Bispecific antigen binding compositions for targeting solid tumors

Abstract

Provided herein are bispecific antigen binding compositions and pharmaceutical compositions thereof. Further provided are nucleotide sequences encoding said compositions, as well as methods and uses therefor for the treatment of diseases, such as cancer.

Description

W0571.70068WQ00BISPECIFIC ANTIGEN BINDING COMPOSITIONS FOR TARGETING SOLID TUMORSRELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 63 / 709,397, filed October 18, 2024, which is incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number R01CA279259 awarded by the National Institutes of Health. The government has certain rights in this invention.REFERENCE TO AN ELECTRONIC SEQUENCE

[0003] The contents of the electronic sequence listing (W057170068WO00-SEQ-FL.xml; Size: 156,387 bytes; and Date of Creation: October 17, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0004] Macrophages are often the most common infiltrating immune cells in tumors, and they can be provoked to attack cancer under certain conditions1, 2'3'4. Just like T cells, macrophages are regulated by immune checkpoints that constrain their ability to recognize cancer cells as foreign and eliminate them2, 3’4’5. Cancer cells are known to hijack these interactions to indicate “don’t eat me” signals. For these reasons, there exists an explicit need for therapeutics which can harness macrophages against tumor cells.SUMMARY

[0005] In one aspect, the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and / or the second antigen-binding domain binds to CD20, CD47, EGFR, SIRPa, CD24, PD-1, EPCAM, HER2, PD-L1, Trop-2, Folate Receptor Alpha, Nectin-4, Netrin-1, or CD71.

[0006] In some embodiments, the first antigen-binding domain is fused to a first Fc fragment and the second antigen-binding domain is fused to second Fc fragment, optionally wherein the first Fc fragment and / or the second Fc fragment comprise a CH2 and a CH3 domain.1 / 155#14489013vlW0571.70068WQ00

[0007] In some embodiments, the first Fc fragment comprises an amino acid substitution relative to the second Fc fragment, optionally wherein the first Fc fragment and / or the second Fc fragment comprises a CH3 domain and the amino acid substitution is in the CH3 domain.

[0008] In some embodiments, the first Fc fragment and the second Fc fragment heterodimerize.

[0009] In some embodiments, the first antigen-binding domain comprises WTa2dl (SEQ ID NO: 214) or comprises the following light chain CDR sequences: SEQ ID NOs: 102, 107, and 112 and the following heavy chain CDR sequences: SEQ ID NOs: 122, 127, and 132 and the second antigen-binding domain targets EGFR or TROP-2, optionally wherein the second antigen-binding domain comprises: a) the following light chain CDRs: SEQ ID NOS: 143, 152, and 160 and the following heavy chain CDRs: SEQ ID NOs: 178, 187, and 196; or b) the following light chain CDRs: SEQ ID NO: 147, 155, and 164 and the following heavy chain CDRs: SEQ ID NOs: 182, 191, and 200.

[0010] In some embodiments, the first Fc fragment and / or the second Fc fragment comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 215 or 216. In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 213 or 214.[Oil] In another aspect, provided is a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigenbinding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen and / or a tumor associated antigen. In some embodiments, the tumor specific antigen and / or tumor associated antigen comprises CD20, EGFR, PD-1, EPC AM, HER2, PD-L1, Trop-2, Folate Receptor Alpha, Nectin-4, Netrin-1, or CD71.

[0012] In another aspect, provided is a pharmaceutical composition comprising the bispecific antigen binding composition disclosed herein and a pharmaceutically acceptable excipient.

[0013] In another aspect, the present disclosure provides one or more nucleic acid constructs encoding one or more proteins of the bispecific antigen binding composition described herein.

[0014] In yet another aspect, provided is a vector comprising the one or more nucleic acid constructs described here.2 / 155#14489013vlW0571.70068WQ00

[0015] In one aspect, provided herein is a cell comprising one or more bispecific antigen binding composition, the one or more nucleic acid constructs, or the vector disclosed herein.

[0016] In another aspect, the present disclosure provides a method of treating disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell disclosed herein.

[0017] In one aspect the present disclosure provides use of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell described herein for the treatment of a disease, optionally wherein the disease is cancer.

[0018] In another aspect the present disclosure provides a method of reducing tumor size in a subject in need thereof, the method comprising administering to the subject of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell described herein.

[0019] In one aspect the present disclosure provides a method of eliciting phagocytosis of tumor cells, the method comprising contacting the tumor cells with the bispecific antigen binding composition or pharmaceutical composition described herein.

[0020] In another aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for reducing tumor size in a subject in need thereof or killing tumor cells in vitro.

[0021] In one aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for eliciting phagocytosis of tumor cells in a subject in need thereof or in vitro.

[0022] In another aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for treating disease in a subject in need thereof, wherein the disease is cancer.

[0023] In yet another aspect provided herein is a method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen- 3 / 155#14489013vlW0571.70068WQ00 binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising: means for reducing tumor size in the subject.

[0024] In one aspect the present disclosure provides a method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigenbinding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising: means for eliciting phagocytosis of tumor cells in the subject.

[0025] In another aspect, provided is a kit the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell disclosed herein, and instruction for use.

[0026] The foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosure and together with the description, provide non-limiting examples.

[0028] FIGs. 1A-1I show bispecific antigen binding compositions can maximize anti-tumor responses by macrophages while minimizing binding to healthy cells. FIG. 1A is a diagram showing process for high-throughput development and functional evaluation of bispecific antigen binding compositions targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgGl Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antigen binding compositions (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. FIG. IB shows growth of StayGold+ DLD-1 cells in co- culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells. FIG. 1C shows anti-tumor efficacy of bispecific antigen binding compositions at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-4 / 155#14489013vlW0571.70068WQ00 way ANOVA with Dunnett’s multiple comparisons test. FIGs. 1D-1F show growth curves for each of the WTa2dl constructs ( FIG. ID), CD24-3 constructs ( FIG. IE), or CV1 constructs ( FIG. IF). FIG. 1G shows representative whole-well imaging of co-cultures treated with different bispecific antigen binding compositions at approximately t = 6.5 day. Growth of StayGold+ DLD-1 cells is depicted. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. FIG. 1H shows scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. Data for CV 1 clustered in the upper right corner of the graph. Data for CD24-3 clustered in the lower right corner of the graph. Data for WTa2dl also clustered together. FIG. II shows representative histograms showing binding of the indicated bispecific antigen binding compositions to human neutrophils and red blood cells.

[0029] FIG. 2 is a diagram showing the CD47 / SIRPa and CD24 / Siglec-10 interaction, a macrophage immune checkpoint.

[0030] FIGs. 3A-3C show dual targeting of CD47 and CD24 maximizes anti-tumor responses by macrophages. FIG. 3A shows histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. FIG. 3B shows quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. For each cell line, data is shown in the following order: WT, CD47 KO, CD24 KO, and Double knockout. FIG. 3C shows primary mouse macrophages were co-cultured in 384- well plates with GFP+ KPCA cells (a murine ovarian cancer cell line) at baseline or in the presence of an anti-CD47 (MIAP410) or anti-CD24 (Ml 69) antibody. Cells were co-cultured for 7 days and the GFP+ area was quantified over time. Representative whole-well images from the Sartorius Incucyte S3 live-cell imaging system at seven days are presented for vehicle control, anti- CD47, anti-CD24, and combination anti-CD47 with anti-CD24. (Ordinary one-way ANOVA, Tukey’s multiple comparisons test, n= 3 per group).

[0031] FIGs. 4A-4B show unbiased CRISPR screen identifies CD24 as a target of opsonization rather than a macrophage immune checkpoint. FIG. 4A is a schematic of a genome- wide CRISPR screening strategy in murine lung cancer cell line 238N1 identifying differential responses to macrophage co-culture and treatment with anti-CD24. FIG. 4B shows differentially enriched GO Biological Process pathways comparing treatment with macrophages and anti-CD24 against treatment with macrophages alone. Top 5 enriched and depleted pathways are shown (p-adjusted <0.05).5 / 155#14489013vlW0571.70068WQ00

[0032] FIGs. 5A-5D show genetic ablation of CD24 does not influence anti-tumor responses in vitro. FIG. 5A shows quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells treated with the indicated antibodies. FIG. 5B shows quantification of phagocytosis using CFSE+ MC38 cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. FIG. 5C shows phagocytosis assays using primary human macrophages and CD24 knockdown variants of NCI-H3122. FIG. 5D shows phagocytosis assays using CD24 overexpression lines deriving from PC9.

[0033] FIGs. 6A-6D show CD24 exhibits limited activity as an immune checkpoint in immunocompetent models and poses risks for hematological toxicity. FIG. 6A shows tumor volume growth curve for KPCA.C cells with the indicated knockout engrafted subcutaneously into C57BL / 6J mice. FIG. 6B shows tumor volumes for MC38 overexpressing CD24 that were engrafted subcutaneously into C57BL / 6J mice. FIG. 6C shows phagocytosis of CFSE+ human neutrophils by human macrophages following 2 hour co-culture with the indicated therapies. FIG. 6D shows quantification of receptor occupancy of anti-CD24 on the surface of red blood cells.

[0034] FIG. 7A-7C shows perturbations of human CD24 minimally influence tumor growth in xenograft models but alter immune activation. FIG. 7A shows tumor volume growth curves of NSG mice engrafted with control or CD24 overexpression lines deriving from PC9. FIG. 7B shows tumor volume growth curves of NSG mice engrafted subcutaneously with control or CD24 knockdown tumors deriving from NCI-H3122. FIG. 7C shows UMAP showing identified cell clusters from immune cells sorted from CD24- negative versus CD24-positive tumors. Representative datapoints are labeled.DEFINITIONS

[0035] As used herein, the “knob” and “hole” technology is used to create a hetero or homo dimeric molecule wherein the “knob” mutated region of the CH3 antibody domain associates with the “hole” mutated region of the other CH3 antibody domain.

[0036] Without wishing to be bound by theory, “close proximity” as used herein defines the reduced distance between the tumor cell and the immune cell when the binding of the bispecific antigen binding molecule brings the tumor cell and the immune cell closer together for therapeutic benefit. The term “bispecific antigen binding molecule” is used interchangeably with the term “bispecific antigen binding composition” and “bispecific antigen-binding composition.”6 / 155#14489013vlW0571.70068WQ00

[0037] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

[0038] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0039] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Proteins preferably contain only natural amino acids, although non-natural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and / or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these.

[0040] The term “inhibit” or “inhibition” in the context of modulating level (e.g., expression and / or activity) of a target is not limited to only total inhibition. Thus, in some embodiments, partial inhibition or relative reduction is included within the scope of the term “inhibition.” In some embodiments, the term refers to a reduction of the level (e.g., expression, and / or activity) of a target to a level that is reproducibly and / or statistically significantly lower than an initial or other appropriate reference level, which may, for example, be a baseline level of a target. In some embodiments, the term refers to a reduction of the level (e.g., expression and / or activity) of a target to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of a target.7 / 155#14489013vl

[0041] As used herein, the term "inhibitor" refers to an agent whose presence or level correlates with decreased level or activity of a target to be modulated. In some embodiments, an inhibitor may act directly (in which case it exerts its influence directly upon its target, for example by binding to the target); in some embodiments, an inhibitor may act indirectly (in which case it exerts its influence by interacting with and / or otherwise altering a regulator of a target, so that level and / or activity of the target is reduced). In some embodiments, an inhibitor is one whose presence or level correlates with a target level or activity that is reduced relative to a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known inhibitor, or absence of the inhibitor as disclosed herein, etc.).

[0042] The terms “composition” and “formulation” are used interchangeably. In some embodiments, the composition is a bispecific antigen binding composition.

[0043] A “subject” refers to a human (i.e., male or female of any age group, e.g., pediatric subject e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. A “patient” refers to a human subject in need of treatment of a disease.

[0044] The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample.

[0045] The terms “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described 8 / 155#14489013vlherein, or a composition thereof, in or on a subject. The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and / or lymph supply, and / or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for intravenous or subcutaneous injection into a subject.

[0046] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay and / or prevent recurrence.

[0047] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0048] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0049] An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a9 / 155#14489013vlW0571.70068WQ00 prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses.

[0050] A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0051] A “prophylactically effective amount” of a compound described herein is an amount effective to prevent a condition, or one or more symptoms associated with the condition and / or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0052] A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0053] The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and 10 / 155#14489013vlW0571.70068WQ00 in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and / or is associated with a disease.

[0054] The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’ s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[0055] The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, 11 / 155#14489013vlW0571.70068WQ00 hematological malignancies. The term “hematological malignancy” refers to tumors that affect blood, bone marrow, and / or lymph nodes. Exemplary hematological malignancies include, but are not limited to, leukemia, such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B- cell NHL, such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL, e.g., activated B-cell (ABC) DLBCL (ABC-DLBCL))), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B- cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, Waldenstrom’s macroglobulinemia (WM, lymphoplasmacytic lymphoma), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, central nervous system (CNS) lymphoma (e.g., primary CNS lymphoma and secondary CNS lymphoma); and T-cell NHL, such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); lymphoma of an immune privileged site (e.g., cerebral lymphoma, ocular lymphoma, lymphoma of the placenta, lymphoma of the fetus, testicular lymphoma); a mixture of one or more leukemia / lymphoma as described above; myelodysplasia; and multiple myeloma (MM). Additional exemplary cancers include, but are not limited to, lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, a.k.a. Wilms’ tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma;12 / 155#14489013vlW0571.70068WQ00 chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the13 / 155#14489013vlthyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0056] The term “immunotherapy” refers to a treatment of disease by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress an immune response are classified as suppression immunotherapies.Immunotherapy may encompass treatment with a molecular entity (e.g., immunotherapeutic agent) and / or a non-molecular entity (e.g., adoptive cell transfer).

[0057] The term “macrophage- directed immunotherapy” refers to an immunotherapy that derives its therapeutic effect by stimulating macrophages. Such stimulation can mobilize macrophage and myeloid components to destroy a tumor and its stroma, including the tumor vasculature. Macrophages can be induced to secrete antitumor cytokines and / or to perform phagocytosis, including antibody-dependent cellular phagocytosis.

[0058] The term “immunotherapeutic agent” refers to a molecular entity that induces, enhances, or suppresses an immune response. Immunotherapeutic agents include, but are not limited to, monoclonal antibodies, cytokines, chemokines, vaccines, small molecule inhibitors, and small molecule agonists.

[0059] The term “immune checkpoint inhibitor” refers to an agent that blocks certain proteins made by some types of immune system cells (e.g., T cells, macrophages) and some cancer cells. These proteins function to keep immune responses in check and can also function to keep immune system cells (e.g., T cells, macrophages) from killing cancer cells. When these proteins are blocked, immune system function is restored and the immune system is released enabling the desired immune system cells to kill cancer cells. Some immune checkpoint inhibitors are useful in treating cancer. A “macrophage immune checkpoint inhibitor” functions to stimulate macrophage phagocytosis of cancer cells. For example, CD47 is associated with a macrophage immune checkpoint (CD47 / SIRPa as described herein). CD47-blocking therapies thus stimulate macrophage phagocytosis of cancer cells and are effective in treating cancer.

[0060] The terms “biologic,” “biologic drug,” and “biological product” refer to a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, nucleic acids, and proteins. Biologies may include sugars, proteins, or nucleic acids, or complex combinations of these substances, or may be living entities such as cells and tissues. Biologies may be isolated from a variety of natural sources (e.g., human,14 / 155#14489013vlW0571.70068WQ00 animal, microorganism) and / or may be produced by biotechnological methods and / or other technologies.

[0061] The term “antibody” refers to a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulins) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, or antigens. Antibodies are also known collectively as immunoglobulins. An antibody may be of human or non-human (for example, rodent such as murine, dog, camel, etc.) origin (e.g., may have a sequence originally developed in a human or non-human cell or organism), or may be or comprise a chimeric, humanized, reshaped, or reformatted antibody based, e.g., on a such a human or non-human antibody (or, in some embodiments, on an antigen-binding portion thereof).

[0062] All natural antibodies have the same overall basic structure of an immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Such mutants, variants, or derivatives may be a single-domain antibody, a Fab fragment, scFV, affibody, nanobody, peptide, DARPin, adnectin, anticalin, or fragment thereof.

[0063] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as ECVR or VE) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass.15 / 155#14489013vlW0571.70068WQ00

[0064] The term “antigen-binding domain” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi- specific formats; specifically binding to two or more different antigens. Multispecific, dual specific, and bispecific antibody constructs are well known in the art and described and characterized in Kontermann (ed.), Bispecific Antibodies, Springer, NY (2011), and Spiess et al., Mol. Immunol. 67(2):96- 106 (2015). In some embodiments, an antigen-binding domain comprises the extracellular domain of a protein that binds to an antigen. In some embodiments, an antigen is a ligand.

[0065] Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90 / 05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigenbinding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).16 / 155#14489013vlW0571.70068WQ00

[0066] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0067] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0068] The term “synthetic antibody” as used herein, refers an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a viral vector. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0069] In some embodiments, the term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full- length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0070] In some embodiments, as will be clear from context, the term “antibody” as used herein encompasses formats that include epitope-binding sequences of an antibody, which such formats include, for example chimeric and / or single chain antibodies (e.g., a nanobody or Fcab), as well as binding fragments of antibodies, such as Fab, Fv fragments or single 17 / 155#14489013vlW0571.70068WQ00 chain Fv (scFv) fragments, as well as multimeric forms such as dimeric IgA molecules or pentavalent IgM molecules. Also included are bispecific antibodies, bispecific T cell engagers (BiTEs), immune mobilixing monoclonal T cell receptors against cancer (ImmTACs), dual-affinity re-targeting (DART); alternative scaffolds or antibody mimetics (e.g., anticalins, FN3 monobodies, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Fynomers, Im7, VLR, VNAR, Trimab, CrossMab, Trident); nanobodies, binanobodies, F(ab’)2, Fab’, di-sdFv, single domain antibodies, trifunctional antibodies, diabodies, and minibodies. In some embodiments, an antibody is a bispecific antigen binding composition disclosed herein. In some embodiments, an antibody is a bispecific antibody. In some embodiments, a bispecific antibody is a bispecific antigen binding composition disclosed herein.

[0071] The term “therapeutic agent” refers to an agent having one or more therapeutic properties that produce a desired, usually beneficial, effect. For example, a therapeutic agent may treat, ameliorate, and / or prevent disease. In some embodiments, a therapeutic agent may be or comprise a biologic, a small molecule, or a combination thereof.

[0072] The term “chemotherapeutic agent” refers to a therapeutic agent known to be of use in chemotherapy for cancer.

[0073] The term “targeted agent” refers to an anticancer agent that blocks the growth and spread of cancer by interfering with specific proteins ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted agents are sometimes called “targeted therapies,” “targeted cancer therapies,” "molecularly targeted drugs," "molecularly targeted therapies," or "precision medicines." Targeted agents differ from standard chemotherapy in that targeted agents act on specific molecular targets that are associated with cancer, whereas many chemotherapeutic agents act on all rapidly dividing cells (e.g., whether or not the cells are cancerous). Targeted agents are deliberately chosen or designed to interact with their target, whereas many standard chemotherapies are identified because they may indiscriminately kill cells.

[0074] A “cell” or “target cell” as used herein, may be present in a population of cells (e.g., in a tissue, an organ, or an organoid). In some embodiments, a population of cells is composed of a plurality of cell types. Cells for use in the methods of the present disclosure can be present within an organism, a single cell type derived from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, etc. Virtually any cell type and size can be accommodated in the methods and systems described herein. Suitable cells 18 / 155#14489013vlW0571.70068WQ00 include bacterial, fungal, plant, and animal cells. In some embodiments, the cells are mammalian cells (e.g., complex cell populations such as naturally occurring tissues). In some embodiments, the cells are from a human. In certain embodiments, the cells are collected from a subject (e.g., a human) through a medical procedure such as a biopsy. Alternatively, the cells may be a cultured population (e.g., a culture derived from a complex population or a culture derived from a single cell type where the cells have differentiated into multiple lineages).

[0075] In particular, the term “target cell” refers to a cell (e.g., inflicted cells such as cancer cells, infected cells, etc.) that is targeted for phagocytosis by a macrophage. The target cell can be any cell as described herein. Examples of target cells include but are not limited neoplastic cells, such as cancer cells, and disease associated cells. Examples of cancers include but are not limited to solid tumors, breast cancer, pancreatic cancer, colon cancer, non-small cell lung cancer, leukemias, lymphomas, such as mantle cell lymphoma, and myelomas, such as multiple myeloma. The term “cancer cells” can also be used to refer to cancer or tumor cell lines.

[0076] The terms “assessing”, “determining”, “evaluating”, and “assaying” are used interchangeably herein to refer to any form of detection or measurement, and include determining whether a substance, signal, enzymatic activity, disease, condition, etc., is present or not. The result of an assessment may be expressed in qualitative and / or quantitative terms. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something that is present or determining whether it is present or absent.

[0077] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0078] Inflammatory disease or condition.

[0079] The terms “inflammatory disease” and “inflammatory condition” are used interchangeably herein, and refer to a disease or condition caused by, resulting from, or resulting in inflammation. Inflammatory diseases and conditions include those diseases, disorders or conditions that are characterized by signs of pain (dolor, from the generation of noxious substances and the stimulation of nerves), heat (calor, from vasodilatation), redness (rubor, from vasodilatation and increased blood flow), swelling (tumor, from excessive inflow or restricted outflow of fluid), and / or loss of function (functio laesa, which can be partial or complete, temporary or permanent. Inflammation takes on many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, 19 / 155#14489013vlW0571.70068WQ00 disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and / or ulcerative inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and / or T-lymphocytes leading to abnormal tissue damage and / or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren’s syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto’s thyroiditis, Graves’ disease, Goodpasture’s disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener’s granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host- versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, 20 / 155#14489013vlW0571.70068WQ00 transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis. An ocular inflammatory disease includes, but is not limited to, post-surgical inflammation.

[0080] Additional exemplary inflammatory conditions include, but are not limited to, inflammation associated with acne, anemia (e.g., aplastic anemia, hemolytic autoimmune anemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu’s arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter’s arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, Type II diabetes mellitus), a skin condition (e.g., psoriasis, eczema, bums, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infection, ischemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious anemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, scleroderma, sarcoidosis, spondyloarthopathies, Sjogren’s syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scarring, burns, physical injury), vasculitis, vitiligo and Wegener’s granulomatosis. In certain embodiments, the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis and prostatitis. In certain embodiments, the inflammatory condition is an acute inflammatory21 / 155#14489013vlcondition (e.g., for example, inflammation resulting from infection). In certain embodiments, the inflammatory condition is a chronic inflammatory condition e.g., conditions resulting from asthma, arthritis and inflammatory bowel disease). The compounds may also be useful in treating inflammation associated with trauma and non-inflammatory myalgia. The compounds disclosed herein may also be useful in treating inflammation associated with cancer

[0081] Liver disease

[0082] The term “liver disease” or “hepatic disease” refers to damage to or a disease of the liver. Non-limiting examples of liver disease include intrahepatic cholestasis (e.g., alagille syndrome, biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye’s syndrome), hepatic vein thrombosis, hepatolenticular degeneration (i.e., Wilson’s disease), hepatomegaly, liver abscess (e.g., amebic liver abscess), liver cirrhosis (e.g., alcoholic, biliary, and experimental liver cirrhosis), alcoholic liver diseases (e.g., fatty liver, hepatitis, cirrhosis), parasitic liver disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis, chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced chronic hepatitis), toxic hepatitis, viral human hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, varices, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastases, cystic liver metastases, fibrolamellar hepatocarcinoma, hepatic adenoma, hepatoma, hepatic cysts (e.g., Simple cysts, Polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst), mesenchymal tumors (mesenchymal hamartoma, infantile hemangioendothelioma, hemangioma, peliosis hepatis, lipomas, inflammatory pseudotumor), epithelial tumors (e.g., bile duct hamartoma, bile duct adenoma), focal nodular hyperplasia, nodular regenerative hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi’s sarcoma, hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma, peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (e.g., acute intermittent porphyria, porphyria cutanea tarda), and Zellweger syndrome.

[0083] Immune disorder22 / 155#14489013vlW0571.70068WQ00

[0084] Immune disorders, such as auto-immune disorders, include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren’s syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet’s disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, bums, dermatitis, pruritus (itch)), enuresis, eosinophilic disease, gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), and disorders ameliorated by a gastroprokinetic agent (e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies and other functional bowel disorders, such as non-ulcerative dyspepsia (NUD) and noncardiac chest pain (NCCP, including costo-chondritis)).

[0085] In certain embodiments, the inflammatory disorder and / or the immune disorder is a gastrointestinal disorder. In some embodiments, the gastrointestinal disorder is selected from gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)). In certain embodiments, the gastrointestinal disorder is inflammatory bowel disease (IBD).

[0086] In certain embodiments, the inflammatory condition and / or immune disorder is a skin condition. In some embodiments, the skin condition is pruritus (itch), psoriasis, eczema, bums or dermatitis. In certain embodiments, the skin condition is psoriasis. In certain embodiments, the skin condition is pruritis.

[0087] Lung disease23 / 155#14489013vlW0571.70068WQ00

[0088] The term “lung disease” or “pulmonary disease” refers to a disease of the lung. Examples of lung diseases include, but are not limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema, cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis, aspergilloma, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia, bronchial pneumonia, interstitial pneumonia), pulmonary fibrosis, pulmonary tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., nonsmall-cell lung carcinoma (e.g., adenocarcinoma, squamous-cell lung carcinoma, large-cell lung carcinoma), small-cell lung carcinoma).

[0089] Autoimmune disease

[0090] An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture’s disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture’s syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, , psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, and cardiomyopathy.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0091] The aspects described herein are not limited to specific embodiments, methods, uses, or configurations, and as such can, of course, vary. The terminology used herein is for the24 / 155#14489013vlW0571.70068WQ00 purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0092] Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown.

[0093] As presented herein the therapeutic potential of targeting two distinct macrophage immune checkpoints, CD47 and CD24, was evaluated. It was found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, surprisingly, via unbiased genome-wide CRISPR screens, it was found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, it was found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity that was lifethreatening in mice.

[0094] To overcome these challenges and leverage these findings for therapeutic purposes, a collection of 77 novel bispecific antigen binding compositions that bind to a tumor antigen with one arm and engage macrophages with the second arm were engineered. Multiple novel bispecifics were discovered that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, the findings presented herein indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics presented herein may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors

[0095] To date, the best-characterized macrophage checkpoint is the CD47 / SIRPa interaction2. CD47 is a surface antigen that is expressed on many cancers, and it functions by binding the inhibitory receptor, SIRPa, expressed on macrophages2. Multiple drugs targeting this interaction have shown efficacy in preclinical models of both solid tumors and hematologic malignancies6, 7'8'9. Clinical trials using CD47-blocking therapies are ongoing and show promise for multiple types of cancer10, indicating that activation of macrophage- mediated cytotoxicity can be an effective therapeutic strategy.

[0096] Based on the promise of targeting the CD47 / SIRPa interaction, research efforts have focused on identifying additional macrophage immune checkpoints, or “don’t eat me” signals. Another surface antigen on cancer cells, CD24, is thought to act in this manner. In small cell lung cancer, the combination of CD47 blockade with anti-CD24 antibodies was 25 / 155#14489013vlW0571.70068WQ00 found to enhance macrophage phagocytosis9. A subsequent study proposed that CD24 on cancer cells functions by transmitting a direct inhibitory signal to macrophages, suggesting it acts as a macrophage immune checkpoint11. However, the conservation of CD24 as a macrophage checkpoint across different cancers is unclear since unbiased genomic screens did not identify CD24 as a robust hit across multiple different human cancer types12.

[0097] CD24 is a short, GPI-linked cell surface protein that is heavily glycosylated13. It has been reported to interact with a number of ligands, including Siglec-10 (Siglec-G in mice), which can transduce inhibitory signals to some immune cell populations14. In certain contexts, CD24 may also play an inhibitory role in B cell development15. In cancer, prior studies examining the therapeutic potential of targeting CD24 have primarily relied upon xenograft models, which are limited by a lack of an adaptive immune system11, 16. Furthermore, the anti-CD24 antibodies used in these studies target human CD24 and do not cross-react with the murine CD24 homolog. Therefore, these models do not accurately reflect the therapeutic window for targeting CD24 since they underestimate toxicity and overrepresent selectivity to the tumor microenvironment. These pharmacologic properties can be better evaluated using syngeneic, immunocompetent mouse models. As presented herein, these shortcomings may be addressed by evaluating the therapeutic potential of targeting CD24 in immunocompetent mouse models of cancer. It was found that dualtargeting of CD47 and CD24 could indeed elicit maximal levels of macrophage phagocytosis. However, via a series of genetic and pharmacologic experiments, it is demonstrated that CD24 predominantly acts as a target of opsonization rather than a macrophage immune checkpoint. These findings provide insight into therapeutic targeting of CD24 in patients with cancer, and guided the development of novel bispecific antigen binding compositions that maximally activate macrophage anti-tumor functions.Bispecific antigen binding compositions

[0098] In one aspect, the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain and / or second antigenbinding domain is a protein.

[0099] In some embodiments, the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen.

[0100] In some embodiments, the tumor specific antigen is EGFR or TROP2. In some embodiments, the tumor specific antigen is EGFR. In some embodiments, the tumor specific antigen is TROP2.26 / 155#14489013vlW0571.70068WQ00

[0101] In certain embodiments, the first antigen-binding domain binds CD47.

[0102] In some embodiments, the first antigen-binding domain binds CD24.

[0103] In some embodiments, a tumor associated antigen and / or tumor specific antigen comprises any antigen derived from genes overexpressed in tumors, differentiation antigens, and / or cancer germline / cancer testis antigens as described in Feola et al. “Uncovering the tumor antigen landscape: What we know about the Discovery Process,” Cancers (Basel). 2020 Jun 23; 12(6): 1660 and Ilyas et al., “Landscape of Tumor Antigens in T-cell Immunotherapy,” J. Immunol. 2015 Dec 1; 195(11):5117-5122 the contents of which are both incorporated herein in their entirety. In some embodiments, the tumor-associated antigens comprise EGFR, hTERT, p53, carbonic anhydrase IX, renal antigen 1 (RAGE-1), HER2 / NEU, MART-1, gplOO, CEA, CD19, NY-ES01, MAGE-A3, hTERT, EGFR, mesothelin, HPV, EBV, MUC-1, MCC, Mum-1, B-catenin, CDK4, and ERBB2IP.

[0104] In some embodiments, a tumor specific antigen and / or a tumor associated antigen comprises CD20, CD47, EGFR, CD24, PD-1, EPCAM, HER2, PD-L1, Trop-2, Folate Receptor Alpha, Nectin-4, Netrin-1, or CD71.

[0105] In some embodiments, a first antigen-binding domain and / or a second antigen-binding domain comprises a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a variable heavy (VH) domain, a variable light (VL) domain, and / or a single-domain antibody (VHH, VNAR).

[0106] In some embodiments, a first antigen-binding domain and / or second antigen-binding domain comprises an extracellular domain of a protein. In some embodiments, the protein is a transmembrane protein. In some embodiments, the protein is a receptor protein. In some embodiments, a first antigen-binding domain and / or second-antigen binding domain comprises a natural or engineered ligand, an enzymes, a cytokine, or a synthetic protein scaffold designed for specific binding activity. Exemplary embodiments of antigen-binding domains, include but are not limited to, (i) all or part of the extracellular region of a protein; (ii) proteins, peptides, or other molecules that bind to a receptor or target protein; (iii) signaling proteins capable of receptor binding; and (iv) engineered protein frameworks capable of specific binding, including, but not limited to, anticalins, FN3 monobodies, DARPins, affibodies, affilins, affimers, affitins, alphabodies, avimers, fynomers, variable lymphocyte receptors (VLR), and VNAR domains.

[0107] In some embodiments, the first antigen-binding domain and / or anti-antigen binding domain is capable of binding CD47. In some embodiments, a first antigen-binding domain and / or a second antigen-binding domain comprises a domain that corresponds to the 27 / 155#14489013vlW0571.70068WQ00 extracellular domain of signal regulatory protein alpha. In some embodiments, a first antigen-binding domain and / or a second antigen-binding domain comprises a domain with one or more mutations, insertions, and / or deletions relative to the extracellular domain of signal regulatory protein alpha.

[0108] Non-limiting examples of a domain that corresponds to the extracellular domain of signal regulatory protein alpha include WTa2dl and CV1. WTa2dl is provided as SEQ ID NO: 214. CV1 is provided as SEQ ID NO: 213. In some embodiments, a first antigenbinding domain and / or second antigen-binding domain comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 213 or SEQ ID NO: 214. See also, e.g., Weiskopf et al., Science. 2013 Jul 5;341(6141):88-91.

[0109] In some embodiments, a first antigen-binding domain and / or a second antigen-binding domain binds to CD20, CD47, EGFR, SIRPa, CD24, PD-1, EPCAM, HER2, PD-L1, Trop-2, Folate Receptor Alpha, Nectin-4, Netrin-1, or CD71. In some embodiments, a bispecific antigen binding composition binds to a target disclosed in any one of Tables 1-7. In some embodiments, a bispecific antigen binding composition binds to a combination of targets disclosed in any one of Tables 1-5. In some embodiments, the combination of targets for a bispecific antigen binding composition is a combination of targets disclosed in any one of Tables 1-5.

[0110] In some embodiments, a bispecific antigen binding composition comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to any one of the amino acid sequences set forth in any one of Tables 1-7. In some embodiments, a bispecific antigen binding composition comprises a sequence set forth in any one of Tables 1-7. from any one of Tables 1-8.

[0111] In some embodiments, a bispecific antigen binding composition comprises a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 amino acid substitutions, insertions, and / or deletions relative to a sequence set forth in any one of Tables 1-8.

[0112] Aspects of the present disclosure provide bispecific antigen binding compositions comprising two distinct antigen-binding domains capable of interacting with different parts of the same target or are capable of interacting with two different targets, wherein each antigen-binding domain is fused to an Fc fragment that comprises a CH3 domain. In some embodiments, the Fc fragment further comprises a CH2 domain. See also the Methods of Assembling Bispecific Antigen Binding Compositions section below.28 / 155#14489013vlW0571.70068WQ00

[0113] In some embodiments, the bispecific antigen binding composition disclosed herein comprises a “knob” sequence and a “hole” sequence.

[0114] In certain embodiments, the “knob” sequence comprises the first antigen-binding domain or the second antigen-binding domain.

[0115] In some embodiments, the “knob” sequence comprises the first antigen-binding domain.

[0116] In certain embodiments, the “knob” sequence comprises the second-antigen binding domain.

[0117] In some embodiments, the “knob” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 8-23.

[0118] In some embodiments, the “knob” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12.

[0119] In certain embodiments, the “knob” sequence comprises a sequence identical to any one of the amino acid sequences set forth in SEQ ID NOs: 8-23.

[0120] In some embodiments, the “knob” sequence comprises a sequence identical to any one of the amino acid sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12.

[0121] In certain embodiments, the “hole” sequence comprises the first antigen-binding domain or the second antigen-binding domain.

[0122] In some embodiments, the “hole” sequence comprises the first antigen-binding domain.

[0123] In certain embodiments, the “hole” sequence comprises the second-antigen binding domain.

[0124] In some embodiments, the “hole” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-7.

[0125] In certain embodiments, the “hole” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 6.29 / 155#14489013vlW0571.70068WQ00

[0126] In some embodiments, the “hole” sequence comprises a sequence identical to any one of SEQ ID NOs: 1-7.

[0127] In some embodiments, the “hole” sequence comprises a sequence identical to any one of SEQ ID NO: 2 and SEQ ID NO: 6.

[0128] In some embodiments, a) the “knob” sequence comprises a sequence identical to SEQ ID NO: 8 and the “hole” sequence comprises a sequence identical to of SEQ ID NO: 2, b) the “knob” sequence comprises a sequence identical to SEQ ID NO: 8 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 6, c) the “knob” sequence comprises a sequence identical to SEQ ID NO: 12 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 2, or d) the “knob” sequence comprises a sequence identical to SEQ ID NO: 12 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 6.

[0129] In some embodiments,(a) a heavy chain complementarity determining region 1 (CDRH1) comprising any one of SEQ ID NOs: 119-123 or 178-186;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising any one of SEQ ID NOs: 124-128 or 187-195;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising any one of SEQ ID NOs: 129-133 or 196-204;(d) a light chain complementarity determining region 1 (CDRL1) comprising any one of SEQ ID NO: 99-103 or 143-151 ;(e) a light chain complementarity determining region 2 (CDRL2) comprising any one of SEQ ID NOs: 104-108 or 152-159; and(f) a light chain complementarity determining region 3 (CDRL3) comprising any one of SEQ ID NOs: 109-113 or 160-169.

[0130] In some embodiments,(a) a heavy chain complementarity determining region 1 (CDRH1) comprisingSEQ ID NO: 182;(b) a heavy chain complementarity determining region 2 (CDRH2) comprisingSEQ ID NO: 191;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 200;30 / 155#14489013vlW0571.70068WQ00(d) a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 147;(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 155; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NOs: 164.

[0131] In some embodiments, the bispecific antigen binding composition disclosed herein comprises:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 178;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 187;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 196;(d) a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 143;(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 152; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NOs: 160.

[0132] In some embodiments, the bispecific antigen binding composition comprises:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 122;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 127;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 132;(d) a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 102;(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 107; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NOs: 112.

[0133] In some embodiments, a bispecific antigen binding composition comprises one or more linkers. In some embodiments, the linkers join one or more domains of the bispecific 31 / 155#14489013vlW0571.70068WQ00 antigen binding composition. For example, in some embodiments, an antigen binding domain comprises a linker. In some embodiments, the antigen binding domain comprises an scFv comprising a VL domain and a VH domain. In some embodiments, the VL domain and the VH domain are fused by a linker. In some embodiments, a linker fuses an antigen binding domain to an Fc fragment. In some embodiments, the Fc fragment comprises a CH3 domain. In some embodiments, the Fc fragment comprises a CH2 domain and a CH3 domain. In some embodiments, a linker is used to fuse one or more other domains to the bispecific antigen binding composition.

[0134] Any suitable linker may be used to fuse one or more domains of any of the bispecific antigen binding compositions disclosed herein. In some embodiments, the linker comprises the amino acid sequence AAA, AAAA, (A)n, or GGGGSGGGGSGGGGS (SEQ ID NO: 217).

[0135] In another aspect, provided is a pharmaceutical composition comprising the bispecific antigen binding composition disclosed herein and a pharmaceutically acceptable excipient.

[0136] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutical agent.

[0137] In another aspect, the present disclosure provides one or more nucleic acid constructs encoding one or more proteins of the bispecific antigen binding composition described herein.

[0138] In some embodiments, the nucleic acid is DNA or RNA.

[0139] In yet another aspect, provided is a vector comprising the one or more nucleic acid constructs described here.

[0140] In one aspect, provided herein is a cell comprising one or more bispecific antigen binding composition, the one or more nucleic acid constructs, or the vector disclosed herein.

[0141] In another aspect, the present disclosure provides a method of treating disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell disclosed herein.

[0142] In some embodiments, the disease is a proliferative disease.

[0143] In certain embodiments, the proliferative disease is cancer.

[0144] In some embodiments, the cancer is breast, colon, liver, lung, ovarian, melanoma, prostate, skin, renal, thyroid, or uterine cancer. In some embodiments, the cancer is adenocarcinoma.

[0145] In certain embodiments, the cancer is therapy resistant.32 / 155#14489013vlW0571.70068WQ00

[0146] In certain embodiments, the disease is psoriasis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), asthma, inflammatory bowel disease (IBD), ulcerative colitis and Crohn's disease, liver cirrhosis, renal fibrosis, atherosclerosis, keloids and hypertrophic scars, epidermal growth disorders such as seborrheic keratosis, fibrotic diseases such as lung fibrosis, wound healing disorders including chronic wounds, and autoimmune skin disorders like pemphigus.

[0147] In one aspect the present disclosure provides use of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell described herein for the treatment of a disease.

[0148] In some embodiments, the disease is a proliferative disease.

[0149] In certain embodiments, the proliferative disease is cancer.

[0150] In some embodiments, the cancer is breast, colon, liver, lung, ovarian, melanoma, prostate, skin, renal, thyroid, or uterine cancer.

[0151] In another aspect the present disclosure provides a method of reducing tumor size in a subject in need thereof, the method comprising administering to the subject of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell described herein.

[0152] In some embodiments, the method comprises in bringing into close proximity a tumor cell with an immune cell.

[0153] In certain embodiments, the immune cell is a macrophage.

[0154] In some embodiments, the tumor or tumor cell is TROP2+ or EGFR+. In some embodiments, the tumor is a solid tumor.

[0155] In certain embodiments, the immune cell is a CD47+ cell or a CD24+ cell.

[0156] In one aspect the present disclosure provides a method of eliciting phagocytosis of tumor cells, the method comprising contacting the tumor cells with the bispecific antigen binding composition or pharmaceutical composition described herein.

[0157] In another aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for reducing tumor size in a subject in need thereof or killing tumor cells in vitro.

[0158] In one aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding 33 / 155#14489013vlW0571.70068WQ00 domain binds a tumor specific antigen with means for eliciting phagocytosis of tumor cells in a subject in need thereof or in vitro.

[0159] In another aspect the present disclosure provides a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for treating disease in a subject in need thereof, wherein the disease is cancer.

[0160] In yet another aspect provided herein is a method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigenbinding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising: means for reducing tumor size in the subject.

[0161] In yet another aspect provided herein is a method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigenbinding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising: means for eliciting phagocytosis of tumor cells in the subject.In some embodiments, an antigen-binding domain that binds CD47 on tumor cells provides a means for eliciting phagocytosis of the tumor cells. In some embodiments, an antigenbinding domain that binds CD24 on tumor cells provides a means for eliciting phagocytosis of the tumor cells.

[0162] In some embodiments, the bispecific antigen binding compositions disclosed herein disrupt the CD47-SIRPa and / or CD24-Siglec-10 signaling pathways, which may be useful in overcoming immune resistance.

[0163] In one aspect, provided is a kit the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell described herein, and instruction for use.Pharmaceutical Compositions

[0164] The present disclosure provides pharmaceutical compositions comprising the bispecific antigen binding composition disclosed herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises a compound of disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.34 / 155#14489013vl

[0165] In certain embodiments, the bispecific antigen binding compositions described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a hematological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a hematological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a neurological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a neurological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a in a painful condition subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a painful condition in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a psychiatric disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a psychiatric disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a metabolic disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a metabolic disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.

[0166] In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), 35 / 155#14489013vldog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

[0167] In certain embodiments, the cell is present in vitro. In certain embodiments, the cell is present ex vivo.

[0168] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include bringing the bispecific antigen binding compositions described herein (z.e., the “active ingredient”) into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi- dose unit.

[0169] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0170] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0171] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents or fillers, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0172] Exemplary diluents or fillers include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, starches (such as dry starch, cornstarch), sugars (such as powdered sugar), calcium trisulfate, carboxymethylcellulose calcium, dextrate, dextrin, dextrose,36 / 155#14489013vlW0571.70068WQ00 fructose, lactitol, lactose, magnesium carbonate, magnesium, maltitol, maltodextrin, maltose, sucrose, glucose, mannitol, silicic acid, xylitol, and mixtures thereof.

[0173] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0174] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, poly aery lie acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F- 68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.37 / 155#14489013vl

[0175] Exemplary disintegrating agents or disintegrants include agar, algin, alginic acid, sodium alginate, silicates, sodium carbonate, calcium carbonate, carboxymethylcellulose, cellulose, clay, colloidal silicon dioxide, croscarmellose sodium, crospovidone, rubber, magnesium silicate, methylcellulose, potassium krillin, hydroxypropylcellulose (e.g., low substituted Hydroxypropylcellulose), crosslinked polyvinylpyrrolidone, hydroxypropylcellulose, and starch (e.g., sodium glycolate starch, potato or tapioca starch).

[0176] Exemplary binding agents include starch (e.g., glycolate starch, cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0177] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0178] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0179] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.

[0180] Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.38 / 155#14489013vlW0571.70068WQ00

[0181] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0182] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0183] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0184] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, NeoIone®, Kathon®, and Euxyl®.

[0185] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer’ s solution, ethyl alcohol, and mixtures thereof.

[0186] Exemplary lubricating agents include agar, ethyl oleate, ethyl laurate, glycerin, blyceryl palmitostearate, magnesium oxide, magnesium stearate, mannitol, poloxamer, glycol, sodium stearyl, sorbitol, zinc stearate, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0187] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, 39 / 155#14489013vlW0571.70068WQ00 jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0188] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0189] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0190] In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a ready-to-use (“RTU”) preparation that can be directly administered to a subject. In some embodiments, the RTU preparation is a suspension. In some embodiments, 40 / 155#14489013vlW0571.70068WQ00 the RTU preparation is a solution. In some embodiments, the RTU preparation is an emulsion. In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a solid that is reconstituted prior to administration. In some embodiments, the solid is a lyophilized solid. In some embodiments, injectable preparations of the compositions disclosed herein are in the form of a liquid or suspension that is diluted prior to administration.

[0191] In some embodiments, the pharmaceutical compositions disclosed herein comprise a bulking agent. Bulking agents can be used, e.g., to improve the appearance of a solid composition, to provide visible “bulk” to demonstrate product quality or to facilitate preparation, e.g., of a solid composition prepared for reconstitution prior to administration. Bulking agents can be used for low dose (high potency) drugs that do not have the necessary bulk to support their own structure or provide a visible composition in a unit dosage form. Bulking agents are used in lyophilized formulations. Bulking agents provide a desirable structure for a lyophilized cake comprising pores that provide the means for vapor to escape from the product during lyophilization cycles, and facilitate dissolution on reconstitution. In some embodiments, the bulking agent is mannitol, lactose, sucrose, dextran, trehalose, povidone, dextran, glycine, isoleucine, methionine, or a cyclodextrin (e.g., (2- hydroxypropyl)-P-cyclodextrin).

[0192] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0193] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

[0194] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.41 / 155#14489013vlW0571.70068WQ00

[0195] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0196] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[0197] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active42 / 155#14489013vlW0571.70068WQ00 ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

[0198] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin.Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and / or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis are suitable. Ballistic powder / particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

[0199] A pharmaceutical composition described herein can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and / or using a self-propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0200] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).43 / 155#14489013vlW0571.70068WQ00

[0201] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation.

[0202] Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0203] The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and / or lymph supply, and / or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.44 / 155#14489013vlW0571.70068WQ00

[0204] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein.45 / 155#14489013vlW0571.70068WQ00In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

[0205] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0206] A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and / or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and / or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and / or in inhibiting the activity of an abherant process in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and / or modify metabolism, inhibit excretion, and / or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and / or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves different effects.

[0207] The composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, 46 / 155#14489013vlW0571.70068WQ00RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and / or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and / or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and / or with the compound or composition described herein in a single dose or composition or administered separately in different doses or compositions. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0208] The additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal antiinflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretics, hormones, and prostaglandins. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is an binder or inhibitor of a protein kinase. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the compounds 47 / 155#14489013vlW0571.70068WQ00 described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy. Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells.

[0209] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

[0210] Thus, in one aspect, provided are kits including a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.

[0211] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) 48 / 155#14489013vlW0571.70068WQ00 in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.Methods of Assembling Bispecific Antigen Binding Compositions

[0212] Aspects of the present disclosure provide bispecific antigen binding compositions comprising two distinct antigen-binding domains capable of interacting with different epitopes of the same target or with two different targets, wherein each antigen-binding domain is fused to an Fc fragment that comprises a CH3 domain. In some embodiments, each antigen-binding domain is fused to a CH3 domain. In some embodiments, each antigen-binding domain is fused to a CH2 domain and a CH3 domain. In some embodiments, a Fc fragment comprises a CH2 domain and CH3 domain. In some embodiments, bispecific antigen binding compositions comprising a first antigen-binding domain, a second antigen-binding domain, and Fc fragment may be assembled using heterodimerization and pairing strategies disclosed herein and known in the art. The first and second antigen-binding domains may be the same or different. In some embodiments, the methods described herein are suitable for assembling heterodimeric bispecific construct in which distinct fusion polypeptides each comprising an antigen binding domain and CH3 domain are joined through engineered CH3 interactions.

[0213] In some embodiments, an Fc fragment comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 215 or 216.

[0214] In some embodiments: a) the first antigen-binding domain comprises CV1 (SEQ ID NO: 213) and the second antigen binding domain targets EGFR, EPCAM, HER2, PDL1, TROP2, FOLR1, Nectin-4, Netrin-1, or CD71; b) the first antigen-binding domain comprises WTa2dl (SEQ ID NO: 214) and the second antigen-binding domain targets EGFR, HER2, TROP2, Nectin-4, or CD71;49 / 155#14489013vlW0571.70068WQ00 c) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 99, 104, and 109 and the following heavy chain CDR sequences: SEQ ID NOs: 119, 124, and 129 and the second antigen-binding domain targets TROP2; d) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 100, 105, and 110 and the following heavy chain CDR sequences: SEQ ID NOs: 120, 125, and 130 and the second antigen-binding domain targets TROP2; e) the first antigen-binding domain comprises the following light chain sequences: SEQ ID NOs: 101, 106, and 111 and the following heavy chain CDR sequences: SEQ ID NOs: 121, 126, and 131 and the second antigen-binding domain targets EGFR, HER2, TROP2, Nectin-4, or CD-71; f) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 102, 107, and 112 and the following heavy chain CDR sequences: SEQ ID NOs: 122, 127, and 132 and the second antigen-binding domain targets EGFR, HER2, PD-L1, TROP2, FOLR1, Nectin-4, Netrin-1, or CD71; or g) the first antigen-binding domain targets PD-1 and the second antigen-binding domain targets TROP2.

[0215] In some embodiments, the first antigen-binding domain comprises WTa2dl (SEQ ID NO: 214) or comprises the following light chain CDR sequences: SEQ ID NOs: 102, 107, and 112 and the following heavy chain CDR sequences: SEQ ID NOs: 122, 127, and 132 and the second antigen-binding domain targets EGFR or TROP-2, optionally wherein the second antigen-binding domain comprises: a) the following light chain CDRs: SEQ ID NOS: 143, 152, and 160 and the following heavy chain CDRs: SEQ ID NOs: 178, 187, and 196; or b) the following light chain CDRs: SEQ ID NO: 147, 155, and 164 and the following heavy chain CDRs: SEQ ID NOs: 182, 191, and 200.Fc-Mediated Heterodimerization

[0216] In some embodiments, the assembly of bispecific antigen binding compositions comprising two distinct antigen-binding domains, each fused to a Fc fragment is achieved through heterodimerization of the Fc fragments. When used in the context of a bispecific antigen binding composition, each Fc fragment comprises CH3 domain and may further comprise a CH2 domain. A bispecific antigen binding composition may comprise two Fc50 / 155#14489013vlW0571.70068WQ00 fragments with complementary mutations that promote formation of heterodimers. In some embodiments, formation of homodimers is suppressed.

[0217] In some embodiments, a “knob-into-hole” configuration is employed, wherein one Fc fragment comprises a bulky amino acid substitution in the CH3 domain, and the complementary Fc fragment comprises smaller or complementary substitutions in the CH3 domain (e.g., knob Fc fragment and hole Fc fragment). In some embodiments, the bulky amino acid is tyrosine (Y) and the smaller or complementary amino acid is threonine (T). In some embodiments, the bulkier or smaller amino acid is bulkier or smaller relative to the corresponding amino acid in a wild-type CH3 domain. In some embodiments, the bulkier or smaller amino acid is bulkier or smaller relative to the corresponding amino acid in the other Fc fragment in a heterodimer.

[0218] Without being bound by any particular theory, the steric complementarity between the “knob” and “hole” Fc fragments may promote preferential heterodimer formation. In some embodiments, the Fc fragment is derived from an IgG, IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM antibody.

[0219] In some embodiments, a “knob” sequence comprises an antigen-binding domain and a CH3 domain with a bulky amino acid substitution in the CH3 domain. In some embodiments, a “hole” sequence comprises an antigen-binding domain and a CH3 domain with a smaller or complementary substitution in the CH3 domain. In some embodiments, a “knob” sequence and / or “hole” sequence further comprises a CH2 domain.

[0220] In some embodiments, a knob Fc fragment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bulkier amino acids relative to a hole Fc fragment. In some embodiments, a hole Fc fragment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 smaller amino acids relative to a knob Fc fragment.

[0221] In some embodiments, heterodimerization is promoted through electrostatic steering interactions, in which opposite-charge residues are introduced into corresponding positions on each Fc fragment. In some embodiments, the Fc fragment comprises a CH3 domain. In some embodiments, the Fc fragment comprises a CH2 and a CH3 domain. In some embodiments, the knob-into-hole and electrostatic steering approaches are combined to further enhance heterodimer formation. Additional Fc engineering methods known in the art, such as strand-exchanged engineered domains (SEED), charge-pair optimization, or engineered interchain disulfide bonds, may likewise be employed to generate heterodimeric Fc regions suitable for assembling bispecific antigen binding compositions comprising two distinct antigen-binding domains each operably linked to an Fc fragment.51 / 155#14489013vlW0571.70068WQ00Expression and Co-Assembly

[0222] In some embodiments, a complementary pair of antigen binding domain-Fc fragment polypeptides (e.g., IgG-Hole chain or “hole” sequence and IgG-Knob or “knob” sequence) are co-expressed in a single host cell under conditions permitting assembly of the heterodimeric Fc region. In other embodiments, each antigen binding domain-Fc fragment is expressed separately and subsequently combined in vitro to allow spontaneous or assisted heterodimerization through the engineered Fc interfaces. Assembly may occur during or following oxidative refolding, and reaction conditions such as pH, ionic strength, temperature, and redox potential may be adjusted to favor heterodimer formation.Chemical or Enzymatic Conjugation

[0223] In some embodiments, bispecific antigen binding compositions comprising two distinct antigen-binding domains each operably linked to a Fc fragment are assembled through covalent or enzymatic conjugation of separately produced Fc fragment-comprising polypeptides. Exemplary approaches include site-specific ligation using sortase A, enzymatic coupling using peptide tag systems (for example, SpyTag / SpyCatcher), or chemical conjugation through thiol- or amine-reactive linkers. Disulfide engineering may also be employed to form covalent heterodimers by introducing cysteine residues at specific positions within the Fc interface.Purification and Isolation

[0224] Following assembly, the bispecific heterodimers may be isolated from unassembled or mispaired species using protein A or protein G affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or combinations thereof. In certain embodiments, purification may include selective capture of Fc fragment-comprising species followed by elution and re-equilibration under conditions favoring the recovery of correctly assembled heterodimeric antigen binding domain-Fc fragment molecules.General Considerations

[0225] The foregoing assembly techniques may be used individually or in combination to promote the formation of heterodimeric bispecific antigen binding compositions comprising first and second antigen-binding domains and Fc fragments. The Fc fragment may be derived from IgG or any immunoglobulin isotype or subclass known in the art, including, but not limited to, IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, and IgM, or engineered variants thereof. Variations, substitutions, insertions, deletions, and optimizations in the sequence 52 / 155#14489013vlW0571.70068WQ00 design, expression system, or assembly conditions may be made by one of ordinary skill in the art without departing from the scope of the present disclosure. The methods described herein are intended to encompass all known approaches suitable for producing heterodimeric antigen binding domain-Fc fragment bispecific constructs, including, but not limited to, any Fc fragment derived from naturally occurring or engineered immunoglobulins. As a nonlimiting example, any of the antigen-binding domains disclosed herein may be paired with any of the Fc fragments disclosed herein or known in the art. Any other heterodimerization domain or motif that is known in the art can be used to produce a bispecific antigen binding composition comprising any of the first antigen-binding domains disclosed herein and any of the second antigen-binding domains disclosed herein.EXAMPLES

[0226] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Results

[0227] To evaluate the therapeutic potential of targeting CD47 and CD24, a compendium of novel bispecific antigen binding compositions were developed. It was reasoned that a bispecific format that targets CD47 or CD24 with one arm and a tumor- specific antigen with the other arm could enhance specificity to the tumor microenvironment and minimize toxicity to normal cells ( FIG. 1A). Therefore a library was created of scFvs and binding domains to a variety of tumor antigens (e.g., EGFR, HER2, TROP2, among others) and macrophage immune checkpoint molecules (CD47, CD24, SIRPa, and PD-1). Two different binding domains for targeting CD47 were tested: CV1 (a high-affinity binder) and WTa2dl (a low- affinity binder)18.

[0228] Three different CD24-binding domains (CD24-1, CD24-2, CD24-3) were tested. Each construct was engineered to be expressed as a human IgGl knob-into-hole heterodimer24. Each macrophage checkpoint arm was crossed with each tumor antigen arm to create a total of 77 novel bispecific antibodies. The functional anti-tumor effects of each of these antibodies was individually tested in co-culture assays using primary human macrophages and fluorescent DLD-1 cells (FIGs. 1B-1G). It was found that the majority of bispecific antigen binding compositions containing CV1, WTa2dl, or CD24-3 exhibited significant functional anti-tumor effects (FIGs. 1B-1G).). CV 1 exhibited the greatest anti-tumor effects, nearly eliminating all cancer cells across all formats (FIGs. 1C and IF). WTa2dl exhibited robust anti-tumor efficacy when paired with an EGFR, TROP2, or CD71 binding arms (53 / 155#14489013vlW0571.70068WQ00FIG. ID). The CD24-3 bispecifics exhibited substantial anti-tumor effects when paired with most tumor-targeting arms ( FIG. IE).

[0229] An ideal bispecific antibody should not only exert a robust anti-tumor response, but should also avoid on-target toxicity to the hematologic system. It was therefore determined whether any of the bispecific antigen binding compositions could decrease this risk. It was found that all constructs containing CV1, the high-affinity CD47-binding domain, exhibited intense binding to red blood cells and neutrophils (FIGs. 1H and II). CD24-3 did not bind to red blood cells, but intensely labeled neutrophils (FIGs. 1H and II), again indicating the risk of hematologic toxicity for targeting CD24 in the human system. The WTa2dl x WTa2dl homodimer also exhibited substantial binding to red blood cells (FIGs. 1H and II). In contrast, the heterodimeric WTa2dl bispecifics exhibited significantly reduced binding to both red blood cells and neutrophils (FIGs. 1H and II). Thus, bispecific antigen binding compositions including a WTa2dl domain appear to be optimal to maximize the therapeutic index, particularly WTa2dl x EGFR, WTa2dl x TROP2, and WTa2dl x CD71. In sum, these efforts indicate that CD47 again predominates over CD24 for therapeutic targeting and further illuminate the most effective strategies to maximize anti-tumor responses by macrophages.Discussion

[0230] The bispecific antibody engineering efforts disclosed herein indicate that CD47 may be a better target than CD24 in a bispecific format. Bispecific antigen binding compositions that target CD47 with low affinity may achieve maximal macrophage activation while enhancing specificity to the tumor microenvironment. These antibodies warrant further investigation as therapeutics for patients with cancer. Overall, the results presented herein provide insight into the relative contributions of CD47 and CD24 in regulating anti-tumor immunity by macrophages, informs the best therapeutic strategies for targeting these molecules, and identifies strategies to overcome liabilities. It is expected that these findings will further advance the field of macrophage immune checkpoints and will provide insight into the design of better macrophage- directed therapies for cancer.

[0231] Methods

[0232] Cell lines and culture

[0233] KPCA.C (Weinberg lab) was cultured in DMEM (Thermo Fisher) with 10% heat inactivated ultra-low IgG FBS (Thermo Fisher), lx penicillin / streptomycin / glutamine54 / 155#14489013vl(Gibco), lx insulin-transferrin- selenium (Gibco), and 0.2% human epidermal growth factor25. 238N1 (J. Weissman lab), MC38 (Koch Institute), TUBO-EGFR (I. Weissman lab, Stanford University), 3LL ANRAS (J. Downward lab, Francis Crick Institute), and EL4 (I. Weissman Lab, Stanford University) were cultured in DMEM (Thermo Fisher) with 10% heat inactivated ultra low-IgG FBS (Thermo Fisher), lx penicillin / streptomycin / glutamine (Gibco). NCI-H358, PC9, NCI-H3122 (A. Hata lab, MGH Center for Molecular Therapeutics), KPI (I. Weissman lab, Stanford University), DLD-1 (I. Weissman lab, Stanford University), control and knockout variants of DLD-1 (Synthego), control and knockout variants of A375 (Synthego), and unmodified A375 (Koch Institute) were cultured in RPMI (Thermo Fisher) with 10% heat inactivated ultra low-IgG FBS (Thermo Fisher), lx penicillin / streptomycin / glutamine (Gibco).

[0234] Control and knockout variants of NIH-OVCAR3 (Synthego) were cultured in RPMI (Thermo Fisher) with 20% heat inactivated ultra low-IgG FBS (Thermo Fisher), lx penicillin / streptomycin / glutamine (Gibco) .

[0235] Bone-marrow derived mouse macrophage generation

[0236] Primary mouse macrophages were derived from bone marrow as previously described18, 26. Briefly, forelimb and hind leg bones were collected from C57BL / 6J or B6129SF1 / J mice (Jackson Labs), and bone marrow was extracted using a mortar and pestle. Unfractionated bone marrow was washed with PBS (Thermo Fisher). Red blood cells were lysed using ACK Lysis Buffer (Thermo Fisher) and washed with PBS. Cells were cultured on Petri dishes (Corning) in IMDM (Gibco) containing 10% heat inactivated ultra-low IgG FBS, lx penicillin / streptomycin / glutamine (Gibco) with 20 ng / ml murine M-CSF (Peprotech) for 7 days. Macrophages were collected for further experimental analysis by incubating with TrypLE Express Enzyme (IX) no phenol red (Thermo Fisher) followed by cell lifting and replating as needed. Macrophages were generally used for experiments between days 7-21 of culture.

[0237] Human macrophage derivation

[0238] Primary human macrophages were differentiated ex vivo as previously described17, 27. Briefly, leukocyte reduction system chambers containing heparinized blood were obtained from discarded specimens of anonymous blood donors (Crimson Core Biobank, Brigham and Women’s Hospital). Monocytes were isolated using Straight From Whole Blood CD 14+ microbeads (Miltenyi) with whole blood purification performed using an autoMACS Pro Separator or autoMACS NEO Separator (Miltenyi). Purified CD 14+ monocytes were55 / 155#14489013vlW0571.70068WQ00 cultured in IMDM (Gibco) containing 10% heat inactivated ultra-low IgG FBS with lx penicillin / streptomycin / glutamine (Gibco) and 20 ng / mL human M-CSF (Peprotech) for 7 days. Macrophages were collected for further experimental analysis by incubating with TrypLE Express Enzyme (IX) no phenol red (Thermo Fisher) followed by cell lifting and replating as needed. Macrophages were generally used for experiments between days 7-21 of culture.

[0239] In vitro phagocytosis assays

[0240] Cancer cells were collected, washed with PBS and removed using TrypLE, then fluorescently labeled with CellTrace CFSE Cell Proliferation Kit for flow cytometry (Thermo Fisher). Macrophages were collected as described above. Cancer cells and macrophages were combined at a ratio of 4:1 in each well of an ultra-low cluster U-bottom 96- well plate (Corning 7007) in IMDM (Gibco 12440061) with the indicated therapeutic antibodies. The co-culture was then incubated for 2 hours at 37°C in a humidified incubator with 5% carbon dioxide. At the end of the incubation, cells were washed with cold autoMACS Running Buffer (Miltenyi), and for human experiments stained with a fluorophore-conjugated anti-human CD45 antibody (BioLegend) to identify the macrophages. For murine experiments, cells were stained with a fluorophore-conjugated anti-mouse CD45 or F4 / 80 antibody (BioLegend) to identify macrophages. Cells were stained with DAPI to exclude dead cells. Phagocytosis was quantified as the percentage of macrophages that had engulfed CFSE-positive cancer cells. All therapeutic antibodies were used at a final concentration of 10 ug / ml.

[0241] Long -term co-culture assays

[0242] Cancer cells and macrophages were collected as described above. Macrophages, antibodies, then cancer cells were plated sequentially in 20 ul each of IMDM, no phenol red (Gibco 21056023) with 10% heat inactivated ultra-low IgG FBS (Gibco 16- 250-078), 1% penicillin-streptomycin-glutamine (100X) (Gibco 10-378-016), and 20 ng / ml recombinant human M-CSF (PeproTech 300-25) or recombinant murine M-CSF (PeproTech 315-02) into a Coming 384-well microplate (Coming CLS3764). Monoclonal antibodies were used at a final concentration of 10 ug / ml. Plates were allowed to sit at room temperature for 15 minutes before being transferred to an Incucyte S3 Live-Cell Analysis System (Sartorius) in a humidified incubator with 5% carbon dioxide maintained at 37° C. Whole-well images of phase contrast, green fluorescence, and / or red fluorescence were acquired every four to eight hours for up to 7 days using a 4X objective. The growth of cancer cells was analyzed by 56 / 155#14489013vlW0571.70068WQ00 automated image analysis using the Incucyte Base Analysis Software (Sartorius) to quantify the fluorescent area per well as previously described17. Statistical comparisons were generally performed at t = 6.5 days as a reference endpoint unless otherwise indicated.

[0243] Lentiviral production and transduction

[0244] GFP-luciferase+ lines were transduced by plating 75,000 cells in their respective medium in a 12-well plate (Coming) with 5 uL CMV-GFP-T2A- Luciferase lentivirus (Systems Biosciences) and 8 ug / mL polybrene (Milipore Sigma). Medium was changed after 3 days to allow cells to recover. GFP+ populations were sorted at least two independent times on a BDFACS Aria II machine for GFP+ purity. For generation of CRISPRi-mCherry lines, plasmid pJB109_pHR-UCOE-EFla-Zim3-NLS-dCas9-HA-2xNLS-P2A-mCherry28was co-transfected with delta8.2 and VSV-G into HEK-293T to make CRISPRi-mCherry lentivirus. Stable cell lines expressing CRISPRi-mCherry were generated by infecting cells with the CRISPRi-mCherry lentivirus with 8 ug / ml polybrene. mCherry+ cells were purified by FACS and then confirmed by post-sort analysis of mCherry expression. Lentivirus for mouse or human CD24 overexpression was produced by VectorBuilder using the pLV expression vector with an EF1A promoter driving mCd24a (NM_009846.2) or human CD24(NM_001291738.1) expression and a puromycin resistance gene. CRISPRi sgRNA, control, and CD24 overexpression lines were created by resuspending 500,000 cells in 1 mL vims with 8 ug / ml polybrene. Medium was changed after 24 hours and cells were allowed to recover before subjecting to puromycin selection. Cells were maintained and passaged in 3 ug / ml puromycin (Selleckchem) after establishment of the lines, and cells were generally used for experiments after at least 5 days of selection. Lentivims for StayGold29expression was produced by VectorBuilder using the pLV expression vector with an EF1A promoter driving hStayGold (Genbank LC593679.1) and contained a puromycin resistance gene. Transduced cells were selected for StayGold expression using puromycin.

[0245] RNP transfection

[0246] Knockout cell lines were made using Synthego Gene Knockout Kit v2 and according to the Synthego Immortalized Cell Lipofection Protocol in 24- well plates. In brief, sgRNA and recombinant Cas9 were diluted in OptiMem, and Cas9fectamine was diluted in Optimem in a separate tube. The tubes were mixed together and incubated at room temperature for 5- 10 minutes to allow RNP complexes to form. RNP complexes were then added to cells in culture. Medium was changed after 2 days. Cells were passaged and upscaled for further selection by cell sorting. Fluorophore-conjugated antibodies were used to stain and sort for 57 / 155#14489013vlW0571.70068WQ00 polyclonal populations of cells that exhibited loss of cell-surface CD24 or CD47 protein expression. Cells were sorted at least twice to achieve homogeneous loss of cell-surface expression.

[0247] CRISPR screen

[0248] Genome-wide CRISPR screens were performed using the murine lung adenocarcinoma cell line 238N1. Cancer cells were transduced with the mouse Gouda genome- wide sgRNA library30at low multiplicity of infection (MOI-O.3) and selected with puromycin to ensure each cancer cell carries a single and unique sgRNA. Puromycin- selected cancer cells were subjected to 3 distinct conditions: (i) cancer cells alone, (ii) cancer cells and macrophages,

[0249] (iii) cancer cells, macrophages, and an anti-CD24 antibody. Cancer cells treated with macrophages were co-cultured with primary mouse bone marrow-derived macrophages (BMDMs) at 1:4 ratio for 4 days. Cancer cells were treated with anti-CD24 antibody clone MI / 69 at a concentration of 10 ug / mL. Cells were grown at minimum library coverage of 500x for the screen. Genomic DNA from each sample was extracted, the cassette encoding the sgRNA was amplified by PCR, and relative sgRNA abundance was determined by next generation sequencing as previously described30.

[0250] CRISRPR screen analysis: Sequencing FASTQ files from CRISPR screens were aligned, processed, and counted using custom Python-based scripts (based on github.com / mhorlbeck / ScreenProcessing), as previously described31, 32. sgRNAlevel phenotypes were normalized as log2(enrichment) between sample conditions. Genelevel phenotypes were scored based on the average sgRNA-level phenotype of the 2 sgRNAs targeting a gene. Mann- Whitney test p-values were calculated by comparing all sgRNAs targeting a given gene to the full set of negative control sgRNAs. Discovery scores were calculated by the absolute value of gene-level log 2 (enrichment) over the standard deviation of all evaluated phenotypes multiplied by the -loglO(p-value). Screen hits were defined as genes with a discovery score greater than 5 (such that FDR is < 0.05). All analyses were performed in Python.

[0251] CRISPR screen Gene Set Enrichment Analysis

[0252] Gene set enrichment analysis (GSEA) was performed using FGSEA from the R Bioconductor Suite (v.3.18). Gene Ontology (GO) Biological Process and KEGG pathways were used for analysis. GPI-AP related genes were identified based on previous studies33.58 / 155#14489013vlW0571.70068WQ00

[0253] Therapeutic antibodies

[0254] Antibodies used for experiments included: InVivoMAb anti- mouse / human / rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone MI / 69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019- 1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000). For FcR blocking experiments, anti- mouse Truestain clone 93 (BioLegend 101320) and anti-mouse CD16 / CD32 clone 2.4G2 (BioXCell BE0307) were used.

[0255] Syngeneic tumor models

[0256] C57BL / 6J (Jackson Laboratory #000664) sex and age matched mice were engrafted with appropriate cancer cell lines. Mice were used for engraftment when they were generally 6-12 weeks of age. 1 million cells in 100 uL of sterile PBS per subcutaneous engraftment were used. 238N1 cancer cells were engrafted into B6129SF1 / J mice (Jackson Laboratory #101043) at 1 million cells in 100 uL sterile PBS subcutaneously. Tumor dimensions were measured by caliper twice per week and used to calculate tumor volumes according to an ellipsoid formula: length x width x width x jt / 6. For treatment experiments, therapeutic antibodies were administered by intraperitoneal injection in sterile PBS three times per week or as otherwise indicated. For an intraperitoneal model, 1 million KPCA.C cells were engrafted into the peritoneal cavity in 100 uL sterile PBS. Mice were euthanized according to humane experimental endpoints including tumor size and body conditioning scores.

[0257] Xenograft tumor models

[0258] NOD.Cg-PrkdcscldI12rgtmlWjl / SzJ (NSG) (Jackson Laboratory #005557) sex and age matched male or female mice were engraftment subcutaneously with cancer cells in a 1 : 1 ratio of PBS and Matrigel Matrix hESC-Qualified Matrix (Corning 354277). Mice were used for engraftment when they were approximately 6-12 weeks of age. Mice were engrafted with 1 million cancer cells in 100 uL sterile PBS subcutaneously. Tumor volumes were measured and calculated as described above. Mice were euthanized according to human experimental endpoints including tumor size and body conditioning scores.

[0259] Mouse blood analysis

[0260] Venous blood samples from mice were collected via retro-orbital sampling into Microvette 100 EDTA tubes (Sarstedt). Blood was analyzed for hematologic parameters by IDEXX CBC (MIT DCM Diagnostics Lab). Receptor occupancy was examined by flow59 / 155#14489013vlW0571.70068WQ00 cytometry using an anti-mouse IgG secondary antibody conjugated to AlexaFluor647 (BioLegend). Red blood cells were prepared for in vitro phagocytosis assays by CFSE- labeling, then the cells were co-cultured with primary mouse macrophages as described above. For quantification of internalization of red blood cells, red blood cells were labeled with pHrodo (Thermo Fisher) according to manufacturer's instructions. Blood cells were then incubated with primary mouse macrophages and imaged using a Incucyte S3 Live Imaging System (Sartorius). pHrodo-positive macrophages were quantified over time and evaluated as a measure of phagocytosis using Incucyte Base Analysis Software (Sartorius).

[0261] Human blood analysis

[0262] Unfractionated blood from LRS chambers containing anticoagulant, or EDTA- anticoagulated peripheral blood (Miltenyi Biotec) was used for analysis. Blood specimens were depleted of red blood cells using EasySep RBC Depletion Reagent and “The Big Easy” EasySep Magnet according to the manufacturer’s protocol (STEMCELL Technologies). For viability assays, cells were then counted and plated in ultra-low retention 96-well U-bottom plates (Coming #7007) at 200k cells per well in IMDM medium containing 10% heat- inactivated ultra-low IgG FBS and lx pen / strep / glutamine and 10 ug / ml of the indicated antibodies. Cells were incubated at 37°C for 24 hours and stained for the indicated markers and assessed for viability using a BD LSRFortessa Cell Analyzer (BD Biosciences). For neutrophil phagocytosis assays, primary neutrophils were isolated from RBC depleted blood using MACSxpress Whole Blood Neutrophil Isolation Kit, human (Miltenyi Biotec) and purified according to the manufacturer’s protocol. Neutrophils were labeled using CellTrace CFSE Cell Proliferation Kit (Thermo Fisher). 200,000 neutrophils were co-cultured with 50,000 primary human macrophages with 10 ug / ml of the indicated antibodies as appropriate for 2 hours at 37 °C in 100 ul serum-free IMDM. Cells were then stained with APC antihuman CD45 as previously described and analyzed on a BD LSRFortessa Cell Analyzer. Macrophages were distinguished from neutrophils based on forward and side scatter as well as high expression of CD45. Phagocytosis was quantified as the percentage of macrophages that had engulfed CFSE+ neutrophils.

[0263] Cytokine Analysis

[0264] Cell culture supernatants from unfractionated leukocytes were collected after 24 hours of culture by centrifugation followed by freezing at -80 °C. Supernatants were subjected to analysis using a Human Cytokine 71-Plex Discovery Assay (Eve Technologies). Data were analyzed by creating a matrix of observed cytokine concentrations, setting any out- of-range 60 / 155#14489013vlW0571.70068WQ00 measurements to the maximum observed level of that cytokine (if above the calibration range) or 0 (if below). Cytokines were dropped from the analysis if they were within range in at most one condition. Cytokine levels were compared to the control condition using oneway ANOVA followed by Dunnett's method for multiple comparisons. To generate a heatmap of cytokine levels relative to the control mean, cytokines were clustered by uncentered correlation, with clusters merged by pairwise average-linkage.

[0265] Flow Cytometry

[0266] Cells were stained in autoMACS Running Buffer (Miltenyi Biotec 130-091- 221) at the recommended concentrations and were analyzed using a BD LSRFortessa Cell Analyzer. When necessary, anti-mouse or anti-human TrueStain (Biolegend) was used to block Fc receptors. In general, cells were stained on ice for 30 minutes with primary antibodies. Cells were then washed twice and resuspended in 100 ng / mL DAPI (Millipore-Sigma). Antibodies used include APC anti-mouse CD24 (BioLegend 101814), APC anti-mouse CD47 (BioLegend 127513), APC anti-mouse CD45 (BioLegend 103112), AlexFluor647 antimouse F4 / 80 (Biolegend), APC anti-human CD45 (Biolegend), APC anti-human CD24 (Biolegend), and APC anti-human CD47 (Biolegend). As appropriate, compensation was performed using Ultracomp beads(BD). Analysis was generally performed by excluding debris and doublets based on forward and side scatter and excluding dead cells based on DAPI staining. Analysis of geometric mean fluorescence intensity and population percentages were performed using FlowJo version versions 9-10 (TreeStar).

[0267] Single-cell RNA Sequencing Sample Preparation

[0268] Tumors were obtained from euthanized mice and put into 6-well plates (Coming) with PBS. Tumors were weighed and placed into gentleMACS C tubes (Miltenyi Biotech) containing RPMI (Gibco) with 1% collagenase (Sigma Aldrich) and 1% hyalunorase (Sigma Aldrich) on ice. Tumors were then dissociated in an OctoDissociator (Miltenyi Biotech) for 1 minute. The tubes were then incubated at 37°C for 30 minutes on a shaker. Homogenized cell suspensions were filtered through 70 um (Falcon), then 40 um (Falcon) cell strainers and centrifuged. The supernatant was removed and RBCs lysed using ACK lysis buffer (Thermo Fisher) for 1 minute. Cells were centrifuged again and stained with anti-mouse CD45.2 APC (Biolegend) or anti-human CD45 APC (Biolegend) for 20 minutes on ice. Cells were centrifuged and resuspended in DAPI (Sigma Aldrich) and sorted for DAPI negative APC positive cells. Sorted cells were centrifuged and aspirated, then resuspended in cell staining buffer (Biolegend) with anti-mouse or human TrueStain and incubated on ice for 10 minutes.61 / 155#14489013vlW0571.70068WQ00To each cell population, 1 pg hashtagging antibody (Biolegend) was added and cells were incubated on ice for 30 min. Cells were washed with Cell Staining Buffer twice and counted. Cells were brought to 1000 cells / pL and pooled appropriately for sequencing.

[0269] lOx Library Preparation and Sequencing

[0270] Cells were processed using the 10X Genomics Chromium Controller with the Single Cell 3' v3.1 Reagent Kit, according to manufacturer’s directions. Briefly, a target number of cells per library was roughly achieved by loading 1.65 times the target number of cells in suspension, along with barcoded beads and partitioning oil, into the Chromium Controller, in order to create GEMs (Gel Beads in Emulsion). The Chromium Controller combines individual cells, first strand master mix, and gel beads containing barcoded oligonucleotides into single-cell droplets for first strand cDNA synthesis, so that each cell is marked with its own unique barcode during reverse transcription. The 3’ beads contain a poly(dT) oligo that enables the production of barcoded, full-length cDNA from poly-adenylated mRNA. After first strand synthesis is complete, the emulsion is dissolved and the cDNA is pooled for bulk processing as a single sample. The sample is fragmented, end-repaired, A-tailed and ligated with universal adapters. A second sample barcode is then added during the PCR step, allowing for unique library identification. The end result is a single library representing one cell suspension, containing data for each individual cell. qPCR was performed on all libraries using KAPA qPCR library quant kit as per manufacturer's protocol. The samples are loaded on the NovaSeq 6000 based on qPCR concentrations. The demultiplexing and fastq generation is performed using illumina's BCL to FASTQ file converter bcl2fastq v2.20.0.422.

[0271] Single-cell RNA Sequencing Analysis: The same processing and analysis pipeline was used across all samples and all batches. Raw read processing was performed using the 10X Genomics workflow. The CellRanger Single-Cell Software Suite (v7.0.0) was used to perform barcode assignment and unique molecular identifier (UMI) quantification. The reads were aligned to the 10X reference genome mmlO (ref-2020-A). Seurat package (v5.1.0) was used for demultiplexing cells based on TotalSeq barcodes. Any cells which were not associated with TotalSeq barcode (negative) or assigned to more than 1 TotalSeq barcode (doublet) were discarded. Data for all singlet cells were normalized and scaled using default parameters and then integrated using "Harmonyintegration" method implemented in seurat. The AUCell bioconductor package was used to identify immune specific marker sets (defined in Panglaodb - panglaodb.se / ) that are highly expressed in each cell for cell type62 / 155#14489013vlW0571.70068WQ00 determination. FindMarkers function implemented in seurat was used for identifying differentially expressed genes. A functional Gene Set Enrichment Analysis (GSEA) was also performed for all genes using hallmark genesets defined in msigDB (gsea- msigdb.org / gsea / msigdb / ) and the fgsea R package.

[0272] Plasmid Preparation for scFv Library

[0273] Antibody and binding domain sequences were curated from publicly available databases and literature sources and included the following: EGFR (cetuximab, IMGT 7906), EpCAM (US Patent No.: US 9,777,073 B2), HER2 (trastuzumab, IMGT ln8z), PD-L1 (atezolizumab, KEGG D 10773), TROP2 (sacituzumab, KEGG D 10984), FOLR1 (mirvetuximab, KEGG D10953), Nectin-4 (enfortumab, KEGG DI 1524), Netrin-1 (NP13734), CD71 (delpacibart, IMGT 1374), CV118, WTa2dl18, SIRPa (KWAR2335), CD24- ]36

[0274] CD24-2 (US Patent Application Pub. No.: 20210213055 Al), CD24-3 (US Patent No.: US 8,614,301 B2), PD-1 (nivolumab, KEGG D10316). The sequences were reverse- translated, codon-optimized for Homo sapiens and then engineered with flanking 5' and 3 ' cloning sites. Gene fragments containing the sequences were obtained from Twist Biosciences and were then cloned into a modified pFuse vector (Invivogen) containing a human IgGl knob or hole construct. Cloning was performed using New England Biolabs restriction enzymes for DNA digestion followed by T4 DNA ligase fusion and bacterial transformation with chemically competent E.coli TOP 10 cells. Transformed cells were selected on LB agar plates containing 40 pg / ml zeocin. Single colonies were then used to inoculate 5 mL of LB medium containing the same concentration of antibiotic. The culture was used for plasmid purification using the ZymoPure Plasmid Purification kit following manufacturer instructions. The concentration of the plasmid product was assessed using a ThermoFisher Nanodrop by taking the absorbance at 260 nm and the correct insertion was verified by whole plasmid DNA sequencing (Quintara Biosciences).

[0275] Expression and Purification of Recombinant Antibodies

[0276] Purified pairs of IgGl -Knob and IgGl -Hole plasmids were co-transfected to a final concentration of 1 ug / ml of each into 900 pl of Expi293F cells at 3 x 10 cells / ml (Thermo Fisher Scientific, A14528) in 96 deep-well plate (USA Scientific, 1896-2110) with ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific, A14524) following the manufacturer’s recommendation and incubated at 37°C, 8% CO2 withshaking at 900 rpm63 / 155#14489013vlW0571.70068WQ00 for 7 days. The antibody supernatants were diluted 2-fold with PBS and used for further analysis.Example 2.

[0277] Macrophages are the most common immune cell to infiltrate tumors, but are often thought of as promoters of cancer progression. It has been shown that by targeting macrophage-specific immune checkpoints, such as the CD47 / SIRPa axis, macrophage function can be directed towards cancer cell phagocytosis ( FIG. 2). Specifically, CD47 on cancer cells binds to SIRPa on macrophages and prevents them from performing phagocytosis. Antibodies targeting CD47 promote phagocytosis both by blocking the interaction with SIRPa, and by opsonizing cancer cells.

[0278] CD24 / Siglec-10 is a new macrophage-specific immune checkpoint which can also prevent macrophages from performing phagocytosis. It is present on the surface of developing B cells and is expressed on a variety of hematological and solid tumors.

[0279] As presented herein, the therapeutic potential of targeting two distinct macrophage immune checkpoints, CD47 and CD24, was evaluated. It was found that antibodies to these antigens could elicit maximal levels of phagocytosis in vitro. However, surprisingly, via an unbiased CRISPR screen and a series of in vitro and in vivo genetic validation studies, it was found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, CD24 primarily functions as a target of opsonization, which creates a risk for significant on-target hematologic toxicity in vivo. These findings informed the development of 77 novel bispecific antigen binding compositions to maximally activate macrophages while minimizing binding to healthy cells. Overall, these findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer.

[0280] Results are outlined in the following figures:FIGs. 3A-3C show dual targeting of CD47 and CD24 maximizes anti-tumor responses by macrophages;FIGs. 4A-4B show unbiased CRISPR screen identifies CD24 as a target of opsonization rather than a macrophage immune checkpoint;FIGs. 5A-5D show genetic ablation of CD24 does not influence anti-tumor responses in vitro',FIGs. 6A-6D show CD24 exhibits limited activity as an immune checkpoint in immunocompetent models and poses risks for hematological toxicity; and 64 / 155#14489013vlW0571.70068WQ00FIGs. 1A-1I show bispecific antigen binding composition scan maximize antitumor responses by macrophages while minimizing binding to healthy cells.These results suggest:1) Across mouse and human systems, in the majority of cases, CD24 is neither necessary nor sufficient to protect cancer cells from macrophage attack.2) Anti-CD24 antibodies primarily act in an Fc-dependent manner to opsonize cancer cells.3) The effects of CD47 ablation were greater than genetic perturbations of CD24.4) Bispecific antigen binding compositions that target CD47 with low affinity may achieve maximal macrophage activation while enhancing specificity to the tumor microenvironment.5) CD47 predominates over CD24 as a macrophage immune checkpoint in cancer.65 / 155#14489013vlW0571.70068WQ00SEQUENCESTable 1. Antibody Sequences66 / 155#14489013vlW0571.70068WQ0067 / 155#14489013vlW0571.70068WQ0068 / 155#14489013vlW0571.70068WQ0069 / 155#14489013vlW0571.70068WQ0070 / 155#14489013vlW0571.70068WQ0071 / 155#14489013vlW0571.70068WQ0072 / 155#14489013vlW0571.70068WQ0073 / 155#14489013vlW0571.70068WQ0074 / 155#14489013vlW0571.70068WQ0075 / 155#14489013vlW0571.70068WQ0076 / 155#14489013vlW0571.70068WQ0077 / 155#14489013vlW0571.70068WQ0078 / 155#14489013vlW0571.70068WQ0079 / 155#14489013vlW0571.70068WQ0080 / 155#14489013vlW0571.70068WQ0081 / 155#14489013vlW0571.70068WQ0082 / 155#14489013vlW0571.70068WQ0083 / 155#14489013vlW0571.70068WQ0084 / 155#14489013vlW0571.70068WQ0085 / 155#14489013vlW0571.70068WQ0086 / 155#14489013vlW0571.70068WQ0087 / 155#14489013vlW0571.70068WQ0088 / 155#14489013vlW0571.70068WQ0089 / 155#14489013vlW0571.70068WQ0090 / 155#14489013vlW0571.70068WQ0091 / 155#14489013vlW0571.70068WQ0092 / 155#14489013vlW0571.70068WQ0093 / 155#14489013vlW0571.70068WQ0094 / 155#14489013vlW0571.70068WQ0095 / 155#14489013vlW0571.70068WQ0096 / 155#14489013vlW0571.70068WQ0097 / 155#14489013vlW0571.70068WQ0098 / 155#14489013vlW0571.70068WQ0099 / 155#14489013vlW0571.70068WQ00100 / 155#14489013vlW0571.70068WQ00101 / 155#14489013vlW0571.70068WQ00102 / 155#14489013vlW0571.70068WQ00103 / 155#14489013vlW0571.70068WQ00104 / 155#14489013vlW0571.70068WQ00105 / 155#14489013vlW0571.70068WQ00106 / 155#14489013vlW0571.70068WQ00107 / 155#14489013vlW0571.70068WQ00108 / 155#14489013vlW0571.70068WQ00109 / 155#14489013vlW0571.70068WQ00110 / 155#14489013vlW0571.70068WQ00111 / 155#14489013vlW0571.70068WQ00112 / 155#14489013vlW0571.70068WQ00113 / 155#14489013vlW0571.70068WQ00114 / 155#14489013vl***Note: SirpA is not the target; it is the Wta2dl SirpA chain. CV1 is not target; it a protein modified from SirpA. Both proteins targets CD47. Kwar is the clone of antibody that binds to SirpA115 / 155#14489013vlW0571.70068WQ00Table 2. IgG-Hole (Chain 1) Light Chain Sequences116 / 155#14489013vlW0571.70068WQ00117 / 155#14489013vlW0571.70068WQ00118 / 155#14489013vlW0571.70068WQ00119 / 155#14489013vlW0571.70068WQ00*** Note: SirpA is not the target; it is the Wta2dl SirpA chain. CV1 is not target; it a protein modified from SirpA. Both proteins targets CD47. Kwar is the clone of antibody that binds to SirpATable 3. IgG-Hole (Chain 1) Heavy Chain Sequences120 / 155#14489013vlW0571.70068WQ00121 / 155#14489013vlW0571.70068WQ00122 / 155#14489013vlW0571.70068WQ00123 / 155#14489013vlW0571.70068WQ00124 / 155#14489013vlW0571.70068WQ00*** Note: SirpA is not the target; it is the Wta2dl SirpA chain. CV1 is not target; it a protein modified from SirpA. Both proteins targets CD47. Kwar is the clone of antibody that binds to SirpATable 4. IgG-Knob (Chain 2) Light Chain Sequences125 / 155#14489013vlW0571.70068WQ00126 / 155#14489013vlW0571.70068WQ00127 / 155#14489013vlW0571.70068WQ00128 / 155#14489013vlW0571.70068WQ00129 / 155#14489013vlW0571.70068WQ00130 / 155#14489013vlW0571.70068WQ00*** Note: SirpA is not the target; it is the Wta2dl SirpA chain. CV1 is not target; it a protein modified from SirpA. Both proteins targets CD47. Kwar is the clone of antibody that binds to SirpA.Table 5. IgG-Knob (Chain 2) Heavy Chain Sequences131 / 155#14489013vlW0571.70068WQ00132 / 155#14489013vlW0571.70068WQ00133 / 155#14489013vlW0571.70068WQ00134 / 155#14489013vlW0571.70068WQ00135 / 155#14489013vlW0571.70068WQ00136 / 155#14489013vlW0571.70068WQ00137 / 155#14489013vlW0571.70068WQ00*** Note: SirpA is not the target; it is the Wta2dl SirpA chain. CV1 is not target; it a protein modified from SirpA. Both proteins targets CD47. Kwar is the clone of antibody that binds to SirpATable 6. Light Chain Single Sequences138 / 155#14489013vlW0571.70068WQ00Table 7. Heavy Chain Single Sequences139 / 155#14489013vlW0571.70068WQ00140 / 155#14489013vlW0571.70068WQ00Table 8. Additional sequences associated with the disclosureREFERENCES1. Weiskopf, K. & Weissman, I.L. Macrophages are critical effectors of antibody therapies for cancer. mAbs 7, 303-310 (2015).2. Weiskopf, K. Cancer immunotherapy targeting the CD47 / SIRPalpha axis. Eur. J. Cancer 76, 100-109 (2017).3. Cassetta, L. & Pollard, J.W. Targeting macrophages: therapeutic approaches in cancer.Nat. Rev. Drug Discov. 17, 887-904 (2018).4. Pathria, P., Louis, T.L. & Varner, J.A. Targeting Tumor-Associated Macrophages in Cancer. Trends Immunol. 40, 310-327 (2019).5. Mantovani, A., Allavena, P., Marchesi, F. & Garlanda, C. Macrophages as tools and targets in cancer therapy. Nat. Rev. Drug Discov. 21, 799-820 (2022).6. Majeti, R. et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 138, 286-299 (2009).7. Willingham, S.B. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. U. S. A. 109, 6662-6667 (2012).8. Edris, B. et al. Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma. Proc. Natl. Acad. Sci. U. S. A. 109, 6656-6661 (2012).9. Weiskopf, K. et al. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer. J. Clin. Invest. 126, 2610-2620 (2016).10. Advani, R. et al. CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma. N. Engl. J. Med. 379, 1711-1721 (2018).11. Barkal, A.A. et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392-396 (2019).141 / 155#14489013vlW0571.70068WQ0012. Kamber, R.A. et al. Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature 597, 549-554 (20 1).13. Fang, X., Zheng, P., Tang, J. & Liu, Y. CD24: from A to Z. Cell. Mol. Immunol. 1, 100-103 (2010).14. Chen, G.Y., Tang, J., Zheng, P. & Liu, Y. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science 323, 1722-1725 (2009).15. Nielsen, P.J. et al. Altered erythrocytes and a leaky block in B-cell development in CD24 / HSA-deficient mice. Blood 89, 1058-1067 (1997).16. Salnikov, A.V. et al. Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas. Br. J. Cancer 108, 1449-1459 (2013).17. Vaccaro, K. et al. Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction. J. Clin. Invest. 134 (2024).18. Weiskopf, K. et al. Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341, 88-91 (2013).19. Eischen, A. et al. Human resident peritoneal macrophages: phenotype and biology. Br.J. Haematol. 88, 712-722 (1994).20. Yamaguchi, T. et al. Tumor-associated macrophages of the M2 phenotype contribute to progression in gastric cancer with peritoneal dissemination. Gastric Cancer 19, 1052- 1065 (2016).21. Xia, H. et al. Autophagic adaptation to oxidative stress alters peritoneal residential macrophage survival and ovarian cancer metastasis. JCI Insight 5 (2020).22. Chen, G.Y. et al. Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. Nat. Biotechnol. 29, 428-435 (2011).23. Shultz, L.D. et al. Human lymphoid and myeloid cell development in NOD / LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J. Immunol.174, 6477-6489 (2005).24. Ridgway, J.B., Presta, L.G. & Carter, P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 9, 617-621 (1996).25. Iyer, S. et al. Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy. Cancer Discov 11, 384-407 (2021).26. Chhabra, A. et al. Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci. Transl. Med. 8, 351ral05 (2016).27. Phadnis, V.V. et al. MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis. Cell Rep 42, 113023 (2023).28. Replogle, J.M. et al. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. Elife 11 (2022).29. Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat.Biotechnol. 40, 1132-1142 (2022).30. DeWeirdt, P.C. et al. Genetic screens in isogenic mammalian cell lines without single142 / 155#14489013vlW0571.70068WQ00 cell cloning. Nat. Commun. 11, 752 (2020).31. Chen, J. et al. Pervasive functional translation of noncanonical human open reading frames. Science 367, 1140-1146 (2020).32. Horlbeck, M.A. et al. Compact and highly active next-generation libraries for CRISPRmediated gene repression and activation. Elife 5 (2016).33. Liu, S.S. et al. A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins. Commun Biol 4, 777 (2021).34. Cassier, P.A. et al. Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer. Nature 620, 409-416 (2023).35. Ring, N.G. et al. Anti-SIRPalpha antibody immunotherapy enhances neutrophil and macrophage antitumor activity. Proc. Natl. Acad. Sci. U.S.A. 114, E10578-E10585 (2017).36. Sun, F. et al. Engineering a high-affinity humanized anti-CD24 antibody to target hepatocellular carcinoma by a novel CDR grafting design. Oncotarget 8, 51238-51252 (2017).EQUIVALENTS AND SCOPE

[0281] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0282] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the invention or aspects of the invention143 / 155#14489013vlW0571.70068WQ00 consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0283] It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0284] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0285] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.144 / 155#14489013vl

Claims

W0571.70068WQ00CLAIMSWhat is claimed is:

1. A bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen.

2. The bispecific antigen binding composition of claim 1, wherein the tumor specific antigen is EGFR or TROP2.

3. The bispecific antigen binding composition of claim 1 or 2, wherein the first antigenbinding domain binds CD47.

4. The bispecific antigen binding composition of claim 1 or 2, wherein the first antigenbinding domain binds CD24.

5. The bispecific antigen binding composition of any one of claims 1-4, wherein the bispecific antigen binding composition comprises comprising a “knob” sequence and a “hole” sequence.

6. The bispecific antigen binding composition of claim 5, wherein the “knob” sequence comprises the first antigen-binding domain or the second antigen-binding domain.

7. The bispecific antigen binding composition of claim 5 or 6, wherein the “knob” sequence comprises the first antigen-binding domain.

8. The bispecific antigen binding composition of claim 5 or 6, wherein the “knob” sequence comprises the second antigen-binding domain.

9. The bispecific antigen binding composition of any one of claims 5-8, wherein the “knob” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 8-23.145 / 155#14489013vlW0571.70068WQ0010. The bispecific antigen binding composition of claim 9, wherein the “knob” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12.

11. The bispecific antigen binding composition of any one of claims 5-8, wherein the “knob” sequence comprises a sequence identical to any one of the amino acid sequences set forth in SEQ ID NOs: 8-23.

12. The bispecific antigen binding composition of claim 11, wherein the “knob” sequence comprises a sequence identical to any one of the amino acid sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12.

13. The bispecific antigen binding composition of claim 5, wherein the “hole” sequence comprises the first antigen-binding domain or the second antigen-binding domain.

14. The bispecific antigen binding composition of claim 5 or 13, wherein the “hole” sequence comprises the first antigen-binding domain.

15. The bispecific antigen binding composition of claim 5 or 13, wherein the “hole” sequence comprises the second antigen-binding domain.

16. The bispecific antigen binding composition of any one of claims 5-15, wherein the “hole” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-7.

17. The bispecific antigen binding composition of any one of claims 5-15, wherein the “hole” sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identical to any one of the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 6.146 / 155#14489013vlW0571.70068WQ0018. The bispecific antigen binding composition of any one of claims 5-15, wherein the “hole” sequence comprises a sequence identical to any one of SEQ ID NOs: 1-7.

19. The bispecific antigen binding composition of claim 18, wherein the “hole” sequence comprises a sequence identical to any one of SEQ ID NO: 2 and SEQ ID NO: 6.

20. The bispecific antigen binding composition of any one of claims 5-19 wherein:(a) the “knob” sequence comprises a sequence identical to SEQ ID NO: 8 and the “hole” sequence comprises a sequence identical to of SEQ ID NO: 2,(b) the “knob” sequence comprises a sequence identical to SEQ ID NO: 8 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 6,(c) the “knob” sequence comprises a sequence identical to SEQ ID NO: 12 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 2, or(d) the “knob” sequence comprises a sequence identical to SEQ ID NO: 12 and the “hole” sequence comprises a sequence identical to SEQ ID NO: 6.

21. The bispecific antigen binding composition of any one of claims 1-20 comprising:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising any one of SEQ ID NOs: 119-123 or 178-186;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising any one of SEQ ID NOs: 124-128 or 187-195;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising any one of SEQ ID NOs: 129-133 or 196-204;(d) a light chain complementarity determining region 1 (CDRL1) comprising any one of SEQ ID NOs: 99-103 or 143-151;(e) a light chain complementarity determining region 2 (CDRL2) comprising any one of SEQ ID NOs: 104-108 or 152-159; and(f) a light chain complementarity determining region 3 (CDRL3) comprising any one of SEQ ID NOs: 109-113 or 160-169.

22. The bispecific antigen binding composition of any one of claims 1-20 comprising:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 182;147 / 155#14489013vlW0571.70068WQ00(b) a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 191;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 200;(d) a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 147;(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 155; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NO: 164.

23. The bispecific antigen binding composition of any one of claims 1-20 comprising:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 178;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 187;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 196;(d) a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 143;(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 152; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NO: 160.

24. The bispecific antigen binding composition of any one of claims 1-20 comprising:(a) a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 122;(b) a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 127;(c) a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 132;(d) a light chain complementarity determining region 1 (CDRL1) comprisingSEQ ID NO: 102;148 / 155#14489013vlW0571.70068WQ00(e) a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 107; and(f) a light chain complementarity determining region 3 (CDRL3) SEQ ID NO: 112.

25. A pharmaceutical composition comprising the bispecific antigen binding composition of any one of claims 1-24 and a pharmaceutically acceptable excipient.

26. The pharmaceutical composition of claim 25 further comprising an additional pharmaceutical agent.

27. One or more nucleic acid constructs encoding one or more proteins of the bispecific antigen binding composition of any one of claims 1-24.

28. The one or more nucleic acid constructs of claim 27, wherein the nucleic acid is DNA or RNA.

29. A vector comprising the one or more nucleic acid constructs of claim 27 or 28.

30. A cell comprising one or more bispecific antigen binding composition of any one of claims 1-24, the one or more nucleic acid constructs of claim 27 or 28, or the vector of claim 29.

31. A method of treating disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the bispecific antigen binding composition of any one of claims 1-24, the pharmaceutical composition of claim 25 or 26, the nucleic acid construct of claim 27 or 28, the vector of claim 29, or cell of claim 30.

32. The method of claim 31, wherein the disease is a proliferative disease.

33. The method of claim 32, wherein the proliferative disease is cancer.149 / 155#14489013vlW0571.70068WQ0034. The method of claim 33, wherein the cancer is breast, colon, liver, lung, ovarian, melanoma, prostate, skin, renal, thyroid, or uterine cancer, adenocarcinoma35. The method of any one of claims 31-34, wherein the cancer is therapy resistant.

36. Use of the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell of any one of claims 1-30 for the treatment of a disease.

37. The use of claim 36, wherein the disease is a proliferative disease.

38. The use of claim 37, wherein the proliferative disease is cancer.

39. The use of claim 38, wherein the cancer is breast, colon, liver, lung, ovarian, melanoma, prostate, skin, renal, thyroid, or uterine cancer.

40. A method of reducing tumor size in a subject in need thereof, the method comprising administering to the subject of the bispecific antigen binding composition of any one of claims 1-24, the pharmaceutical composition of claim 25 or 26, the nucleic acid construct of claim 27 or 28, the vector of claim 29, or cell of claim 30.

41. The method of claim 40, wherein the method comprises in bringing into close proximity a tumor cell with an immune cell.

42. The method of claim 41, wherein the immune cell is a macrophage.

43. The method of any one of claims 40-42, wherein the tumor or tumor cell is TROP2+ or EGFR+.

44. The method of any one of claims 40-42, wherein the immune cell is a CD47+ cell or a CD24+ cell.150 / 155#14489013vlW0571.70068WQ0045. A method of eliciting phagocytosis of tumor cells, the method comprising contacting the tumor cells with the bispecific antigen binding composition of any one of claims 1-24 or the pharmaceutical composition of claim 25 or 26.

46. A bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for reducing tumor size in a subject in need thereof or in vitro.

47. A bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for eliciting phagocytosis of tumor cells in a subject in need thereof or in vitro.

48. A bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen with means for treating disease in a subject in need thereof, wherein the disease is cancer.

49. A method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigenbinding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising a means for reducing tumor size in the subject.

50. A method of treating disease in a subject in need thereof by administering a bispecific antigen binding composition comprising a first antigen-binding domain and a second antigenbinding domain, wherein the first antigen-binding domain binds CD47 or CD24, and the second antigen-binding domain binds a tumor specific antigen comprising a means for eliciting phagocytosis of tumor cells in the subject.

51. A kit the bispecific antigen binding composition, pharmaceutical composition, nucleic acid construct, vector, or cell of any one of claims 1-30, and instruction for use.151 / 155#14489013vlW0571.70068WQ0052. A bispecific antigen binding composition comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and / or the second antigen-binding domain binds to CD20, CD47, EGFR, SIRPa, CD24, PD-1, EPCAM, HER2, PD-L1, Trop-2, Folate Receptor Alpha, Nectin-4, Netrin-1, or CD71.

53. The bispecific antigen binding composition of claim 52, wherein the first antigenbinding domain is fused to a first Fc fragment and the second antigen-binding domain is fused to second Fc fragment, optionally wherein the first Fc fragment and / or the second Fc fragment comprise a CH2 and a CH3 domain.

54. The bispecific antigen binding composition of claim 53, wherein the first Fc fragment comprises an amino acid substitution relative to the second Fc fragment, optionally wherein the first Fc fragment and / or the second Fc fragment comprises a CH3 domain and the amino acid substitution is in the CH3 domain.

55. The bispecific antigen binding composition of claim 53 or 54, wherein the first Fc fragment and the second Fc fragment heterodimerize.

56. The bispecific antigen binding composition of any one of claims 52-55, wherein: a) the first antigen-binding domain comprises CV1 (SEQ ID NO: 213) and the second antigen binding domain targets EGFR, EPCAM, HER2, PDE1, TROP2, FOER1, Nectin-4, Netrin-1, or CD71; b) the first antigen-binding domain comprises WTa2dl (SEQ ID NO: 214) and the second antigen-binding domain targets EGFR, HER2, TROP2, Nectin-4, or CD71; c) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 99, 104, and 109 and the following heavy chain CDR sequences: SEQ ID NOs: 119, 124, and 129 and the second antigen-binding domain targets TROP2; d) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 100, 105, and 110 and the following heavy chain CDR sequences: SEQ ID NOs: 120, 125, and 130 and the second antigen-binding domain targets TROP2; e) the first antigen-binding domain comprises the following light chain sequences: SEQ ID NOs: 101, 106, and 111 and the following heavy chain CDR sequences: SEQ ID152 / 155#14489013vlW0571.70068WQ00NOs: 121, 126, and 131 and the second antigen-binding domain targets EGFR, HER2, TROP2, Nectin-4, or CD-71; f) the first antigen-binding domain comprises the following light chain CDR sequences: SEQ ID NOs: 102, 107, and 112 and the following heavy chain CDR sequences: SEQ ID NOs: 122, 127, and 132 and the second antigen-binding domain targets EGFR, HER2, PD-L1, TROP2, FOLR1, Nectin-4, Netrin-1, or CD71; or g) the first antigen-binding domain targets PD-1 and the second antigen-binding domain targets TROP2.

57. The bispecific antigen binding composition of any one of claims 52-56, wherein the first antigen-binding domain comprises WTa2dl (SEQ ID NO: 214) or comprises the following light chain CDR sequences: SEQ ID NOs: 102, 107, and 112 and the following heavy chain CDR sequences: SEQ ID NOs: 122, 127, and 132 and the second antigenbinding domain targets EGFR or TROP-2, optionally wherein the second antigen-binding domain comprises: a) the following light chain CDRs: SEQ ID NOS: 143, 152, and 160 and the following heavy chain CDRs: SEQ ID NOs: 178, 187, and 196; or b) the following light chain CDRs: SEQ ID NO: 147, 155, and 164 and the following heavy chain CDRs: SEQ ID NOs: 182, 191, and 200.

58. The bispecific antigen binding composition of any one of claims 53-57, wherein the first Fc fragment and / or the second Fc fragment comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 215 or 216.

59. The bispecific antigen binding composition of any one of claims 52-58, wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% identical to SEQ ID NO: 213 or 214.

60. A pharmaceutical composition comprising the bispecific antigen binding composition of any one of claims 52-59 and a pharmaceutically acceptable excipient.

61. One or more nucleic acid constructs encoding the bispecific antigen binding composition of any one of claims 52-58.153 / 155#14489013vlW0571.70068WQ0062. The one or more nucleic acid constructs of claim 61, wherein the nucleic acid is DNA or RNA.

63. A vector comprising the one or more nucleic acid constructs of claim 61 or 62.

64. A cell comprising the bispecific antigen binding composition of any one of claims 52- 59, the pharmaceutical composition of claim 60, the one or more nucleic acid constructs of claim 61 or 62, or the vector of claim 63.

65. A method of treating disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the bispecific antigen binding composition of any one of claims 52-59, the pharmaceutical composition of claim 60, the one or more nucleic acid constructs of claim 61 or 62, or the vector of claim 63.154 / 155#14489013vl

Images