKIFSFSNDFTIQKLIETRTNKESKHKMSYSGRVK TLCLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQKEIQSCLT VWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE (SEQ ID NO: 139)), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079-4088, 1998;DQELKHLILEAADGFLFIVSCETGRWYVSDSVTPVLNQQQSEWFGSTLYDQVHPDDVDKLREQLSTSENALTGR(SEQ ID NO: 140)) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014;LSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES (SEQ ID NO: 141)).

[0147] In particular embodiments, the dimerization domain can be a dimerization and docking domain (DDD) on one binding domain and an anchoring domain (AD) on another binding domain to facilitate a stably tethered structure. In particular embodiments, the DDD (DDD1 and DDD2) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) are derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. In particular embodiments, DDD1 includes the amino acid sequence: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 142). In particular embodiments, DDD2 includes the amino acid sequence: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 143). In particular embodiments, AD1 includes the amino acid sequence: QIEYLAKQIVDNAIQQA (SEQ ID NO: 144). In particular embodiments, AD2 includes the amino acid sequence: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 145). However, one skilled in the art will realize that other DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains may be obtained from p53, DCoH (pterin 4 alpha carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1)) and HNF-1 (hepatocyte nuclear factor 1). Other AD sequences of potential use may be found in Patent Publication No. US2003 / 0232420A1.

[0148] The X-type four-helix bundle dimerization motif that is a structural characteristic of the DDD (Newlon, et al. EMBO J. 2001; 20: 1651-1662; Newlon, etal. Nature Struct Biol. 1999; 3: 222-227) is found in other classes of proteins, such as the S100 proteins (for example, S100B and calcyclin), and the hepatocyte nuclear factor (HNF) family of transcriptional factors (for example, HNF-1 a and HNF-1 ). Over 300 proteins that are involved in either signal transduction or transcriptional activation also contain a module of 65-70 amino acids termed the sterile a motif (SAM) domain, which has a variation of the X-type four-helix bundle present on its dimerization interface. For S100B, this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951-1960). Similarly, the N-terminal dimerization domain of HNF-1a (HNF-p1) was shown to associate with a dimer of DCoH (dimerization cofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). In alternative embodiments, these naturally occurring systems can also be used to provide stable multimeric structures with multiple functions or binding specificities. Other binding events such as those between an enzyme and its substrate / inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generate the two associating components (the “docking” step), which are subsequently stabilized covalently (the “lock” step).

[0149] In particular embodiments, dimerization of binding domains can be induced by a chemical inducer. This method of dimerization requires one binding domain to contain a chemical inducer of dimerization binding domain 1(CBD1) and the second binding domain to contain 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). If the CID is rapamycin, CBD1 and CBD2 can be the rapamycin binding domain of FK-binding protein 12 (FKBP12) and the FKBP12-Rapamycin Binding (FRB) domain of mTOR. In particular embodiments, FKBP12 includes the sequence: MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 146).

[0150] In particular embodiments, FRB includes the sequence:MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLTQAWDLYYHVFRRISKLES (SEQ ID NO: 147). If the CID is FK506 / cyclosporin fusion protein or a derivative thereof, CBD1 and CBD2 can be the FK506 (Tacrolimus) binding domain of FK-binding protein 12 (FKBP12) and the cyclosporin binding domain of cylcophilin A. If the CID is estrone / biotin fusion protein or a derivative thereof, CBD1 and CBD2 can be an oestrogen-binding domain (EBD) and a streptavidin binding domain. If the CID is dexamethasone / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain. If the CID is 06-benzylguanine derivative / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be an 06-alkylguanine-DNA alkyltransferase (AGT) binding domain and a dihydrofolate reductase (DHFR) binding domain. If the CID is RSL1 or a derivative thereof, CBD1 and CBD2 can be a retinoic acid receptor domain and an ecodysone receptor domain. If the CID is AP1903 or a derivative thereof, CBD1 and CBD2 can be the FK506 binding protein (FKBP12) binding domains including a F36V mutation. Use of the CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin: KTW has high, KHF intermediate and PLW is low (Bayle et al, Chemistry & Biology 13, 99-107, January 2006).

[0151] In particular embodiments, binding domains can multimerize using a transmembrane polypeptide derived from a FceRI chain. In particular embodiments, a binding domain can include a part of a FcsRI alpha chain and another binding domain can include a part of an FCERI beta chain or variant thereof such that said FCERI chains spontaneously dimerize together to form a dimeric binding domain. In particular embodiments, binding domains can include a part of a FCERI alpha chain and a part of a FCERI gamma chain or variant thereof such that said FCERI chains spontaneously trimerize together to form a trimeric binding domain, and in another embodiment the multi-chain binding domain can include a part of FCERI alpha chain, a part of FCERI beta chain and a part of FCERI gamma chain or variants thereof such that said FCERI chains spontaneously tetramerize together to form a tetrameric binding domain.

[0152] In particular embodiments, additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in a binding domain could include: replacing the C-terminus domain with murine counterparts; generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both binding domains; swapping interacting residues in each of the binding domains in the C-terminus domains ("knob-in-hole”); and fusing the variable domains of the binding domains directly to CD3 (CD3 fusion)(Schmitt et al., Hum. Gene Ther. 2009. 20:1240-1248).

[0153] In particular embodiments, an ICEm can be formed by linking binding domains (e.g., single-domain antibodies) together with linkers. In certain aspects, multimerization is achieved by binding domains in a fusion protein with protein linkers. Fusion proteins include different protein domains (e.g., single-domain antibodies) linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0154] Commonly used flexible linkers include linker sequences 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: 148), (Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 149), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 150), and (Gly3Ser)n(Gly4Ser)1 (SEQ ID NO: 151). In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 152), (Gly4Ser)3 (SEQ ID NO: 153), (Gly4Ser)2 (SEQ ID NO: 154), (Gly4Ser)1 (SEQ ID NO: 155), (Gly3Ser)2 (SEQ ID NO: 156), (Gly3Ser)1 (SEQ ID NO: 157), (Gly2Ser)2 (SEQ ID NO: 158) or (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 159), GGSGGGSGSG (SEQ ID NO: 160), or GGSGGGSG (SEQ ID NO: 161).

[0155] In particular embodiments, a linker includes the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 162).

[0156] 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 prolinerich 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).

[0157] Linkers can also include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkerscan be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0158] Tri-specific antibodies are artificial proteins that simultaneously bind to three different types of antigens. Trispecific antibodies are described in, for example, WO2016 / 105450, WO 2010 / 028796; WO 2009 / 007124; WO 2002 / 083738; US 2002 / 0051780; and WO 2000 / 018806.

[0159] In some embodiments, a multi-domain binding molecule includes a basic immunoglobulin structure such as an IgM domain. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g, Harlow eta / ., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0160] 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 IgM constant regions asa 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.

[0161] Multi merized antibodies and antibody-like molecules such as 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 US20180265596 A1, and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 165340.

[0162] Particular embodiments include using IgM constant region domains to allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow etal., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0163] IgM possesses an 18-amino acid extension in the C terminus called the "tailpiece" (tp). The IgM tp is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. tp contain an N-linked carbohydrate addition site, the presence of which is required for pentamer formation in IgM.

[0164] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0165] 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 C|u4-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.

[0166] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the C|d4 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 C|u4 and / or tp domains (also referred to herein collectively as C|u4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the C|j4-tp domains. An IgM heavy chain constant region can additionally include a C 3 domain or a fragment thereof, a Cpi2 domain or a fragment thereof, a C i 1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0167] 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 p chains before IgM is secreted from antibody-producing 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 etal. 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 mushroom-shaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: 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.

[0168] 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.

[0169] 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 Cpi1), an antibody heavy chain constant domain 2 (CM2 or Cpi2), an antibody heavy chain constant domain 3 (CM3 or Cpi3), and an antibody heavy chain constant domain 4 (CM4 or Cpi4) that can include a tp, as indicated above.

[0170] 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 Cpi4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM Cpi3 domain situated N-terminal to the IgM Cpi4 and IgM tp domains.

[0171] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cpi2 domain situated N-terminal to the IgM Cpi3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cpi2, Cpi3, and Cpi4-tp domains as follows:VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTST LTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWT RQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAR EQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCW AHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 163).

[0172] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include,instead of, or in addition to an IgM C 2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM C 3 domain. An exemplary variant human lgG1 hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine isVEPKSSDKTHTCPPCPAP (SEQ ID NO: 164). An exemplary IgM constant region of this type includes the variant human IgG 1 hinge region fused to a multimerizing fragment of the human IgM constant region including the Cpi3, C 4, and tp domains, and includes the amino acid sequence:VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHP NATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFS PADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKST GKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 165).

[0173] 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: 166 or SEQ ID NO: 167 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.

[0174] The human IgM heavy chain constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 166; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1). Referring to this SEQ ID NO: 166, the human Cpi1 region ranges from amino acid 5 to amino acid 102; the human Cpi2 region ranges from amino acid 114 to amino acid 205, the human Cpi3 region ranges from amino acid 224 to amino acid 319, the Cpi4 region ranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0175] In particular embodiments, an IgM heavy chain constant region includes the sequence:GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 167; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 166 by one amino acid at position 191.

[0176] 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: 166 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.

[0177] 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: 166. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 166 corresponds to S524 of Kabat; E402 of SEQ ID NO: 166 corresponds to E525 of Kabat; E403 of SEQ ID NO: 166 corresponds to E526 of Kabat; R344 of SEQ ID NO: 166 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 166 corresponds to E468 of Kabat.

[0178] In particular embodiments, “corresponds to” means the designated position of SEQ ID NO: 166 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.

[0179] In particular embodiments, P311 of SEQ ID NO: 166 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 166 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 166 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL ASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 168).

[0180] In certain aspects, S401 of SEQ ID NO: 166 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 166 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVAEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 169).

[0181] In certain aspects, E402 of SEQ ID NO: 166 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 166 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSAEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 170).

[0182] In certain aspects, E403 of SEQ ID NO: 166 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 166 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEAEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 171).

[0183] In certain aspects, R344of SEQ ID NO: 166 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 166 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 172).

[0184] In certain aspects, E345 of SEQ ID NO: 166 can be substituted with any amino acid. In certain aspects, E345of SEQ ID NO: 166 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 173).

[0185] 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:MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRT RFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD(SEQ ID NO: 174). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 174 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 174.

[0186] The mature human J-chain includes the amino acid sequence QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 175).

[0187] 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: 175. 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.

[0188] 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: 175.

[0189] By “an amino acid corresponding to” a position of SEQ ID NO: 175 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: 175 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 175 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: 175 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.

[0190] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 175 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 175 can be substituted with alanine (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCATYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 176),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCSTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 177),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCRTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 178).

[0191] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 175 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 175 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYAYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 179).

[0192] 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: 175, 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: 175.

[0193] The amino acids corresponding to N49 and S51 of SEQ ID NO: 175 along with the amino acid corresponding to 150 of SEQ ID NO: 175 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: 175 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 175 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: 175 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid seguence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 180).

[0194] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 175 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 175 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 175 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:EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLCKKCDPTEVELDNQI VTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 181).

[0195] 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: 148) wherein n is 1-5.

[0196] A 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 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: 175 between cysteine residues 92 and 101 of SEQ ID NO: 175. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 175 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: 175 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0197] In particular embodiments, the binding domain is introduced into the native human J-chain sequence of SEQ ID NO: 175 by chemical or chemo-enzymatic derivatization. In particular embodiments, the binding domain is introduced into the native human J-chain sequence of SEQ ID NO: 175 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-translationally attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

[0198] 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.

[0199] 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 simum, 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, Chrysemyspicta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0200] In particular embodiments, an IgM multimerization domain includes the sequence: PLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTIS WTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLP PAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYT CWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 182) and a J-chain including the sequence of SEQ ID NO: 175.

[0201] Additional scaffolds for multimerization are in FIG. 25D.

[0202] (V) Variants & Nucleic Acid Sequences. Amino acid and nucleic acid sequence variations of the binding domains or ICEm disclosed herein are contemplated. Variations can include additions, deletions or substitutions of residues within the amino acid sequences. Variations of sequences disclosed herein include CDR variants, variant Fc regions, or humanized antibodies

[0203] In particular embodiments, variants of the sequences disclosed herein leave CDR sequences unchanged. In particular embodiments, variants of the sequences disclosed herein change the CDR sequences by less than 80%, less than 85%, less than 90%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% compared to the sequence disclosed herein. In particular embodiments, variants of the sequences disclosed herein replace one or more tyrosines with CDRs to a less hydrophobic amino acid.

[0204] Variants of the sequences disclosed and referenced herein are also included. In certain examples, SEQ ID NOs. should be interpreted to include the entirety of the sequence associated with the number. In other embodiments, SEQ ID NOs. reference only the portion of the sequence between an N-terminal signal peptide and a C-terminal tag. Such signal peptides and tags are readily identifiable by one of ordinary skill in the art. Sequences with less than 100% sequence identity to a reference sequence can include those in which an N-terminal signal peptide and / or a C-terminal tag is removed.

[0205] 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.

[0206] 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, TheBenjamin / 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 (non-polar): 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.

[0207] 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).

[0208] It is known in the art that certain amino acids may be substituted by other amino acids having 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.

[0209] 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.

[0210] In particular embodiments, a binding domain of the present disclosure can be derived from or based on a binding domain of a known antibody (e.g., single-domain antibody) and can 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), ora combination of the above-noted changes, when compared with the binding domain of the known antibody. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain including the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0211] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid sidechain 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.

[0212] Variants of the protein and nucleic acid 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 and nucleic acid sequences described or disclosed herein.

[0213] In particular embodiments, a binding domain includes or is a 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 a known amino acid sequence of a heavy chain variable region (VH), or both.

[0214] 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 pig / 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. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0215] “ % 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 and nucleic acidsequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including (but not limited to) 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). Preferred 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, 111-20. 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. "Default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0216] CDR sequences within binding domains can be based on LlamaMagic or based on other methods known in the art. For example, definitive delineation of a CDR and identification of residues including the binding site of a binding domain can be accomplished by solving the structure of the binding domain and / or solving the structure of the binding domain-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography.

[0217] In addition to LlamaMagic, CDR sets can be based on, for example, 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.

[0218] Software programs and bioinformatic tools, such as ABodyBuilder and Paratome can also be used to determine CDR sequences.

[0219] 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 lgG1, lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. An "Fc region variant” includes an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from one to ten amino acid substitutions, and preferably from one to five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably at least 90% homology therewith, more preferably at least 95% homology therewith. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described elsewhere herein.

[0220] Binding domains can be humanized. Humanized binding domains have lowered immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-humanized binding domains.

[0221] A “humanized” antibody refers to a chimeric antibody including amino acid residues from non-human CDRs and amino acid residues from human FRs In particular embodiments, a humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0222] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008, and are further described, e.g., in Riechmann et al., Nature 332:323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033, 1989; U. S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60,2005 (describing “FR shuffling’’); and Osbourn et al., Methods 36:61-68, 2005 and Klimka et al., Br. J. Cancer, 83:252-260, 2000 (describing the “guided selection” approach to FR shuffling). EP-B-0239400 provides additional description of “CD R-g rafting”, in which one or more CDR sequences of a first antibody is / are placed within a framework of sequences not of that antibody, for instance of another antibody.

[0223] Human framework regions that may be used for humanization include: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); framework regions derived from the consensussequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc Natl. Acad. Sci. USA, 89:4285, 1992; and Presta et al., J. Immunol., 151:2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.13:1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.272:10678-10684, 1997; and Rosok et al, J. Biol. Chem. 271:22611-22618, 1996).

[0224] In particular embodiments, variants have the same or decreased binding to a targeted antigen as the variant’s original reference sequence. "Bind" means that the binding domain associates with its target epitope with a dissociation constant (1(D) of 108M or less, in particular embodiments of from 10-5M to 10-13M, in particular embodiments of from 105M to 10'10M, in particular embodiments of from 10-5M to 10 M, in particular embodiments of from 108M to 10-13M, or in particular embodiments of from 10'9M to 10’13M. The term can be further used to indicate that the binding molecule does not bind to other biomolecules present, (e.g, it binds to other biomolecules with a dissociation constant (KD) of 10'4M or more, in particular embodiments of from 10’4M to 1 M). A targeted epitope is one that will be bound by its corresponding ICEm under relevant in vitro conditions and in in vivo conditions as described herein. In particular embodiments, relevant in vitro conditions for binding can include a buffered salt solution approximating physiological pH (7.4) at room temperature or 37°C.

[0225] (VI) Expression of Proteins. The present disclosure includes methods of producing the ICEm disclosed herein. In particular embodiments, the method includes vector construction and expression within a host. In particular embodiments, the method includes nucleic acid synthesis and codon optimization, vector construction, expression within a host cell, and purification.

[0226] Particular embodiments utilize genetic constructs (e.g, chimeric genes, expression cassettes, expression vectors, recombination vectors, etc.) including a nucleic acid sequence encoding the protein or proteins of interest (i.e, coding sequence) operatively linked to appropriate expression control sequences. These genetic constructs are not naturally-occurring DNA molecules and are useful for introducing DNA into host-cells to express selected proteins of interest.

[0227] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g, Maniatiset al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y, 1982; and Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y, 1989.

[0228] Expression control sequences are nucleic acid (e.g, DNA) sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expression control sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure,such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361, 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0229] The promoter may include, or be modified to include, one or more enhancer elements. In particular embodiments, the promoter will include a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0230] For efficient expression, the coding sequences can be operatively linked to a 3' untranslated sequence. In particular embodiments, the 3' untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3' untranslated region can be obtained, for example, from the flanking regions of genes.

[0231] In particular embodiments, a 5' untranslated leader sequence can also be employed. The 5' untranslated leader sequence is the portion of a nucleic acid sequence that extends from the 5' CAP site to the translation initiation codon.

[0232] In particular embodiments, the genetic construct includes sequences encoding linkers. Commonly used linkers are described elsewhere herein In particular embodiments, the linker includes the sequence GS. In particular embodiments, the linker includes the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 153) or the Whitlow linker (SEQ ID NO: 162).

[0233] In particular embodiments, the genetic construct includes a sequence encoding a signal peptide sequence. In particular embodiments, the signal peptide includes the sequence: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 183).

[0234] In addition to the coding sequence and expression control sequence, genetic constructs can include a sequence encoding a tag. Example tags include tags can include, for example, His tag (HHHHHH (SEQ ID NO: 184)), Flag tag (DYKDDDD (SEQ ID NO: 185), Xpress tag (DLYDDDDK (SEQ ID NO: 186)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 187)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 188)), Polyglutamate tag (EEEEEE (SEQ ID NO: 189)), HA tag (YPYDVPDYA (SEQ ID NO: 190)), Myc tag (EQKLISEEDL (SEQ ID NO: 191)), Strep tag (WRHPQFGG (SEQ ID NO: 192)), STREP® tag II (WSHPQFEK (SEQ ID NO: 193); IBA Institutfur Bioanalytik, Germany; see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 194)), Softag 3 (TQDPSRVG (SEQ ID NO: 195)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 196)) In particular embodiments, the tag includes the sequence AWSHPQFEK (SEQ ID NO: 197). In particular embodiments, the tag includes the sequence SAWSHPQFEK (SEQ ID NO: 198).

[0235] In particular embodiments, a “hisavi" tag can be added to the N-terminus or C-terminus of a gene by the addition of nucleotides coding for the Avitag amino acid sequence (SEQ ID NO: 187), as well as the 6xhistidine tag coding sequence (SEQ ID NO: 184). The Avitag avidity tag can be biotinylated by a biotin ligase to allow for biotinavidin or biotin-streptavidin based interactions for protein purification, as well as for immunobiology (such as immunoblotting or immunofluorescence) using anti-biotin antibodies. The 6xhistidine tag allows for protein purification using Ni-2+ affinity chromatography.

[0236] In particular embodiments, genetic constructs 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 coding sequence and a polynucleotide encoding a selectable marker. Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A; coding sequence: GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCA GGCCGGCGACGTGGAGGAGAACCCCGGCCCCGGAGCTAGCGGA (SEQ ID NO: 278)), Thosea asigna virus (T2A; GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 279)), equine rhinitis A virus (E2A; GSGQCTNYALLKLAGDVESNPGPP (SEQ ID NO: 280)), foot-and-mouth disease virus (F2A; GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 281)). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011)).

[0237] Nucleic acid sequences encoding proteins disclosed herein can be derived by those of ordinary skill in the art. Nucleic acid sequences can also include one or more of various sequence polymorphisms, mutations, and / or sequence variants. In particular embodiments, the sequence polymorphisms, mutations, and / or sequence variants do not affect the function of the encoded protein. The sequences can also include degenerate codons of a native sequence or sequences that may be introduced to provide codon preference.

[0238] In some aspects, the genetic constructs can be introduced by transfection, a technique that involves introduction of genetic constructs into the nucleus of eukaryotic cells. In some aspects, the proteins can be synthesized by transient transfection (DNA does not integrate with the genome of the eukaryotic cells, but the genes are expressed for 24-96 hours). Various methods can be used to introduce the genetic constructs into the host-cells, and transfection can be achieved by chemical-based means including by the calcium phosphate, by dendrimers, by liposomes, and by the use of cationic polymers. Non-chemical methods of transfection include electroporation, sonoporation, optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery. In some embodiments, transfection can be achieved by particle-based methods including gene gun where the genetic construct is coupled to a nanoparticle of an inert solid which is then "shot" directly into the target-cell's nucleus. Other particle-based transfection methods include magnet assisted transfection and impalefection.

[0239] Any cell suitable for expression of a genetic construct can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as mammalian cells. Examples of bacterial host cells include Escherichia coli, Streptomyces, or Salmonella typhimurium. Mammalian host cells can include HEK293 cells or CHO cells.

[0240] Once expressed, ICEm and / or binding domains 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)).

[0241] (VII) Modifications to Provide Administration Benefits. In particular embodiments, the ICEm can be modified to produce an administration benefit. Exemplary administration benefits can include (1) altered binding affinities, (2)altered binding affinity for forming protein complexes, (3) reduced susceptibility to proteolysis, (4) reduced susceptibility to oxidation, (5) reduced immunogenicity; and / or (6) modified (extended or shortened) half-life. While the disclosure below describes these modifications in terms of their application to antibodies, the modifications can also be applied to the ICEm format. In particular embodiments, the ICEm are modified at the nucleic acid level or at the protein level. In particular embodiments, modifications include Fc silencing mutations. Fc silencing provides several benefits including biodistribution of the ICEm, lowers toxicity, and prevents exhaustion of immune cells in the periphery.

[0242] In particular embodiments, ICEm are modified to have reduced binding affinity. For example, one or more of the binding domains of an ICEm are modified to have reduced binding affinity. An example modification to reduce binding affinity includes replacing tyrosines within the CDR of a binding domain with a less hydrophobic amino acid (e.g., an alanine). In particular embodiments, one, more than one or all of the tyrosines can be replaced with a less hydrophobic amino acid. In particular embodiments, the anti-CD19 binding domain is modified to replace tyrosines within the CDR with alanines. In particular embodiments, the anti-CD33 binding domain is modified to replace tyrosines within the CDR with alanines.

[0243] In particular embodiments, modified ICEm include those wherein one or more amino acids have been replaced with a different amino acid, 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, 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. Nucleic acid(s) can be modified to result in an amino acid mutation.

[0244] 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-linked carbohydrate 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 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 cysteinevariants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0245] In particular embodiments, the ICEm can be fused or coupled to an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of an ICEm that contains the altered Fc polypeptide as compared to the halflife of a similar ICEm containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, the Fc polypeptide includes a substitution at positions CH24, CH25, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type lgG1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH2 4, CH2 5, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH24, or CH25, or both. In particular embodiments, the antibody includes both, a CH2 L4A and a CH2 L5A substitution. In one aspect, the substitutions are L234A and L235A (LALA). Such antibodies are referred to herein as a "LALA” variant. Interestingly, a "LALA" 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.

[0246] In particular embodiments, the Fc polypeptide includes a substitution at position L234A, L235A and P329G in an Fc region derived from a human lgG1 Fc region. Such mutations are referred to as ALAPG” mutations. In particular embodiments, the LALAPG mutation contributes to Fc silencing.

[0247] In another aspect, the substitutions are L234A, L235A and D265A in an Fc region derived from a human lgG1 Fc region. This mutation is referred to as a “LALA-DA” mutation.

[0248] 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 ICEm described herein.

[0249] 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 core-hinge region to a proline creates the lgG4_S228P mutant. In particular embodiments, the I gG4_S228P mutant prevents Fab arm exchange.

[0250] In particular embodiments, it may be desirable to create cysteine engineered ICEm in which one or more residues of an ICEm are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the ICEm. By substituting those residues with cysteine, reactive thiol groups are therebypositioned at accessible sites of the ICEm and may be used to conjugate the ICEm 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.

[0251] 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 et al., 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.

[0252] 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 acetylphenylalanine residues can be performed.

[0253] 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).

[0254] 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.

[0255] 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 or ICEm 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 WO 2008 / 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 etal. 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, knockoutCHO cells (see, e.g, Yamane-Ohnuki etal., Biotech. Bioeng. 87: 614, 2004; Kanda etal., Biotechnol. Bioeng, 94(4):680-688, 2006; and W02003 / 085107).

[0256] (VIII) Antibody Conjugates. Antibody conjugates include binding domains disclosed herein (e.g, ICEm) linked to another molecule. Examples of antibody conjugates include antibody immunotoxins, antibody-drug conjugates (ADCs), antibody-detectable label conjugates, antibody radioisotope conjugates, and antibody-particle conjugates.

[0257] Antibody immunotoxins include an ICEm 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 ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, 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.

[0258] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies (e.g, ICEm) and cytotoxic drugs (e.g, chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter and Senter (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 et al. (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 [MMAEj; 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.

[0259] Antibody-detectable label conjugates include an ICEm (e.g., molecule with an ICAE binding domain, a CD19 binding domain, and a CD33 binding domain) linked to a detectable label. Detectable labels can include any suitable label or detectable group 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.

[0260] 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.

[0261] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester

[0262] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0263] 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-Vl-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0264] Antibody-radioisotope conjugates include an ICEm 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 daughter isotope223Ra. Additional examples of useful radioisotopes include228Ac,111Ag,124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,169Dy,166Dy,168Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,263Fm,66Ga,72Ga,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,280Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,1910s,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,163Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,186Tm,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 includes131!,90Y, and / or211At. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0265] Antibody-particle conjugates include an antibody (e.g., ICEm) 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, and / or colored beads, and can be made from organic matter and / or inorganic matter.

[0266] In particular embodiments, an antibody 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).

[0267] (IX) Compositions for Administration. Any of the ICEm described herein in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the ICEm 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). ICEm and / or nucleic acids encoding ICEm are collectively referred to herein as "active ingredients”. Salts and / or pro-drugs of active ingredients can also be used.

[0268] A pharmaceutically acceptable salt includes any salt that retains the activity of the active ingredient 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.

[0269] 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 andphosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0270] 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.

[0271] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of an active ingredient or by hydrolysis of a biologically labile group.

[0272] 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.

[0273] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0274] 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.

[0275] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0276] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0277] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldi methyl benzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0278] 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.

[0279] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation,infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intramuscular, intravesicular, and / or subcutaneous injection. A delivery vehicle refers to any method, apparatus, or system used to administer or introduce an active ingredient. Examples of delivery vehicles include syringes, needles, catheters, infusion pumps, transdermal patches, inhalers, or oral dosage forms.

[0280] 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.

[0281] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid compositions such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0282] 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.

[0283] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.

[0284] 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 chemicalnature, release one or more active ingredients following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0285] Depot compositions can include a variety of bioerodible polymers including poly (lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0286] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0287] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g, F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0288] Excipients that partition into the external phase boundary of nanoparticles or microparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0289] Additional processing of the disclosed sustained release depot formulations can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0290] In particular embodiments, 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.

[0291] Any composition 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 can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U. S. FDA Office of Biological Standards and / or otherrelevant foreign regulatory agencies.

[0292] In particular embodiments, compositions include immunogenic compositions. An immunogenic composition refers to a composition that stimulates an immune response in a subject. The immune response can be, for example, a T-cell response. A T-cell response can be detected, for example, by measuring production of cytokines, such as interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), interleukin (IL)-2, IL-4, IL-10, and IL-17. In particular embodiments, a T-cell response can be detected by measuring production of granzyme B and / or perforin.

[0293] In particular embodiments, compositions include therapeutic compositions. A therapeutic composition refers to a composition that treats a subject. In particular embodiments, efficacy of a treatment can be detected by a reduction in a subject's disease (e.g., cancer) or symptoms as described elsewhere herein.

[0294] (X) Kits. Also disclosed herein are kits including at least one ICEm, sequences encoding at least one ICEm, at least one binding domain, sequence encoding at least one binding domain, and / or compositions disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the method described herein. In particular embodiments, a kit includes a pharmaceutically acceptable carrier and / or a delivery vehicle.

[0295] In particular embodiments, kits can include one or more containers including one or more ICEm or compositions disclosed herein. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. In particular embodiments, ICEm within kits are chosen based on assessment of a particular subject's anticipated disease course.

[0296] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions” can include the address of an internet site which provides instructions.

[0297] (XI) 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 disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0298] An "effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are oftenadministered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model or clinical study relevant to the assessment of a cancer's development or progression. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0299] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer or displays only early signs or symptoms of a cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer further. Thus, a prophylactic treatment functions as a preventative treatment against a cancer.

[0300] A "therapeutic treatment” includes a treatment administered to a subject who displays signs or symptoms of a cancer and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms 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.

[0301] 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.

[0302] The disclosed ICEm are useful for targeting and killing cells co-expressing both CD19 and CD33. Examples of CD19 and CD33 co-expressing cancers include B cell malignancies such as chronic phase leukemia, accelerated phase leukemia, blast phase leukemia, mixed phenotype acute leukemia (MPAL), and subsets of B-cell acute lymphoblastic leukemia (B-ALL). B cell malignancies are often related to problems with the bone marrow such as anemia, tiredness, pale skin, irritability, and faster breathing. Similarly, symptoms of B cell malignancies include weakness, fatigue, fever, night sweats, shortness of breath, swollen lymph nodes, bleeding or bruising, and loss of appetite or weight loss.

[0303] 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.

[0304] Anti-cancer effects can also reduce or eliminate side effects of a cancer, such as anemia, tiredness, weakness, fatigue, pale skin, irritability, fever, night sweats, shortness of breath, swollen lymph nodes, bleeding or bruising, and loss of appetite or weight loss.

[0305] 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. 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, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0306] Useful doses can range from 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 to5 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.

[0307] 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.

[0308] In particular embodiments, therapeutically effective amounts are administered at a time interval to reduce or eliminate cancer recurrence without causing autoimmune toxicity.

[0309] The ICEm described herein can be administered on top of the current standard of care for 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

[0310] The compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration and more particularly by intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual injection.

[0311] 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.

[0312] (XII) Exemplary Embodiments.1. A multi-domain binding molecule including:an immune cell activating epitope binding domain, an anti-CD19 binding domain, and an anti-CD33 binding domain.2. The multi-domain binding molecule of embodiment 1, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding lack an Fc region of an antibody.3. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, and the anti-CD33 binding lack an Fc region of an antibody.4. The multi-domain binding molecule of any of embodiments 1-3, comprising a sequence with at least 90%sequence identity to SEQ ID NO: 334, 335, 337, 338, or 339.5. The multi-domain binding molecule of any of embodiments 1-4, comprising SEQ ID NO: 334, 335, 337, 338, or 339.6. The multi-domain binding molecule of any of embodiments 1-5, wherein the immune cell activating epitope includes an epitope on a T cell, an NK cell, or a macrophage.7. The multi-domain binding molecule any of embodiments 1-6, wherein the immune cell activating epitope includes CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.8. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope includes CD3, CD28, 4-1 BB, or CD8.9. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope includes NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.10. The multi-domain binding of embodiment 1 or 2, wherein the immune cell activating epitope includes CD 11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFNGR2, Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.11. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope binding domain binds and activates a T cell.12. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope binding domain includes an anti-CD3 binding domain.13 The multi-domain binding molecule of embodiment 8, wherein the anti-CD3 binding domain includes a variable heavy chain including complementarity determining region (CDR) heavy (H)1 including the sequence of SEQ ID NO: 308, a CDRH2 including the sequence of SEQ ID NO: 309, and a CDRH3 including the sequence of SEQ ID NO: 310, and a variable light chain including a CDR light (L)1 including the sequence of SEQ ID NO: 311, a CDRL2 including the sequence of SEQ ID NO: 312, and a CDRL3 including the sequence of SEQ ID NO: 313;a CDRH1 including the sequence of SEQ ID NO: 314, a CDRH2 including the sequence of SEQ ID NO: 315, and a CDRH3 including the sequence of SEQ ID NO: 316, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 317, a CDRL2 including the sequence of SEQ ID NO: 318, and a CDRL3 including the sequence of SEQ ID NO: 319;a CDRH1 including the sequence of SEQ ID NO: 320, a CDRH2 including the sequence of SEQ ID NO: 321, and a CDRH3 including the sequence of SEQ ID NO: 322, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 323, a CDRL2 including the sequence of SEQ ID NO: 324, and a CDRL3 including the sequence of SEQ ID NO: 325;a CDRH1 including the sequence of SEQ ID NO: 104, a CDRH2 including the sequence of SEQ ID NO: 105, and a CDRH3 including the sequence of SEQ ID NO: 106, and a variable light chain including a CDRL1 including thesequence of SEQ ID NO: 102, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 103;a CDRH1 including the sequence of SEQ ID NO: 109, a CDRH2 including the sequence of SEQ ID NO: 110, and a CDRH3 including the sequence of SEQ ID NO: 111, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 107, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 108;a CDRH1 including the sequence of SEQ ID NO: 114, a CDRH2 including the sequence of SEQ ID NO: 115, and a CDRH3 including the sequence of SEQ ID NO: 116, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 112, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 113; ora CDRH1 including the sequence of SEQ ID NO: 119, a CDRH2 including the sequence of SEQ ID NO: 120, and a CDRH3 including the sequence of SEQ ID NO: 121, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 117, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 11814. The multi-domain binding molecule of embodiment 8, wherein the anti-CD3 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 53 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 54 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 56 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 57 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 58 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 59 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 60 or a sequence having at least 95% sequence identity thereto.15. The multi-domain binding molecule of embodiment 8, wherein the anti-CD3 binding domain includes a sequence of SEQ ID NO: 61, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 217, or SEQ ID NO: 218 or a sequence having at least 90% sequence identity to SEQ ID NO: 61, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 217, or SEQ ID NO: 218.16 The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence having a CDR1 including a sequence of SEQ ID NO: 95, a CDR2 including a sequence of SEQ ID NO: 96, and a CDR3 including a sequence of SEQ ID NO: 97; ora CDR1 including a sequence of SEQ ID NO: 98, a CDR2 including a sequence of SEQ ID NO: 99, and a CDR3 including a sequence of SEQ ID NO: 100.17. The multi-domain binding molecule of embodiment 1 or 2, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence of SEQ ID NOs: 62-94 or SEQ ID NO: 101, or a sequence having at least 95% sequence identity to SEQ ID NOs: 62-94 or SEQ ID NO: 101.18. The multi-domain binding molecule of any of embodiments 1-13, wherein the anti-CD19 binding domain includes a variable heavy chain including CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 284, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 284, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 288, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 289, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 290, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287; ora CDRH1 including the sequence of SEQ ID NO: 291, a CDRH2 including the sequence of SEQ ID NO: 292, and a CDRH3 including the sequence of SEQ ID NO: 293, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 294; a CDRL2 including the sequence of SEQ ID NO: 295, and a CDRL3 including the sequence of SEQ ID NO: 296.19. The multi-domain binding molecule of any of embodiments 1-13, wherein the anti-CD19 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 5 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 8 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 9 or a sequence having at least 95% sequenceidentity thereto, and a variable light chain including the sequence of SEQ ID NO: 10 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 11 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 12 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 13 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 14 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 34 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 35 or a sequence having at least 95% sequence identity thereto.20. The multi-domain binding molecule of any of embodiments 1-13, wherein the anti-CD19 binding domain includes a sequence having a CDR1 including a sequence of SEQ ID NO: 23 or SEQ ID NO: 24, a CDR2 including a sequence of SEQ ID NO: 25, and a CDR3 including a sequence of SEQ ID NO: 26;a CDR1 including a sequence of SEQ ID NO: 27, a CDR2 including a sequence of SEQ ID NO: 28, and a CDR3 including a sequence of SEQ ID NO: 29; ora CDR1 including a sequence of SEQ ID NO: 30 or 31, a CDR2 including a sequence of SEQ ID NO: 32, and a CDR3 including a sequence of SEQ ID NO: 33.21. The multi-domain binding molecule of any of embodiments 1-13, wherein the anti-CD19 binding domain includes a sequence of SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205, ora sequence having at least 90% sequence identity to one of SEQ ID NOs: 199-205.22. The multi-domain binding molecule of any of embodiments 1-13, wherein the anti-CD19 binding domain includes a sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22 or a sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22.23. The multi-domain binding molecule of any of embodiments 1-18, wherein the anti-CD19 binding domain is modified to have reduced binding affinity.24. The multi-domain binding molecule of any of embodiments 1-18, wherein the anti-CD19 binding domain is modified to have reduced binding affinity by replacing each of one or more tyrosines in the CDRs of the anti-CD19 binding domain with a less hydrophobic amino acid.25. The multi-domain binding molecule of any of embodiments 1-18, wherein the anti-CD19 binding domain is modified to have reduced binding affinity by replacing each tyrosine in the CDRs of the anti-CD 19 binding domain with a less hydrophobic amino acid.26. The multi-domain binding molecule of embodiment 20 or 21, wherein the less hydrophobic amino acid includes alanine.27. The multi-domain binding molecule of any of embodiments 1-22, wherein the anti-CD33 binding domain includes a variable heavy chain including CDRH1 including the sequence of SEQ ID NO: 297, a CDRH2 including the sequence of SEQ ID NO: 298, and a CDRH3 including the sequence of SEQ ID NO: 299, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 300, a CDRL2 including the sequence of SEQ ID NO: 301, and a CDRL3 including the sequence of SEQ ID NO: 302; ora CDRH1 including the sequence of SEQ ID NO: 303, a CDRH2 including the sequence of SEQ ID NO: 304, and a CDRH3 including the sequence of SEQ ID NO: 305, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 306, a CDRL2 including the sequence of SEQ ID NO: 301, and a CDRL3 including the sequence of SEQ ID NO: 307.28. The multi-domain binding molecule of any of embodiments 1-22, wherein the anti-CD33 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 36 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 37 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 38 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 39 or a sequence having at least 95% sequence identity thereto.29. The multi-domain binding molecule any of embodiments 1-22, wherein the anti-CD33 binding domain includes a sequence having a CDR1 including a sequence of SEQ ID NO: 44, a CDR2 including a sequence of SEQ ID NO: 45, and a CDR3 including a sequence of SEQ ID NO: 46;CDR1 including a sequence of SEQ ID NO: 47, a CDR2 including a sequence of SEQ ID NO: 48, and a CDR3 including a sequence of SEQ ID NO: 49; ora CDR1 including a sequence of SEQ ID NO: 50, a CDR2 including a sequence of SEQ ID NO: 51, and a CDR3 including a sequence of SEQ ID NO: 52.30. The multi-domain binding molecule of any of embodiments 1-22, wherein the anti-CD33 binding domain includes a sequence of SEQ ID NO: 206, 207, 208, 209, 210, or 215, or a sequence having at least 90% sequence identity to SEQ ID NO: 206, 207, 208, 209, 210, or 215.31. The multi-domain binding molecule of any of embodiments 1-22, wherein the anti-CD33 binding domain includes a sequence of SEQ ID NO: 40, 41, 42, or 43, or a sequence having at least 90% sequence identity to SEQ ID NO: 40, 41, 42, or 43.32. The multi-domain binding molecule of any of embodiments 1-27, wherein the anti-CD33 binding domain is modified to have reduced binding affinity.33 The multi-domain binding molecule of any of embodiments 1-27, wherein the anti-CD33 binding domain is modified to have reduced binding affinity by replacing each of one or more tyrosines in the CDRs of the anti-CD33 binding domain with a less hydrophobic amino acid.34. The multi-domain binding molecule of any of embodiments 1-27, wherein the anti-CD33 binding domain is modified to have reduced binding affinity by replacing each tyrosine in the CDR of the anti-CD33 binding domain with a less hydrophobic amino acid.35. The multi-domain binding molecule of embodiment 29 or 30, wherein the less hydrophobic amino acid includes alanine.36. The multi-domain binding molecule of any of embodiments 1-31, including a first polypeptide including a first multimerization domain and a second polypeptide including a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.37. The multi-domain binding molecule of embodiment 32, wherein the first multimerization domain includes an lgG1 Fc knob and the second multimerization domain includes an lgG1 Fc hole.38. The multi-domain binding molecule of embodiment 32, wherein the first multimerization domain includes an lgG1 Fc hole and the second multimerization domain includes an lgG1 Fc knob.39. The multi-domain binding molecule of embodiment 33 or 34, wherein the IgG 1 Fc knob includes the sequence of SEQ ID NO: 127 or a sequence having at least 90% sequence identity thereto.40. The multi-domain binding molecule of embodiment 33 or 34, wherein the I gG 1 Fc hole includes the sequence of SEQ ID NO: 128 or a sequence having at least 90% sequence identity thereto.41. The multi-domain binding molecule of embodiment 32, wherein the first polypeptide includes a first binding domain or portion thereof and the second polypeptide includes a second binding domain or portion thereof.42. The multi-domain binding molecule of embodiment 32, wherein the first polypeptide includes a third binding domain or portion thereof.43. The multi-domain binding molecule of embodiment 32, wherein the second polypeptide includes a third binding domain or portion thereof.44. The multi-domain binding molecule of embodiment 38, whereinthe first binding domain includes the immune cell activating epitope binding domain, the second binding domain includes the anti-CD 19 binding domain, and the third binding domain includes the anti-CD33 binding domain;the first binding domain includes the immune cell activating epitope binding domain, the second binding domain includes the anti-CD33 binding domain, and the third binding domain includes the anti-CD 19 binding domain;the first binding domain includes the anti-CD19 binding domain, the second binding domain includes the immune cell activating epitope binding domain, and the third binding domain includes the anti-CD33 binding domain;the first binding domain includes the anti-CD19 binding domain, the second binding domain includes the anti-CD33 binding domain, and the third binding domain includes the immune cell activating epitope binding domain; the first binding domain includes the anti-CD33 binding domain, the second binding domain includes the immune cell activating epitope binding domain, and the third binding domain includes the anti-CD 19 binding domain; orthe first binding domain includes the anti-CD33 binding domain, the second binding domain includes the anti-CD19 binding domain, and the third binding domain includes the immune cell activating epitope binding domain.45. The multi-domain binding molecule of embodiment 32, wherein the first polypeptide includes the anti-CD 19 binding domain and an lgG1 Fc hole and the second polypeptide includes the anti-CD33 binding domain and an lgG1 Fc knob.46. The multi-domain binding molecule of embodiment 41, wherein the first polypeptide includes a sequence of SEQ ID NOs: 226-232, 342, or 343 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343.47. The multi-domain binding molecule of embodiment 41, wherein the first polypeptide includes a sequence of SEQ ID NOs: 342 or 343 or a sequence having at least 90% sequence identity to SEQ ID NO: 342 or 343.48. The multi-domain binding molecule of embodiment 41 or 42, wherein the second polypeptide includes a sequence of SEQ ID NO: 233-238 or 344-347 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 233-238 or 344-347.49 The multi-domain binding molecule of embodiment 41 or 42, wherein the second polypeptide includes a sequence of SEQ ID NOs: 344 or 345 or a sequence having at least 90% sequence identity to SEQ ID NO: 344 or 345.50. The multi-domain binding molecule of any of embodiments 32-43, further including a third polypeptide. 51. The multi-domain binding molecule of embodiment 44, wherein:the first polypeptide includes the anti-CD 19 binding domain and an lgG1 Fc hole,the second polypeptide includes a heavy chain of the anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide includes a light chain of the anti-CD33 binding domain.52. The multi-domain binding molecule of embodiment 45, whereinthe first polypeptide includes a sequence of SEQ ID NO: 226-232, 342, or 343 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343,the second polypeptide includes a sequence of SEQ ID NO: 239 or a sequence having at least 90% sequence identity thereto, andthe third polypeptide includes a sequence of SEQ ID NO: 241 or a sequence having at least 90% sequence identity thereto.53. The multi-domain binding molecule of embodiment 45, whereinthe first polypeptide includes a sequence of SEQ ID NOs: 226-232, 342, or 343 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343,the second polypeptide includes a sequence of SEQ ID NO: 240 or a sequence having at least 90% sequence identity thereto, andthe third polypeptide includes a sequence of SEQ ID NO: 242 or a sequence having at least 90% sequence identity thereto.54. The multi-domain binding molecule of embodiment 45, wherein the first polypeptide includes the immune cell activating epitope binding domain.55. The multi-domain binding molecule of embodiment 45, wherein the second polypeptide includes the immune cell activating epitope binding domain.56. The multi-domain binding molecule of embodiment 48 or 49, wherein the immune cell activating epitope binding domain includes an anti-CD3 binding domain.57. The multi-domain binding molecule of embodiment 50, wherein the anti-CD3 binding domain includes a sequence of SEQ ID NO: 211, 212, 217, or 218 or a sequence having at least 90% sequence identity to SEQ ID NO: 211, 212, 217, or 218.58. The multi-domain binding molecule of embodiment 44, wherein the first polypeptide includes a first binding domain and a first multimerization domain, the second polypeptide includes a heavy chain of a second binding domain and a second multimerization domain, and the third polypeptide includes a light chain of the second binding domain and a third binding domain.59 The multi-domain binding molecule of embodiment 52, wherein the first polypeptide includes an anti-CD19 binding domain and an lgG1 Fc hole, the second polypeptide includes a heavy chain of an anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide includes a light chain of the anti-CD33 binding domain and an anti-CD3 binding domain.60. The multi-domain binding molecule of embodiment 53, wherein the first polypeptide includes a sequence of SEQ ID NO: 226-232, 342, or 343 SEQ ID NO: 40, 41, 42, or 43 to one of SEQ ID NOs: 226-232, 342, or 343.61 The multi-domain binding molecule of embodiment 53, wherein the first polypeptide includes a sequence of SEQ ID NO: 342 or 343 or a sequence having at least 90% sequence identity to SEQ ID NO: 342 or 343.62. The multi-domain binding molecule of embodiment 53, wherein the second polypeptide includes a sequence of SEQ ID NO: 239 or a sequence having at least 90% sequence identity thereto and the third polypeptide includes a sequence of SEQ ID NO: 243, 245, or 247 or a sequence having at least 90% sequence identity to SEQ ID NO: 243, 245, or 247.63 The multi-domain binding molecule of embodiment 53, wherein the second polypeptide includes a sequence of SEQ ID NO: 240 or a sequence having at least 90% sequence identity thereto and the third polypeptide includes a sequence of SEQ ID NO: 244, 246, or 248 or a sequence having at least 90% sequence identity to SEQ ID NO: 244, 246, or 248.64. The multi-domain binding molecule of embodiment 53, wherein the second polypeptide includes a sequence of SEQ ID NO: 344, 345, 346, or 347 or a sequence having at least 90% sequence identity to SEQ ID NO: 344, 345, 346, or 347 and the third polypeptide includes a sequence of SEQ ID NO: 248 or a sequence having at least 90% sequence identity thereto.65. The multi-domain binding molecule of embodiment 44, whereinthe first polypeptide includes a first binding domain, a second binding domain, and an lgG1 Fc hole; the second polypeptide includes a heavy chain of a third binding domain and an lgG1 Fc knob; and the third polypeptide includes a light chain of the third binding domain.66. The multi-domain binding molecule of embodiment 57, whereinthe first polypeptide includes an anti-CD 19 binding domain, an anti-CD33 binding domain, and an lgG1 Fc hole;the second polypeptide includes a heavy chain of an anti-CD3 binding domain and an lgG1 Fc knob; and the third polypeptide includes a light chain of the anti-CD3 binding domain.67. The multi-domain binding molecule of embodiment 58, wherein the first polypeptide includes a sequence of SEQ ID NO: 249-256, 334, 336, 338, 340, 348, or 349 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 249-256, 334, 336, 338, 340, 348, or 349.68. The multi-domain binding molecule of embodiment 58 or 59, wherein the second polypeptide includes a sequence of SEQ ID NO: 214 or a sequence having at least 90% sequence identity thereto and the third polypeptide includes the sequence of SEQ ID NO: 213 or a sequence having at least 90% sequence identity thereto.69. The multi-domain binding molecule of embodiment 44, whereinthe first polypeptide includes a first binding domain and an lgG1 Fc hole;the second polypeptide includes a heavy chain of a second binding domain and an lgG1 Fc knob; and the third polypeptide includes a light chain of the second binding domain and a third binding domain.70. The multi-domain binding molecule of embodiment 61, whereinthe first polypeptide includes an anti-CD19 binding domain and an lgG1 Fc hole,the second polypeptide includes a heavy chain of an anti-CD3 binding domain and an lgG1 Fc knob, and the third polypeptide includes a light chain of the anti-CD3 binding domain and an anti-CD33 binding domain.71. The multi-domain binding molecule of embodiment 62, wherein the first polypeptide includes a sequence of SEQ ID NO: 228 or 229 or a sequence having at least 90% sequence identity to SEQ ID NO: 228 or 229.72. The multi-domain binding molecule of embodiment 62 or 63, wherein the second polypeptide includes a sequence of SEQ ID NO: 214 or a sequence having at least 90% sequence identity thereto; and the third polypeptide includes the sequence of SEQ ID NO: 260-271 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 260-271.73. The multi-domain binding molecule of any of embodiments 1-64, wherein any of the immune cell activating epitope binding domain, the anti-CD 19 binding domain, or the anti-CD33 binding domain are joined by a protein linker.74. The multi-domain binding molecule of embodiment 65, wherein the protein linker is a Gly-Ser linker.75 The multi-domain binding molecule of embodiment 66, 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 10.76. The multi-domain binding molecule of any of embodiments 1-67, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain are linked to an Fc region of an antibody.77. The multi-domain binding molecule of embodiment 68, wherein the Fc region is an IgM Fc region.78. The multi-domain binding molecule of embodiment 68, wherein the Fc region is an IgM Fc region having the sequence of SEQ ID NO: 163-174, or 221 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 163-174, or.79 The multi-domain binding molecule of embodiment 68, wherein the Fc region includes a multimerizing fragment of an IgM Fc region.80. The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region includes an IgM tailpiece.81. The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region includes a C 4 domain and an IgM tailpiece.82 The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region includes a Cpi3 domain, a Cpi4 domain, and an IgM tailpiece.83. The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region includes a Cpi2 domain, a Cpi3 domain, a C i4 domain, and an IgM tailpiece.84. The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region includes a Cpi1 domain, a CJJ2 domain, a Cpj3 domain, a C 4 domain, and an IgM tailpiece.85 The multi-domain binding molecule of embodiment 71, wherein the multimerizing fragment of the IgM Fc region has the sequence of SEQ ID NO: 163-174, or 221 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 163-174, or 221.86. The multi-domain binding molecule of embodiment 69, wherein the IgM Fc region includes a first binding domain and a second binding domain; and a joining (J)-chain includes a third binding domain.87. The multi-domain binding molecule of embodiment 78, wherein the J-chain includes the sequence of SEQ ID NO: 175 or a sequence having at least 90% sequence identity thereto.88. The multi-domain binding molecule of embodiment 78 or 79, wherein the third binding domain includes an immune cell activating epitope binding domain.89. The multi-domain binding molecule of embodiment 80, wherein the immune cell activating epitope binding domain includes an anti-CD3 binding domain.90. The multi-domain binding molecule of embodiment 78, wherein the IgM Fc region includes an anti-CD19 binding domain and an anti-CD33 binding domain; and the J-chain includes an anti-CD3 binding domain.91. The multi-domain binding molecule of embodiment 82, wherein the J-chain including an anti-CD3 binding domain includes a sequence of SEQ ID NO: 257, 258, or 259 or a sequence having at least 90% sequence identity toSEQ ID NO: 257, 258, or 259.92 The multi-domain binding molecule of embodiment 82 or 83, wherein the IgM Fc region including the anti-0019 binding domain and the anti-CD33 binding domain includes a sequence of SEQ ID NO: 326-333 or a sequence having at least 90% sequence identity to one of SEQ ID NOs: 326-333.93. The multi-domain binding molecule of any of embodiments 1-84, further including a fourth binding domain.94. The multi-domain binding molecule of any of embodiments 1-85, wherein at least one of the immune cell activating epitope binding domain, the anti-CD 19 binding domain, or the anti-CD33 binding domain include an antibody or a peptide.95. The multi-domain binding molecule of embodiment 86, wherein the antibody includes an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).96. The multi-domain binding molecule of embodiment 87, wherein the sdAb includes a variable heavy domain of a heavy chain (VHH), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody97 The multi-domain binding molecule of embodiment 86, wherein the peptide includes a miniprotein ora peptide aptamer.98. The multi-domain binding molecule of any of embodiments 32-89, wherein the first polypeptide further includes a linker, a signal peptide, and / or a tag.99. The multi-domain binding molecule of any of embodiments 32-90, wherein the second polypeptide further includes a linker, a signal peptide, and / or a tag.100. The multi-domain binding molecule of any of embodiments 1-91, including Fc silencing mutations.101. The multi-domain binding molecule of embodiment 92, wherein the Fc silencing mutations include aglycosy lation mutations or LALAPG mutations.102. A nucleic acid encoding The multi-domain binding molecule of any of embodiments 1-93.103. A conjugate including The multi-domain binding molecule of any of embodiments 1-93linked to an immunotoxin, a drug, a detectable label, a radioisotope, or a particle.104. The conjugate of embodiment 95, wherein the immunotoxin includes a plant toxin or bacterial toxin.105. The conjugate of embodiment 96, wherein the plant toxin includes ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, saporin, Bryodin 1, bouganin, or gelonin.106. The conjugate of embodiment 96, wherein the bacterial toxin includes diphtheria toxin or Pseudomonas exotoxin.107. The conjugate of embodiment 95, wherein the drug includes a cytotoxic drug.108. The conjugate of embodiment 99, wherein the cytotoxic 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, or vincristine.109. The conjugate of embodiment 95, wherein the detectable label includes a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, or an affinity tag.110. The conjugate of embodiment 95, 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,167Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,3H, ™Hf,471Hf,193Hg,193mHg, i6omHo,13°l,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,1910s,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|188W1i25Xe, i27Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, or97Zr.111. The conjugate of embodiment 95, wherein the radioisotope includes 1311, 90Y, or 211 At.112. The conjugate of embodiment 95, wherein the radioisotope does not emit daughter radionuclides.113. A cell genetically modified to express The multi-domain binding molecule of any of embodiments 1-93. 114. A composition including an immune cell activating epitope binding domain, an anti-CD19 binding domain, an anti-CD33 binding domain and a pharmaceutically acceptable carrier.115. A kit including:an immune cell activating epitope binding domain or a nucleic acid encoding the immune cell activating epitope binding domain,an anti-CD19 binding domain or a nucleic acid encoding the anti-CD19 binding domain, andan anti-CD33 binding domain or a nucleic acid encoding the anti-CD33 binding domain.116. The kit of embodiment 107, wherein the immune cell activating epitope includes an epitope on a T cell, an NK cell, or a macrophage.117. The kit of embodiment 107, wherein the immune cell activating epitope includes CD3, CD2, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.118. The kit of embodiment 107, wherein the immune cell activating epitope includes CD3, CD28, 4-1 BB, or CD8.119. The kit of embodiment 107, wherein the immune cell activating epitope includes NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.120. The kit of embodiment 107, wherein the immune cell activating epitope includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFNGR2, Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.121. The kit of embodiment 107, wherein the immune cell activating epitope binding domain binds and activates a T cell.122. The kit of embodiment 107, wherein the immune cell activating epitope binding domain binds CD3.123. The kit of embodiment 107, wherein the immune cell activating epitope binding domain binds CD3 and includes a variable heavy chain including CDRH1 including the sequence of SEQ ID NO: 308, a CDRH2 including the sequence of SEQ ID NO: 309, and a CDRH3 including the sequence of SEQ ID NO: 310, and a variable light chain including a CDR light (L)1 including the sequence of SEQ ID NO: 311, a CDRL2 including the sequence of SEQ ID NO: 312, and a CDRL3 including the sequence of SEQ ID NO: 313;a CDRH1 including the sequence of SEQ ID NO: 314, a CDRH2 including the sequence of SEQ ID NO: 315, and a CDRH3 including the sequence of SEQ ID NO: 316, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 317, a CDRL2 including the sequence of SEQ ID NO: 318, and a CDRL3 including the sequence of SEQ ID NO: 319;a CDRH1 including the sequence of SEQ ID NO: 320, a CDRH2 including the sequence of SEQ ID NO: 321, and a CDRH3 including the sequence of SEQ ID NO: 322, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 323, a CDRL2 including the sequence of SEQ ID NO: 324, and a CDRL3 including the sequence of SEQ ID NO: 325;a CDRH1 including the sequence of SEQ ID NO: 104, a CDRH2 including the sequence of SEQ ID NO: 105, and a CDRH3 including the sequence of SEQ ID NO: 106, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 102, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 103;a CDRH1 including the sequence of SEQ ID NO: 109, a CDRH2 including the sequence of SEQ ID NO: 110, and a CDRH3 including the sequence of SEQ ID NO: 111, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 107, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 108;a CDRH1 including the sequence of SEQ ID NO: 114, a CDRH2 including the sequence of SEQ ID NO: 115, and a CDRH3 including the sequence of SEQ ID NO: 116, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 112, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 113; ora CDRH1 including the sequence of SEQ ID NO: 119, a CDRH2 including the sequence of SEQ ID NO: 120, and a CDRH3 including the sequence of SEQ ID NO: 121, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 117, a CDRL2 including the sequence KVS, and a CDRL3 including the sequence of SEQ ID NO: 118.124. The kit of embodiment 107, wherein the immune cell activating epitope binding domain includes an anti-CD3 binding domain including a variable heavy chain including the sequence of SEQ ID NO: 53 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 54 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 56 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 57 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 58 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 59 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 60 or a sequence having at least 95% sequence identity thereto.125. The kit of embodiment 107, wherein the anti-CD3 binding domain includes a sequence of SEQ ID NO: 61, 211, 212, 217, or 218 or a sequence having at least 90% sequence identity to SEQ ID NO: 61, 211, 212, 217, or 218.126. The kit of embodiment 107, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence having a CDR1 including a sequence of SEQ ID NO: 95, a CDR2 including a sequence of SEQ ID NO: 96, and a CDR3 including a sequence of SEQ ID NO: 97; ora CDR1 including a sequence of SEQ ID NO: 98, a CDR2 including a sequence of SEQ ID NO: 99, and a CDR3 including a sequence of SEQ ID NO: 100.127. The kit of embodiment 107, wherein the immune cell activating epitope binding domain binds CD3 and includes a sequence of SEQ ID NO: 62-94 or 101, or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 62-94 or 101.128. The kit of any of embodiments 107-119, wherein the anti-CD 19 binding domain includes a variable heavy chain including CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 284, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 284, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 288, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 289, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287;a CDRH1 including the sequence of SEQ ID NO: 282, a CDRH2 including the sequence of SEQ ID NO: 283, and a CDRH3 including the sequence of SEQ ID NO: 290, and a variable light chain including a CDRL1 including thesequence of SEQ ID NO: 285, a CDRL2 including the sequence of SEQ ID NO: 286, and a CDRL3 including the sequence of SEQ ID NO: 287; ora CDRHI including the sequence of SEQ ID NO: 291, a CDRH2 including the sequence of SEQ ID NO: 292, and a CDRH3 including the sequence of SEQ ID NO: 293, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 294; a CDRL2 including the sequence of SEQ ID NO: 295, and a CDRL3 including the sequence of SEQ ID NO: 296.129. The kit of any of embodiments 107-119, wherein the anti-CD 19 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 5 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 8 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 9 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 10 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 11 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 12 or a sequence having at least 95% sequence identity thereto;a variable heavy chain including the sequence of SEQ ID NO: 13 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 14 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 34 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 35 or a sequence having at least 95% sequence identity thereto.130. The kit of any of embodiments 107-119, wherein the anti-CD19 binding domain includes a sequence having a CDR1 including a sequence of SEQ ID NO: 23 or SEQ ID NO: 24, a CDR2 including a sequence of SEQ ID NO: 25, and a CDR3 including a sequence of SEQ ID NO: 26;a CDR1 including a sequence of SEQ ID NO: 27, a CDR2 including a sequence of SEQ ID NO: 28, and a CDR3 including a sequence of SEQ ID NO: 29; ora CDR1 including a sequence of SEQ ID NO: 30 or SEQ ID NO: 31, a CDR2 including a sequence of SEQ ID NO: 32, and a CDR3 including a sequence of SEQ ID NO: 33.131. The kit of any of embodiments 107-119, wherein the anti-CD 19 binding domain includes a sequence of SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205, or a sequence having at least 90% sequence identity to SEQ ID NO: 199,200, 201, 202, 203, 204, or 205.132. The kit of any of embodiments 107-119, wherein the anti-CD 19 binding domain includes a sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22 or a sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22.133. The kit of any of embodiments 107-124, wherein the anti-CD 19 binding domain is modified to have reduced binding affinity.134. The kit of any of embodiments 107-124, wherein the anti-CD 19 binding domain is modified to have reduced binding affinity by replacing each of one or more tyrosines in the CDRs of the anti-CD33 binding domain with a less hydrophobic amino acid.135. The kit of any of embodiments 107-124, wherein the anti-CD 19 binding domain is modified to have reduced binding affinity by replacing each tyrosine in the CDR of the anti-CD 19 binding domain with a less hydrophobic amino acid.136. The kit of embodiment 127, wherein the less hydrophobic amino acid includes alanine.137. The kit of any of embodiments 107-128, wherein the anti-CD33 binding domain includes a variable heavy chain including CDRH1 including the sequence of SEQ ID NO: 297, a CDRH2 including the sequence of SEQ ID NO: 298, and a CDRH3 including the sequence of SEQ ID NO: 299, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 300, a CDRL2 including the sequence of SEQ ID NO: 301, and a CDRL3 including the sequence of SEQ ID NO: 302; ora CDRH1 including the sequence of SEQ ID NO: 303, a CDRH2 including the sequence of SEQ ID NO: 304, and a CDRH3 including the sequence of SEQ ID NO: 305, and a variable light chain including a CDRL1 including the sequence of SEQ ID NO: 306, a CDRL2 including the sequence of SEQ ID NO: 301, and a CDRL3 including the sequence of SEQ ID NO: 307.138. The kit of any of embodiments 107-128, wherein the anti-CD33 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 36 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 37 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain including the sequence of SEQ ID NO: 38 or a sequence having at least 95% sequence identity thereto, and a variable light chain including the sequence of SEQ ID NO: 39 or a sequence having at least 95% sequence identity thereto.139. The kit of any of embodiments 107-128, wherein the anti-CD33 binding domain includes a sequence having a CDR1 including a sequence of SEQ ID NO: 44, a CDR2 including a sequence of SEQ ID NO: 45, and a CDR3 including a sequence of SEQ ID NO: 46;CDR1 including a sequence of SEQ ID NO: 47, a CDR2 including a sequence of SEQ ID NO: 48, and a CDR3 including a sequence of SEQ ID NO: 49; ora CDR1 including a sequence of SEQ ID NO: 50, a CDR2 including a sequence of SEQ ID NO: 51, and a CDR3 including a sequence of SEQ ID NO: 52140. The kit of any of embodiments 107-128, wherein the anti-CD33 binding domain includes a sequence of SEQ ID NO: 206, 207, 208, 209, 210, or 215 or a sequence having at least 90% sequence identity to SEQ ID NO: 206, 207, 208, 209, 210, or 215.141. The kit of any of embodiments 107-128, wherein the anti-CD33 binding domain includes a sequence of SEQ ID NO: 40, 41, 42, or 43, or a sequence having at least 90% sequence identity to SEQ ID NO: 40, 41, 42, or 43.142. The kit of any of embodiments 107-133, wherein the anti-CD33 binding domain is modified to have reduced binding affinity.143. The kit of any of embodiments 107-133, wherein the anti-CD33 binding domain is modified to have reduced binding affinity by replacing each of one or more tyrosines in the CDRs of the anti-CD33 binding domain with a less hydrophobic amino acid.144. The kit of any of embodiments 107-133, wherein the anti-CD33 binding domain is modified to have reduced binding affinity by replacing each tyrosine in the CDRs of the anti-CD33 binding domain with a less hydrophobic amino acid.145. The kit of embodiment 135 or 136, wherein the less hydrophobic amino acid includes alanine.146. The kit of any of embodiments 107-137, further including a multimerization domain or a nucleic acid encoding a multimerization domain.147. The kit of embodiment 138, wherein the multimerization domain is a protein linker.148. The kit of embodiment 138, wherein the multimerization domain includes an IgM Fc region.149. The kit of any of embodiments 107-140, includinga first polypeptide including a first multimerization domain or a nucleic acid encoding the first polypeptide and a second polypeptide including a second multimerization domain or a nucleic acid encoding the second polypeptide wherein when expressed, the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.150. The kit of embodiment 141, wherein the first multimerization domain includes an lgG1 Fc hole and the second multimerization domain includes an lgG1 Fc knob.151. The kit of embodiment 141, wherein the first multimerization domain includes an lgG1 Fc knob and the second multimerization domain includes an lgG1 Fc hole.152. The kit of embodiment 142 or 143, wherein the lgG1 Fc knob includes the sequence of SEQ ID NO: 127 or a sequence having at least 90% sequence identity thereto.153. The kit of embodiment 142 or 143, wherein the lgG1 Fc hole includes the sequence of SEQ ID NO: 128 or a sequence having at least 90% sequence identity thereto.154. The kit of any of embodiments 107-145, further including a linker, a signal peptide, and / or a tag.155. The kit of any of embodiments 107-146, further including a pharmaceutically acceptable carrier.156. The kit of any of embodiments 107-147, further including a delivery vehicle.157. The kit of embodiment 148, wherein the delivery vehicle includes a syringe.158. A method of treating a subject having a cancer that co-expresses CD 19 and CD33, the method including administering a therapeutically effective amount of the composition of embodiment 106 to the subject, thereby treating the subject having the cancer that co-expresses CD 19 and CD33.159. The method of embodiment 150, wherein the cancer that co-expresses CD19 and CD33 includes a B cell malignancy.160. The method of embodiment 150, wherein the cancer that co-expresses CD 19 and CD33 includes chronic phase leukemia.161. The method of embodiment 150, wherein the cancer that co-expresses CD19 and CD33 includes accelerated phase leukemia.162. The method of embodiment 150, wherein the cancer that co-expresses CD19 and CD33 includes blast phase leukemia163. The method of embodiment 150, wherein the cancer that co-expresses CD 19 and CD33 includes mixed phenotype acute leukemia (MPAL).164. The method of embodiment 150, wherein the cancer that co-expresses CD19 and CD33 includes B-cell acute lymphoblastic leukemia (B-ALL).165. The method of embodiment 150, wherein the administering is intravenous administering.166. An embodiment according to any of the preceding embodiments including or using a combination of SEQ ID NOs: 342, 345, and 248; 342, 346, and 248, 343, 344, and 248; or 343, 347, and 248 or sequences having at least 90% sequence identity to SEQ ID NO: 342, 345, 248; 346, 343, 344, or 347, or 248.167. An embodiment according to any of the preceding embodiments including or using one or more of SEQ ID NOs: 215, 334, 335, 336, 337, 338, 339, 340, 341, 348, or 349 or sequences having at least 90% sequence identity to of SEQ ID NOs: 215, 334, 335, 336, 337, 338, 339, 340, 341, 348, or 349.

[0313] (XIII) Experimental Example. Example 1. Affinity-Tuned T-Cell Engager for Dual Targeting of B-Myeloid Mixed-Phenotype Acute Leukemia (B-MPAL).

[0314] Introduction. B-myeloid mixed-phenotype acute leukemia (B-MPAL) is a high-risk leukemia subtype presenting with both lymphoid (e.g., CD19) and myeloid (e.g., CD33) surface antigens. Cure rates in children and adolescents remain lower than for almost all other acute lymphoblastic leukemia (ALL) subtypes, with overall survival ranging from 75-80%. Outcomes for adults are notably poorer, with overall survival ranging from 20-50% for patients over 40 years of age. Induction therapies range from those used for ALL versus those used for acute myeloid leukemia (AML) with a paucity of information from randomized clinical trials due to the uncommon nature of this subtype.

[0315] B-Myeloid MPAL cells frequently express both CD19 and CD33 while non-neoplastic human hematopoietic cells do not express both targets, creating an opportunity to selectively target MPAL cells with minimal off tumor toxicity. Multispecific T-cell engager antibodies (MTEs) disclosed herein are engineered to preferentially bind cells expressing CD19 and CD33 such that they will selectively kill MPAL cells better than a bi-specific antibody that requires CD19 or CD33 expression. MTEs were engineered that bind both CD19 and CD33 on MPAL cells and CD3 on T cells. The affinity of the CD19 and CD33 binding domains were fine-tuned so that the MTE binds poorly to single positive (SP) normal cells that express either CD 19 or CD33 but binds well to double positive (DP) leukemia cells. For this, both affinity and avidity were taken advantage of, the latter of which occurs only when the MTE interacts with both CD19 and CD33 expressed on the same cell. Such therapies can be used to induce deep remissions, improve outcomes when integrated with chemotherapy, and / or serve as a bridge to hematopoietic cell transplantation. Blinatumomab, a bispecific antibody recognizing only CD 19, has been a successful addition to treating both childhood and adult ALL. However, patients require months of intravenous immunoglobulin replacement therapy due to the consequential depletion of healthy B-cells, which share CD19 as a critical surface antigen with most B-cell ALL. The new therapeutic strategy disclosed herein improves safety compared to other currently available immunotherapies.

[0316] Methods. CD19 and CD33 were identified as appropriate targets for B-Myeloid MPAL after analyzing aggregated transcriptome data for 31 MPAL patients. Next, 30 tri-specific antibodies targeting CD19 / CD33 / CD3 were designed and produced. Binding domains were selected because they are either components of FDA approved therapeutics, have gone through human clinical trials, or have been extensively studied to de-risk safety and immunogenicity concerns. The MTE framework was derived from the asymmetric “knob into holes” human lgG1 scaffold incorporating effector silencing mutations. The in vitro pipeline to test those antibodies included binding assays as well as T-cell cytotoxic assays using cell lines expressing both targets, only one, or none. Top candidates were further tested for efficacy in in vivo mouse models. A luciferase labeled JIH-5 cell line was used that innately expresses CD19 and CD33. This cell line was xenografted into NSG-SGM3 immunocompromised mice. Engrafted mice were dosed with both MTE and human T-cells from a healthy donor for repeated weekly cycles. Disease progression was followed every week using In Vivo Imaging System (IVI S) imaging.

[0317] Results. The data demonstrates the initial in vitro success in targeting CD19 / CD33 / CD3 antigens as well as the dramatic improvement of specificity by generating lower affinity variants of known CD19 and CD33 binding domains. For multiple candidates, a much lower cytotoxicity for single positive (SP) cells was observed, with up to a 7 log difference in IC50. This difference in IC50 expands the therapeutic window because double positive (DP) cells are specifically targeted for lysis while SP cells are spared. In addition, initial pre-clinical in vivo data is presented for the top candidates in xenograft mouse models, showing successful control of disease in engrafted mice at treatment doses that are well tolerated.

[0318] Conclusions. Taking advantage of the unique features of MPAL, a novel immunotherapy was developed with improved half-life, potency, selectivity, and safety. This example shows that the combinatorial use of binding domainswith the proper affinity for their respective targets allow for the generation of highly specific immunotherapies with minimal cytotoxicity for normal cells

[0319] (XIV) Prophetic Example. A study will be conducted to translate the observed therapeutic window in vitro into a relevant in vivo humanized model. This experiment will focus on the 0.3 g dose, which showed B-cell sparing. Two cohorts of animals from 2 different donors of CD34+ HSPC will be used. All animals will be engrafted with the B / myeloid MPAL cells JIH-5, those which co-express CD19 and CD33. The cells will also be engineered to express YFP and firefly luciferase (FFLuc) to track disease.

[0320] Each cohort will have 15 animals. 5 treated with vehicle, 5 treated with 0.3ug MTE FMC63:: GemY74A:: MT194 and 5 treated with an equivalent molarity of Blinatumomab (0.1 ug per dose per animal). The study will demonstrate reduced disease at the 0.3ug dose of MTE, while still sparing B cells and CD33+ cells.

[0321] (XV) Closing Paragraphs. Sequence information provided by public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed herein.

[0322] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4th Edition (2012 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).

[0323] 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 or component. 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 cancer cell killing, according to procedures described herein.

[0324] 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 ofthe 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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 allpossible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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 multi-domain binding molecule comprising:an immune cell activating epitope binding domain, an anti-CD 19 binding domain, and an anti-CD33 binding domain whereinthe immune cell activating epitope binding domain has the sequence of SEQ ID NO: 248;the anti-CD 19 binding domain has the sequence of SEQ ID NO: 342, 343, 226, 334, 338, 231, 232, or 230; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 345, 346, 344, 347, or 240.

2. The multi-domain binding molecule of claim 1 wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342 or 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 345, 346, 344, or 347.

3. The multi-domain binding molecule of claim 1 wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 345.

4. The multi-domain binding molecule of claim 1 wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 346.

5. The multi-domain binding molecule of claim 1 wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 344.

6. The multi-domain binding molecule of claim 1 wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 347.

7. A multi-domain binding molecule comprising:an immune cell activating epitope binding domain, an anti-CD19 binding domain, and an anti-CD33 binding domain wherein the multi-domain binding molecule can be administered at a dose that kills cells that co-express CD19 and CD33 but does not kill cells that express CD 19 or CD33.

8. The multi-domain binding molecule of claim 7, wherein the anti-CD19 binding domain is modified from a reference anti-CD 19 binding domain to have reduced binding affinity by substituting a tyrosine with a less hydrophobic amino acid.

9. The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain is modified from a reference anti-CD33 binding domain to have reduced binding affinity by substituting a tyrosine with a less hydrophobic amino acid.

10. The multi-domain binding molecule of claim 7, wherein the anti-CD19 binding domain is modified from a reference anti-CD19 binding domain to have reduced binding affinity and the anti-CD33 binding domain is modified from a reference anti-CD33 binding domain to have reduced binding affinity by substituting a tyrosine with a less hydrophobic amino acid.

11. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding lack an Fc region of an antibody.

12. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, and the anti-CD33 binding lack an Fc region of an antibody13. The multi-domain binding molecule of claim 7, comprising a sequence with at least 95% sequence identity to SEQ ID NO: 334, 335, 337, 338, or 339.

14. The multi-domain binding molecule of claim 7, comprising SEQ ID NO: 334, 335, 337, 338, or 339.

15. The multi-domain binding molecule of claim 7, comprising a first polypeptide comprising a first multimerization domain and a second polypeptide comprising a second multimerization domain wherein the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

16. The multi-domain binding molecule of claim 15, wherein the first multimerization domain comprises an lgG1 Fc knob and the second multimerization domain comprises an lgG1 Fc hole.

17. The multi-domain binding molecule of claim 15, wherein the first multimerization domain comprises an lgG1 Fc hole and the second multimerization domain comprises an IgG 1 Fc knob.

18. The multi-domain binding molecule of claims 16 or 17, wherein the lgG1 Fc knob comprises the sequence of SEQ ID NO: 127 or a sequence having at least 95% sequence identity thereto.

19. The multi-domain binding molecule of claims 16 or 17, wherein the lgG1 Fc hole comprises the sequence of SEQ ID NO: 128 or a sequence having at least 95% sequence identity thereto.

20. The multi-domain binding molecule of claim 15, wherein the first polypeptide comprises the anti-CD19 binding domain and an lgG1 Fc hole and the second polypeptide comprises the anti-CD33 binding domain and an lgG1 Fc knob.21 The multi-domain binding molecule of claim 20, wherein the first polypeptide comprises a sequence of SEQ ID NO: 226-232, 342, or 343 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343.

22. The multi-domain binding molecule of claim 20, wherein the first polypeptide comprises a sequence of SEQ ID NO: 342 or 343 or a sequence having at least 95% sequence identity to SEQ ID NO: 342 or 343.

23. The multi-domain binding molecule of claim 20, wherein the second polypeptide comprises a sequence of SEQ ID NO: 233-238 or 344-347 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 233-238 or 344-347.

24. The multi-domain binding molecule of claim 20, wherein the second polypeptide comprises a sequence ofSEQ ID NO: 344 or 345 or a sequence having at least 95% sequence identity to SEQ ID NO: 344 or 345.25 The multi-domain binding molecule of claim 15, further comprising a third polypeptide.

26. The multi-domain binding molecule of claim 25, wherein:the first polypeptide comprises the anti-CD 19 binding domain and an lgG1 Fc hole,the second polypeptide comprises a heavy chain of the anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide comprises a light chain of the anti-CD33 binding domain.

27. The multi-domain binding molecule of claim 26, whereinthe first polypeptide comprises a sequence of SEQ ID NO: 342 or 343 or a sequence having at least 95% sequence identity to SEQ ID NO: 342 or 343,the second polypeptide comprises a sequence of SEQ ID NO: 344, 345, 346, or 347 or a sequence having at least 95% sequence identity to SEQ ID NO: 344, 345, 346, or 347, andthe third polypeptide comprises a sequence of SEQ ID NO: 248 or a sequence having at least 95% sequence identity thereto.28 The multi-domain binding molecule of claim 26, whereinthe first polypeptide comprises a sequence of SEQ ID NOs: 226-232, 342, or 343 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343,the second polypeptide comprises a sequence of SEQ ID NO: 239 or a sequence having at least 95% sequence identity thereto, andthe third polypeptide comprises a sequence of SEQ ID NO: 241 or a sequence having at least 95% sequence identity thereto.

29. The multi-domain binding molecule of claim 26, whereinthe first polypeptide comprises a sequence of SEQ ID NOs: 226-232, 342, or 343 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343,the second polypeptide comprises a sequence of SEQ ID NO: 240 or a sequence having at least 95% sequence identity thereto, andthe third polypeptide comprises a sequence of SEQ ID NO: 242 or a sequence having at least 95% sequence identity thereto.

30. The multi-domain binding molecule of claim 26, wherein the first polypeptide comprises the immune cell activating epitope binding domain.

31. The multi-domain binding molecule of claim 26, wherein the second polypeptide comprises the immune cell activating epitope binding domain.32 The multi-domain binding molecule of claims 30 or 31, wherein the immune cell activating epitope binding domain comprises an anti-CD3 binding domain.

33. The multi-domain binding molecule of claim 32, wherein the anti-CD3 binding domain comprises a sequenceof SEQ ID NO: 211, 212, 217, or 218 or a sequence having at least 95% sequence identity to SEQ ID NO: 211, 212, 217, or 218.

34. The multi-domain binding molecule of claim 25, wherein the first polypeptide comprises a first binding domain and a first multimerization domain, the second polypeptide comprises a heavy chain of a second binding domain and a second multimerization domain, and the third polypeptide comprises a light chain of the second binding domain and a third binding domain.

35. The multi-domain binding molecule of claim 34, wherein the first polypeptide comprises an anti-CD 19 binding domain and an lgG1 Fc hole, the second polypeptide comprises a heavy chain of an anti-CD33 binding domain and an lgG1 Fc knob, and the third polypeptide comprises a light chain of the anti-CD33 binding domain and an anti-CD3 binding domain.

36. The multi-domain binding molecule of claim 35, wherein the first polypeptide comprises a sequence of SEQ ID NO: 226-232, 342, or 343 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 226-232, 342, or 343.37 The multi-domain binding molecule of claim 35, wherein the second polypeptide comprises a sequence of SEQ ID NO: 239 or a sequence having at least 95% sequence identity thereto and third polypeptide comprises a sequence of SEQ ID NO: 243, 245, or 247 or a sequence having at least 95% sequence identity to SEQ ID NO: 243, 245, or 247.

38. The multi-domain binding molecule of claim 35, wherein the second polypeptide comprises a sequence of SEQ ID NO: 240 or a sequence having at least 95% sequence identity thereto and the third polypeptide comprises a sequence of SEQ ID NO: 244, 246, or 248 or a sequence having at least 95% sequence identity to SEQ ID NO: 244, 246, or 248.

39. The multi-domain binding molecule of claim 35, wherein the second polypeptide comprises a sequence of SEQ ID NO: 344, 345, 346, or 347 or a sequence having at least 95% sequence identity to SEQ ID NO: 344, 345, 346, or 347 and the third polypeptide includes a sequence of SEQ ID NO: 248 or a sequence having at least 95% sequence identity thereto40 The multi-domain binding molecule of claim 15, whereinthe first polypeptide comprises a first binding domain, a second binding domain, and an lgG1 Fc hole; the second polypeptide comprises a heavy chain of a third binding domain and an lgG1 Fc knob; and the third polypeptide comprises a light chain of the third binding domain.

41. The multi-domain binding molecule of claim 40, whereinthe first polypeptide comprises an anti-CD19 binding domain, an anti-CD33 binding domain, and an lgG1 Fc hole;the second polypeptide comprises a heavy chain of an anti-CD3 binding domain and an lgG1 Fc knob; and the third polypeptide comprises a light chain of the anti-CD3 binding domain.

42. The multi-domain binding molecule of claim 41, wherein the first polypeptide comprises a sequence of SEQ ID NO: 249-256, 334, 336, 338, 340, 348, or 349 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 249-256, 334, 336, 338, 340, 348, or 349.

43. The multi-domain binding molecule of claim 41, wherein the second polypeptide comprises a sequence of SEQ ID NO: 214 or a sequence having at least 95% sequence identity thereto and the third polypeptide comprises the sequence of SEQ ID NO: 213 or a sequence having at least 95% sequence identity thereto.

44. The multi-domain binding molecule of claim 25, whereinthe first polypeptide comprises a first binding domain and an lgG1 Fc hole;the second polypeptide comprises a heavy chain of a second binding domain and an lgG1 Fc knob; and the third polypeptide comprises a light chain of the second binding domain and a third binding domain.

45. The multi-domain binding molecule of claim 44, whereinthe first polypeptide comprises an anti-CD19 binding domain and an lgG1 Fc hole,the second polypeptide comprises a heavy chain of an anti-CD3 binding domain and an lgG1 Fc knob, and the third polypeptide comprises a light chain of the anti-CD3 binding domain and an anti-CD33 binding domain.

46. The multi-domain binding molecule of claim 45, wherein the first polypeptide comprises a sequence of SEQ ID NO: 228 or 229 or a sequence having at least 95% sequence identity to SEQ ID NO: 228 or 229.

47. The multi-domain binding molecule of claim 45, wherein the second polypeptide comprises a sequence of SEQ ID NO: 214 or a sequence having at least 95% sequence identity thereto; and the third polypeptide comprises the sequence of SEQ ID NO: 260-271 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 260-271.

48. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope comprises an epitope on a T cell, an NK cell, or a macrophage.

49. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope comprises CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1 BB, 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3.50 The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope comprises CD3, CD28, 4-1 BB, or CD8.

51. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, or NKG2D.

52. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope comprises CD11 b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFNGR2, Toll-like receptors (TLRs) 1-9, IL-4Ra, or MARCO.

53. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope binding domain binds and activates a T cell.

54. The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope binding domain comprises an anti-CD3 binding domain.

55. The multi-domain binding molecule of claim 54, wherein the anti-CD3 binding domain comprises a variable heavy chain comprisingcomplementarity determining region (CDR) heavy (H)1 comprising the sequence of SEQ ID NO: 308, a CDRH2 comprising the sequence of SEQ ID NO: 309, and a CDRH3 comprising the sequence of SEQ ID NO: 310, and a variable light chain comprising a CDR light (L)1 comprising the sequence of SEQ ID NO: 311, a CDRL2 comprising the sequence of SEQ ID NO: 312, and a CDRL3 comprising the sequence of SEQ ID NO: 313;a CDRH1 comprising the sequence of SEQ ID NO: 314, a CDRH2 comprising the sequence of SEQ ID NO: 315, and a CDRH3 comprising the sequence of SEQ ID NO: 316, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 317, a CDRL2 comprising the sequence of SEQ ID NO: 318, and a CDRL3 comprising the sequence of SEQ ID NO: 319;a CDRH1 comprising the sequence of SEQ ID NO: 320, a CDRH2 comprising the sequence of SEQ ID NO: 321, and a CDRH3 comprising the sequence of SEQ ID NO: 322, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 323, a CDRL2 comprising the sequence of SEQ ID NO: 324, and a CDRL3 comprising the sequence of SEQ ID NO: 325;a CDRH1 comprising the sequence of SEQ ID NO: 104, a CDRH2 comprising the sequence of SEQ ID NO: 105, and a CDRH3 comprising the sequence of SEQ ID NO: 106, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 102, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 103;a CDRH1 comprising the sequence of SEQ ID NO: 109, a CDRH2 comprising the sequence of SEQ ID NO: 110, and a CDRH3 comprising the sequence of SEQ ID NO: 111, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 107, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 108;a CDRH1 comprising the sequence of SEQ ID NO: 114, a CDRH2 comprising the sequence of SEQ ID NO: 115, and a CDRH3 comprising the sequence of SEQ ID NO: 116, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 112, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 113; ora CDRH1 comprising the sequence of SEQ ID NO: 119, a CDRH2 comprising the sequence of SEQ ID NO: 120, and a CDRH3 comprising the sequence of SEQ ID NO: 121, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 117, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 118.

56. The multi-domain binding molecule of claim 54, wherein the anti-CD3 binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 53 or a sequence having at least 95% sequenceidentity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 54 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 56 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 57 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 58 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain comprising the sequence of SEQ ID NO: 59 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 60 or a sequence having at least 95% sequence identity thereto.

57. The multi-domain binding molecule of claim 54, wherein the anti-CD3 binding domain comprises a sequence of SEQ ID NO: 61, 211, 212, 217, or 218 or a sequence having at least 95% sequence identity to SEQ ID NO: 61, 211, 212, 217, or 218.

58. The multi-domain binding molecule of claim 54, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence having a CDR1 comprising a sequence of SEQ ID NO: 95, a CDR2 comprising a sequence of SEQ ID NO: 96, and a CDR3 comprising a sequence of SEQ ID NO: 97; ora CDR1 comprising a sequence of SEQ ID NO: 98, a CDR2 comprising a sequence of SEQ ID NO: 99, and a CDR3 comprising a sequence of SEQ ID NO: 100.59 The multi-domain binding molecule of claim 54, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence of SEQ ID NO: 62-94 or SEQ ID NO: 101, or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 62-94 or SEQ ID NO: 101.

60. The multi-domain binding molecule of claim 54, wherein the anti-CD19 binding domain comprises a variable heavy chain comprising CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 284, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 284, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 288, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 289, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprisingthe sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 290, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287; ora CDRH1 comprising the sequence of SEQ ID NO: 291, a CDRH2 comprising the sequence of SEQ ID NO: 292, and a CDRH3 comprising the sequence of SEQ ID NO: 293, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 294; a CDRL2 comprising the sequence of SEQ ID NO: 295, and a CDRL3 comprising the sequence of SEQ ID NO: 296.

61. The multi-domain binding molecule of claim 54, wherein the anti-CD19 binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 5 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 8 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 9 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 10 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 11 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 12 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 13 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 14 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain comprising the sequence of SEQ ID NO: 34 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 35 or a sequence having at least 95% sequence identity thereto.

62. The multi-domain binding molecule of claim 7, wherein the anti-CD19 binding domain comprises a sequence having a CDR1 comprising a sequence of SEQ ID NO: 23 or SEQ ID NO: 24, a CDR2 comprising a sequence of SEQ ID NO: 25, and a CDR3 comprising a sequence of SEQ ID NO: 26;a CDR1 comprising a sequence of SEQ ID NO: 27, a CDR2 comprising a sequence of SEQ ID NO: 28, and a CDR3 comprising a sequence of SEQ ID NO: 29; ora CDR1 comprising a sequence of SEQ ID NO: 30 or 31, a CDR2 comprising a sequence of SEQ ID NO: 32, anda CDR3 comprising a sequence of SEQ ID NO: 33.63 The multi-domain binding molecule of claim 7, wherein the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205, or a sequence having at least 95% sequence identity to SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205.

64. The multi-domain binding molecule of claim 7, wherein the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22 or a sequence having at least 95% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22.65 The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain comprises a variable heavy chain comprising CDRH1 comprising the sequence of SEQ ID NO: 297, a CDRH2 comprising the sequence of SEQ ID NO: 298, and a CDRH3 comprising the sequence of SEQ ID NO: 299, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 300, a CDRL2 comprising the sequence of SEQ ID NO: 301, and a CDRL3 comprising the sequence of SEQ ID NO: 302; ora CDRH1 comprising the sequence of SEQ ID NO: 303, a CDRH2 comprising the sequence of SEQ ID NO: 304, and a CDRH3 comprising the sequence of SEQ ID NO: 305, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 306, a CDRL2 comprising the sequence of SEQ ID NO: 301, and a CDRL3 comprising the sequence of SEQ ID NO: 307.

66. The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 36 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 37 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain comprising the sequence of SEQ ID NO: 38 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 39 or a sequence having at least 95% sequence identity thereto.

67. The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain comprises a sequence having a CDR1 comprising a sequence of SEQ ID NO: 44, a CDR2 comprising a sequence of SEQ ID NO: 45, and a CDR3 comprising a sequence of SEQ ID NO: 46;a CDR1 comprising a sequence of SEQ ID NO: 47, a CDR2 comprising a sequence of SEQ ID NO: 48, and a CDR3 comprising a sequence of SEQ ID NO: 49; ora CDR1 comprising a sequence of SEQ ID NO: 50, a CDR2 comprising a sequence of SEQ ID NO: 51, and a CDR3 comprising a sequence of SEQ ID NO: 52.

68. The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain comprises a sequence of SEQ ID NO: 206, 207, 208, 209, 210, or 215 or a sequence having at least 95% sequence identity to SEQ ID NO: 206, 207, 208, 209, 210, or 215.

69. The multi-domain binding molecule of claim 7, wherein the anti-CD33 binding domain comprises a sequenceof SEQ ID NO: 40, 41, 42, or 43, or a sequence having at least 95% sequence identity to SEQ ID NO: 40, 41, 42, or 4370. The multi-domain binding molecule of claim 7, wherein any of the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain are joined by a protein linker.

71. The multi-domain binding molecule of claim 70, wherein the protein linker is a Gly-Ser linker wherein (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 10.72 The multi-domain binding molecule of claim 7, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain are linked to an Fc region of an antibody.

73. The multi-domain binding molecule of claim 72, wherein the Fc region is an IgM Fc region having the sequence of SEQ ID NO: 163-174 or 221 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 163-174, or 221.

74. The multi-domain binding molecule of claim 73, wherein the Fc region comprises a multimerizing fragment of an IgM Fc region.

75. The multi-domain binding molecule of claim 74, wherein the multimerizing fragment of the IgM Fc region has the sequence of SEQ ID NO: 163-174, or 221 or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 163-174 or 221.

76. The multi-domain binding molecule of claim 73, wherein the IgM Fc region comprises a first binding domain and a second binding domain; and a joining (J)-chain comprises a third binding domain.77 The multi-domain binding molecule of claim 76, wherein the J-chain comprises the sequence of SEQ ID NO: 175 or a sequence having at least 95% sequence identity thereto.

78. The multi-domain binding molecule of claim 76, wherein the third binding domain comprises an immune cell activating epitope binding domain.

79. The multi-domain binding molecule of claim 78, wherein the immune cell activating epitope binding domain comprises an anti-CD3 binding domain.80 The multi-domain binding molecule of claim 7, further comprising a fourth binding domain.

81. The multi-domain binding molecule of claim 7, wherein at least one of the immune cell activating epitope binding domain, the anti-CD 19 binding domain, or the anti-CD33 binding domain comprise an antibody or a peptide.

82. The multi-domain binding molecule of claim 81, wherein the antibody comprises an immunoglobulin G (IgG), a Fab fragment, an Fv fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).

83. The multi-domain binding molecule of claim 82, wherein the sdAb comprises a variable heavy domain of a heavy chain (VHH), a camelid heavy chain antibody, an immunoglobulin new antigen receptor (IgNAR), or a picobody84. The multi-domain binding molecule of claim 81, wherein the peptide comprises a miniprotein or a peptideaptamer.85 The multi-domain binding molecule of claim 7, comprising Fc silencing mutations.

86. The multi-domain binding molecule of claim 85, wherein the Fc silencing mutations comprise aglycosylation mutations or LALAPG mutations.

87. A nucleic acid encoding the multi-domain binding molecule of claim 7.

88. A conjugate comprising the multi-domain binding molecule of claim 7 linked to an immunotoxin, a drug, a detectable label, a radioisotope, or a particle.89 A cell genetically modified to express the multi-domain binding molecule of claim 7.

90. A composition comprising an immune cell activating epitope binding domain, an anti-CD 19 binding domain, an anti-CD33 binding domain and a pharmaceutically acceptable carrier wherein the composition can be administered at a dose that kills cells that co-express CD19 and CD33 but does not kill cells that express CD19 or CD33.

91. A kit comprising a multi-domain binding molecule comprising:an immune cell activating epitope binding domain, an anti-CD 19 binding domain, and an anti-CD33 binding domain whereinthe immune cell activating epitope binding domain has the sequence of SEQ ID NO: 248;the anti-CD19 binding domain has the sequence of SEQ ID NO: 342, 343, 226, 334, 338, 231, 232, or 230; and the anti-CD33 binding domain has the sequence of SEQ ID NO: 345, 346, 344, 347, or 240.

92. The kit of claim 91, wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342 or 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 345, 346, 344, or 347.

93. The kit of claim 91, wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 345.

94. The kit of claim 91, wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 342; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 34695. The kit of claim 91, wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 344.

96. The kit of claim 91, wherein:the anti-CD19 binding domain has the sequence of SEQ ID NO: 343; andthe anti-CD33 binding domain has the sequence of SEQ ID NO: 34797. A kit comprising:an immune cell activating epitope binding domain or a nucleic acid encoding the immune cell activating epitopebinding domain,an anti-CD19 binding domain or a nucleic acid encoding the anti-CD19 binding domain, andan anti-CD33 binding domain or a nucleic acid encoding the anti-CD33 binding domain.

98. The kit of claim 97, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain lack an Fc region of an antibody or the nucleotide encoding the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain lacks a sequence encoding an Fc region of an antibody.99 The kit of claim 97, wherein the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain lack an Fc region of an antibody or the nucleotide encoding the immune cell activating epitope binding domain, the anti-CD19 binding domain, or the anti-CD33 binding domain lacks a sequence encoding an Fc region of an antibody.

100. The kit of claim 97, comprising a sequence with at least 95% sequence identity to SEQ ID NO: 334, 335, 337, 338, or 339.

101. The kit of claim 97, comprising SEQ ID NO: 334, 335, 337, 338, or 339.

102. The kit of claim 97, wherein the immune cell activating epitope binding domain binds CD3.

103. The kit of claim 97, wherein the immune cell activating epitope binding domain binds CD3 and comprises a variable heavy chain comprising CDRH1 comprising the sequence of SEQ ID NO: 308, a CDRH2 comprising the sequence of SEQ ID NO: 309, and a CDRH3 comprising the sequence of SEQ ID NO: 310, and a variable light chain comprising a CDR light (L)1 comprising the sequence of SEQ ID NO: 311, a CDRL2 comprising the sequence of SEQ ID NO: 312, and a CDRL3 comprising the sequence of SEQ ID NO: 313;a CDRH1 comprising the sequence of SEQ ID NO: 314, a CDRH2 comprising the sequence of SEQ ID NO: 315, and a CDRH3 comprising the sequence of SEQ ID NO: 316, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 317, a CDRL2 comprising the sequence of SEQ ID NO: 318, and a CDRL3 comprising the sequence of SEQ ID NO: 319;a CDRH1 comprising the sequence of SEQ ID NO: 320, a CDRH2 comprising the sequence of SEQ ID NO: 321, and a CDRH3 comprising the sequence of SEQ ID NO: 322, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 323, a CDRL2 comprising the sequence of SEQ ID NO: 324, and a CDRL3 comprising the sequence of SEQ ID NO: 325;a CDRH1 comprising the sequence of SEQ ID NO: 104, a CDRH2 comprising the sequence of SEQ ID NO: 105, and a CDRH3 comprising the sequence of SEQ ID NO: 106, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 102, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 103;a CDRH1 comprising the sequence of SEQ ID NO: 109, a CDRH2 comprising the sequence of SEQ ID NO: 110, and a CDRH3 comprising the sequence of SEQ ID NO: 111, and a variable light chain comprising a CDRL1 comprisingthe sequence of SEQ ID NO: 107, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 108;a CDRH1 comprising the sequence of SEQ ID NO: 114, a CDRH2 comprising the sequence of SEQ ID NO: 115, and a CDRH3 comprising the sequence of SEQ ID NO: 116, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 112, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 113; ora CDRH1 comprising the sequence of SEQ ID NO: 119, a CDRH2 comprising the sequence of SEQ ID NO: 120, and a CDRH3 comprising the sequence of SEQ ID NO: 121, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 117, a CDRL2 comprising the sequence KVS, and a CDRL3 comprising the sequence of SEQ ID NO: 118.

104. The kit of claim 97, wherein the immune cell activating epitope binding domain comprises an anti-CD3 binding domain comprisinga variable heavy chain comprising the sequence of SEQ ID NO: 53 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 54 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 55 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 56 or a sequence having at least 95% sequence identity thereto;a variable heavy chain comprising the sequence of SEQ ID NO: 57 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 58 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain comprising the sequence of SEQ ID NO: 59 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 60 or a sequence having at least 95% sequence identity thereto.

105. The kit of claim 102, wherein the anti-CD3 binding domain comprises a sequence of SEQ ID NO: 61, 211, 212, 217, or 218 or a sequence having at least 95% sequence identity to SEQ ID NO: 61, 211, 212, 217, or 218.

106. The kit of claim 97, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence having a CDR1 comprising a sequence of SEQ ID NO: 95, a CDR2 comprising a sequence of SEQ ID NO: 96, and a CDR3 comprising a sequence of SEQ ID NO: 97; ora CDR1 comprising a sequence of SEQ ID NO: 98, a CDR2 comprising a sequence of SEQ ID NO: 99, and a CDR3 comprising a sequence of SEQ ID NO: 100.

107. The kit of claim 97, wherein the immune cell activating epitope binding domain binds CD3 and comprises a sequence of SEQ ID NO: 62-94 or 101, or a sequence having at least 95% sequence identity to one of SEQ ID NOs: 62-94 or 101.

108. The kit of claim 97, wherein the anti-CD19 binding domain comprisesa variable heavy chain comprising CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 284, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 284, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 288, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 289, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287;a CDRH1 comprising the sequence of SEQ ID NO: 282, a CDRH2 comprising the sequence of SEQ ID NO: 283, and a CDRH3 comprising the sequence of SEQ ID NO: 290, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 285, a CDRL2 comprising the sequence of SEQ ID NO: 286, and a CDRL3 comprising the sequence of SEQ ID NO: 287; ora CDRH1 comprising the sequence of SEQ ID NO: 291, a CDRH2 comprising the sequence of SEQ ID NO: 292, and a CDRH3 comprising the sequence of SEQ ID NO: 293, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 294; a CDRL2 comprising the sequence of SEQ ID NO: 295, and a CDRL3 comprising the sequence of SEQ ID NO: 296.

109. The kit of claim 97, wherein the anti-CD19 binding domain comprisesa sequence having a CDR1 comprising a sequence of SEQ ID NO: 23 or SEQ ID NO: 24, a CDR2 comprising a sequence of SEQ ID NO: 25, and a CDR3 comprising a sequence of SEQ ID NO: 26;a CDR1 comprising a sequence of SEQ ID NO: 27, a CDR2 comprising a sequence of SEQ ID NO: 28, and a CDR3 comprising a sequence of SEQ ID NO: 29; ora CDR1 comprising a sequence of SEQ ID NO: 30 or SEQ ID NO: 31, a CDR2 comprising a sequence of SEQ ID NO: 32, and a CDR3 comprising a sequence of SEQ ID NO: 33.

110. The kit of claim 97, wherein the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205, or a sequence having at least 95% sequence identity to SEQ ID NO: 199, 200, 201, 202, 203, 204, or 205.

111. The kit of claim 97, wherein the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22 or a sequence having at least 95% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, or 22.

112. The kit of claim 97, wherein the anti-CD33 binding domain comprisesa variable heavy chain comprising CDRH1 comprising the sequence of SEQ ID NO: 297, a CDRH2 comprising the sequence of SEQ ID NO: 298, and a CDRH3 comprising the sequence of SEQ ID NO: 299, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 300, a CDRL2 comprising the sequence of SEQ ID NO: 301, and a CDRL3 comprising the sequence of SEQ ID NO: 302; ora CDRH1 comprising the sequence of SEQ ID NO: 303, a CDRH2 comprising the sequence of SEQ ID NO: 304, and a CDRH3 comprising the sequence of SEQ ID NO: 305, and a variable light chain comprising a CDRL1 comprising the sequence of SEQ ID NO: 306, a CDRL2 comprising the sequence of SEQ ID NO: 301, and a CDRL3 comprising the sequence of SEQ ID NO: 307.

113. The kit of claim 97, wherein the anti-CD33 binding domain comprisesa variable heavy chain comprising the sequence of SEQ ID NO: 36 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 37 or a sequence having at least 95% sequence identity thereto; ora variable heavy chain comprising the sequence of SEQ ID NO: 38 or a sequence having at least 95% sequence identity thereto, and a variable light chain comprising the sequence of SEQ ID NO: 39 or a sequence having at least 95% sequence identity thereto.

114. The kit of claim 97, wherein the anti-CD33 binding domain comprises a sequence having a CDR1 comprising a sequence of SEQ ID NO: 44, a CDR2 comprising a sequence of SEQ ID NO: 45, and a CDR3 comprising a sequence of SEQ ID NO: 46;CDR1 comprising a sequence of SEQ ID NO: 47, a CDR2 comprising a sequence of SEQ ID NO: 48, and a CDR3 comprising a sequence of SEQ ID NO: 49; ora CDR1 comprising a sequence of SEQ ID NO: 50, a CDR2 comprising a sequence of SEQ ID NO: 51, and a CDR3 comprising a sequence of SEQ ID NO: 52.

115. The kit of claim 97, wherein the anti-CD33 binding domain comprises a sequence of SEQ ID NO: 206, 207, 208, 209, 210, or 215 or a sequence having at least 95% sequence identity to SEQ ID NO: 206, 207, 208, 209, 210, or 215.

116. The kit of claim 97, wherein the anti-CD33 binding domain comprises a sequence of SEQ ID NO: 40, 41, 42, or 43, or a sequence having at least 95% sequence identity to SEQ ID NO: 40, 41, 42, or 43.

117. The kit of claim 97, wherein the anti-CD19 binding domain and / or the anti-CD33 binding domain are modified to have reduced binding affinity by replacing tyrosines of the anti-CD19 binding domain and / or the anti-CD33 binding domain with a less hydrophobic amino acid.

118. The kit of claim 97, comprisinga first polypeptide comprising a first multimerization domain or a nucleic acid encoding the first polypeptide and a second polypeptide comprising a second multimerization domain or a nucleic acid encoding the secondpolypeptide wherein when expressed, the first multimerization domain and the second multimerization domain link the first polypeptide to the second polypeptide.

119. The kit of claim 118, wherein the first multimerization domain comprises an lgG1 Fc hole and the second multimerization domain comprises an lgG1 Fc knob.

120. The kit of claim 118, wherein the first multimerization domain comprises an lgG1 Fc knob and the second multimerization domain comprises an lgG1 Fc hole.

121. The kit of claims 119 or 120, wherein the lgG1 Fc knob comprises the sequence of SEQ ID NO: 127 or a sequence having at least 95% sequence identity thereto.

122. The kit of claims 119 or 120, wherein the lgG1 Fc hole comprises the sequence of SEQ ID NO: 128 or a sequence having at least 95% sequence identity thereto.

123. The kit of claim 97, further comprising a pharmaceutically acceptable carrier.

124. The kit of claim 97, further comprising a delivery vehicle.

125. The kit of claim 124, wherein the delivery vehicle comprises a syringe.

126. A method of treating a subject having a cancer that co-expresses CD 19 and CD33, the method comprising administering a therapeutically effective amount of the composition of claim 90 to the subject, thereby treating the subject having the cancer that co-expresses CD 19 and CD33.

127. The method of claim 126, wherein the cancer that co-expresses CD 19 and CD33 comprises a B cell malignancy.

128. The method of claim 126, wherein the cancer that co-expresses CD 19 and CD33 comprises chronic phase leukemia129. The method of claim 126, wherein the cancer that co-expresses CD 19 and CD33 comprises accelerated phase leukemia.

130. The method of claim 126, wherein the cancer that co-expresses CD19 and CD33 comprises blast phase leukemia.

131. The method of claim 126, wherein the cancer that co-expresses CD 19 and CD33 comprises mixed phenotype acute leukemia (MPAL).

132. The method of claim 126, wherein the cancer that co-expresses CD 19 and CD33 comprises B-cell acute lymphoblastic leukemia (B-ALL).

133. The method of claim 126, wherein the administering is intravenous administering.

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