Therapies for treatment of myeloid malignancies

Abstract

Provided are methods of treating a myeloid malignancy in a subject using antibodies. Also provided are compositions and kits for treating a myeloid malignancy in a subject using antibodies.

Description

Attorney Docket No.: 090723-1472570 MDA24-017PCT THERAPIES FOR TREATMENT OF MYELOID MALIGNANCIES STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under grant No. P50 CA100632 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0002] Treatment for myeloid malignancies, for example in leukemia, have made progress in recent years. Stem cell transplant and other therapies (for example, chimeric antigen receptors) could benefit some patients. However, for acute myeloid leukemia (AML) patients, there exists a large patient group that will be refractory and relapsed from the standard of care. There is urgent need for novel therapy. The present disclosure could provide a new therapeutic avenue for many patients suffering from the effects of myeloid malignancies. BRIEF SUMMARY

[0003] The terms “invention,” “the invention,” “this invention” and “the present invention,” as used in this document, are intended to refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Covered embodiments of the invention are defined by the claims, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures and each claim. Some of the exemplary embodiments of the present invention are discussed below.

[0004] Included among the embodiments of the present invention and described in the present disclosure are methods of treating a myeloid malignancy in a subject, comprising: (a) administering to the subject a therapeutically effective amount of a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibodyAttorney Docket No.: 090723-1472570 MDA24-017PCT corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) administering to the subject a therapeutically effective amount of (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRPα axis, such as an anti- CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking CD47- SIRPα axis. Thes second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622. The therapeutic (b)(2) may comprise one or more molecules shown in Table 3. The myeloid malignancy may be characterized by expression of HLA-A2 allele. For example, the myeloid malignancy may be AML, MDS, or MPN. The myeloid malignancy may be a refractive myeloid malignancy. Also included among the embodiments of the present invention are methods of treating a myeloid malignancy in a subject, comprising: (a) administering to the subject a therapeutically effective amount of a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is capable of specifically binding to SEQ ID NO:45; and (b) administering to the subject a therapeutically effective amount of (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

[0005] In the methods according to the embodiments of the present disclosure, the patient may be a patient who has relapsed. In the methods according to the embodiments of the present disclosure, the steps (a) and (b) may be performed simultaneously or not performed simultaneously. The methods according to the embodiments of the present disclosure may further comprise administering at an additional anti-cancer therapy to the patient. The additional anti-cancer therapy may be a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy or cytokine therapy.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0006] Included among the embodiments of the present invention and described in the present disclosure are pharmaceutical composition, comprising: (a) a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second is an antibody blocking CD47-SIRPα axis, such as an anti- CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking CD47- SIRPα axis The second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622. In the pharmaceutical composition, (a) may be the antigen binding fragment of the first antibody. In the pharmaceutical composition, (b)(1) may be the antigen binding fragment of the second antibody. The antigen binding fragment of the first antibody and / or the antigen binding fragment of the second antibody may be a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody. The therapeutic (b)(2) may comprise one or more molecules shown in Table 3.

[0007] Included among the embodiments of the present invention and described in the present disclosure are pharmaceutical compositions comprising: (a) a nucleic acid encoding a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and, (b) a nucleic acid encoding (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRPα axis, such as an anti-CD47 antibody or an anti-CD172a antibody, , or (2) a therapeutic capable of blocking CD47-SIRPα axis. The second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622. In the above pharmaceutical composition, (a) may be antigen binding fragment of the first antibody. In the above pharmaceutical composition, (b)(1) may the antigen binding fragment of the second antibody. The antigen binding fragment of the firstAttorney Docket No.: 090723-1472570 MDA24-017PCT antibody and / or the antigen binding fragment of the second antibody may be a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody. The therapeutic (b)(2) may comprise one or more molecules shown in Table 3. In the above pharmaceutical composition, (a) and (b) may be incorporated in one or more vectors. In the above pharmaceutical composition, (a) and (b) may be (incorporated in one or more cells.

[0008] Included among the embodiments of the present invention and described in the present disclosure are method of treating a myeloid malignancy in a subject, comprising administering to the subject a pharmaceutical composition according to the present disclosure. Also included among the embodiments of the present invention are kits comprising: (a) a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b)(1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRPα axis, such as an anti-CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking CD47-SIRPα axis. The kits may further comprise one or more of: (c) a pharmaceutical package; (d) a chemotherapeutic agent; (e) a cytotoxic agent; (f) a radiotherapeutic agent, or (g) an immunotherapeutic agent. Also included among the embodiments of the present invention are kits comprising: (a) a nucleic acid encoding a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and, (b) a nucleic acid encoding (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody blocking CD47-SIRPα axis, such as an anti-CD47 antibody or an anti-CD172a antibody, or (2) a therapeutic capable of blocking CD47-SIRPα axis. In the above kit embodiments, (b)(2) may comprise one or more molecules shown in Table 3. In the above lot embodiments, the second antibody may be a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab (also known as Hu5F9-G4), TJ011133, or TTI-622.Attorney Docket No.: 090723-1472570 MDA24-017PCT BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present disclosure includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0010] FIG.1 is a line plot illustrating that the loss of CD47 expression delays the growth of AML. The plot shows that CD47 KO in THP1 AML cells slows the growth of THP1 AML cells in both NSG and NSGS mouse models. NSG™ and NSGS mice were inoculated with THP1 wild type (WT) cells or THP1 KO cells. Bioluminescent (BLI) images were taken weekly. The data shown are mean+ / - standard error of mean (SEM) of treatment groups (n=5 mice per group). From top to bottom, the data lines in the graph correspond to Thp1 NSGS(n=5), Thp1 NSG(n=5), Thp1-CD47 NSGS(n=5), and Thp1-CD47 NSG(n=5).

[0011] FIG.2 is a line plot illustrating that the loss of CD47 expression delays the growth of AML. The plot illustrates the survival in each of the groups (n=5 mice per group) calculated using Log-rank (Mantel-Cox) test. From left to right, the data lines in the graph correspond to Thp1 NSGS(n=5), Thp1 NSG(n=5), Thp1-CD47 NSGS(n=5), and Thp1-CD47 NSG(n=5).

[0012] FIG.3 schematically illustrates the experiments in which NSGS mice were injected with THP1-CD47 KO or THP1-WT cells (5x10^5) and treated with Hu8F4 antibody at 1 mg / kg three times per week for a total of 25 doses starting on day 2 (n=5 mice per group).

[0013] FIG.4 is a line plot illustrating that Hu8F4 antibody eliminated CD47 knock out (KO) THP1 AML cells in NSGS mice (n=5 mice per group). BLI was accessed weekly. Each data point represents Total Flux Signal of one mouse at a timepoint. From left to right, the data lines in the graph correspond to THP1-WT / PBS, THP1-WT / Hu8F4, THP1-CD47 KO / PBS, and THP1-CD47 KO / Hu8F4.

[0014] FIG.5 is a plot illustrating the survival in each of the groups (n=5 mice per group) calculated using Log-rank (Mantel-Cox) test. Interruption of CD47 permitted Hu8F4-mediated elimination of CD47 KO THP1 cells in NSGS model. The data lines in the graph correspond to as follows: the dashed line on the left is THP1-WT / PBS, the next line is THP1-WT / Hu8F4, the next line is THP1-CD47 KO / PBS, and the line across the top is THP1-CD47 KO / Hu8F4.

[0015] FIG.6 schematically illustrates the experiments in which NSG mice, injected with with U937-A2 (5x10^3), were treated with Hu8F4 + / - anti-CD47 F(ab)’2 antibody at 1 mg / kg three times per week for a total of 10 doses starting on day 5 (n=5 mice per group).Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0016] FIG.7 is a line plot summarizing BLI data. Data shown are mean+ / - SEM of treatment groups (n=5 mice per group). From top to bottom, the data lines in the graph correspond to PBS, Hu8F4, a-CD47 F(ab’)2, and Hu8F4 + a-CD47 F(ab’)2.

[0017] FIG.8 is a plot illustrating the survival of mice in each of the groups (n=5 mice per group) calculated using Log-rank (Mantel-Cox) test. Combination therapy of Hu8F4 antibody with CD47 blocking antibody eliminated U937-A2 leukemia in NSG model (p=0.0002). The data lines in the graph correspond to as follows: the dashed line on the left is THP1-WT / PBS, the next line is PBS, the next line is Hu8F4, the next line is CD47 F(ab’)2, and the line across the top is Hu8F4 + a-CD47 F(ab’)2.

[0018] FIG.9A, 9B, 9C, and 9D schematically illustrate the experiments showing that Hu8F4-induced phagocytosis by mouse bone marrow-derived monocytes (BMDM) requires CD47 blockade. Fresh bone marrow cells from NSG mouse (effectors) were labelled with red fluorescent linker PKH26 (Sigma, red) and incubated in presence of mouse M-CSF in 96 well plate for 7 - 10 days. GFP-transfected U937-A2 (FIG.9A) and THP1 (FIG.9B) target cells were treated with Hu8F4 + / - a-CD47 F(ab’)2 for 30 minutes and added to BMDM. CD47 CRISPR / Cas 9 CD47 KO cells U937-A2 (FIG.9C) and THP1 (FIG.9D) cells were incubated with and without Hu8F4 and a-CD47 F(ab’)2 antibodies. Fluorescent and bright field images were taken at various time points (96 hours time point shown). Bar graphs show object area of target cells, analyzed and calculated by using Gen5 software. Each bar shows medium calculated object area of green (target) cells in 4 images, captured from corresponding cells using x4 objective by Cytation 3, each error bar shows SEM.2way ANOVA comparison was conducted, adjusted p values: * p < 0.05; ** p < 0.01; ***p <0.001; ****p<0.0001. The labels 8F4 and Hu8F4 both refer to the Hu8F4 antibody.

[0019] FIG.10 are bar graphs illustrating that anti-CD47 blockade acts synergistically with Hu8F4 to promote efficient phagocytosis of AML. The labels 8F4 and Hu8F4 both refer to the Hu8F4 antibody.

[0020] FIG.11 is a bar graph illustrating that H8F4-induced ADCP of target cells in presence of differentiated NSG mouse bone marrow-derived macrophages (BMDM) required inactivation of CD47.

[0021] FIG.12 are bar graphs illustrating that Blockage with either anti-CD47 or anti- SIRP⍺ showe synergistic ADCP activity with h8F4.

[0022] FIG.13 is a schematic illustration of experimental design. Adult female NSG mice were injected intravenously with 1 million THP-1 Luc / GFP leukemia cells via tail vein. Starting at 7 days post-inoculation, groups of mice (n=5 per group) were treated with Hu8F4Attorney Docket No.: 090723-1472570 MDA24-017PCT at 1 mg / kg, anti-SIRPα (anti-CD172a) at 4 mg / kg or 20 mg / kg, or a combination of Hu8F4 and anti-CD172a, administered three times per week for a total of 10 doses.

[0023] FIG.14 are line plots illustrating that that Anti-SIRPα (anti-CD172a) mediated anti-AML activity in vivo only in combination with Hu8F4. DETAILED DESCRIPTION I. TERMS AND CONCEPTS

[0024] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0025] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0026] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of those certain elements.” As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations were interpreted in the alternative (“or”).

[0027] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the present disclosure or features of the claims. See, for example, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q.461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0028] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as ifAttorney Docket No.: 090723-1472570 MDA24-017PCT it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0029] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0030] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be impartedAttorney Docket No.: 090723-1472570 MDA24-017PCT before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0031] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0032] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (i.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

[0033] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al., 2013, “Protein engineering with unnatural amino acids,” Curr. Opin. Struct.Attorney Docket No.: 090723-1472570 MDA24-017PCT Biol.23(4): 581-87; Xie et al., 2005, “Adding amino acids to the genetic repertoire,” Curr. Opin. Chem. Biol.9(6): 548-54; and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0034] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0035] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0036] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0037] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman, 1981, Add. APL. Math.2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol.48:443, by theAttorney Docket No.: 090723-1472570 MDA24-017PCT search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0038] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-10 and Altschul et al., 1977, Nucleic Acids Res.25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915).

[0039] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-87). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison ofAttorney Docket No.: 090723-1472570 MDA24-017PCT the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20. II. INTRODUCTION

[0040] Phagocytosis of tumor cells by professional phagocytes is regulated by a host of pro- phagocytosis (“eat me”) and anti- phagocytosis (“don’t eat me”) signals, specifically through receptor–ligand interactions at the cell–cell interface. Identified “eat me” signals expressed by tumor cells include tumor- associated antigens, the endoplasmic reticulum chaperone protein calreticulin and the glycoprotein signalling lymphocytic activation molecule family member 7 (SL AMF7; also known as CD319). When bound by antibodies, antigens can be recognized by Fc receptors (FcRs) on phagocytes. Calreticulin, on the other hand, is anchored to the cancer cell surface through binding with membrane glycans, where it then interacts with the prolow- density lipoprotein receptor- related protein 1 (LRP1) receptor expressed on phagocytes. Finally, the precise mechanism of SL AMF7-induced tumour cell phagocytosis is unclear. Nevertheless, it seems that the presence of SL AMF7 on target cells and macrophage-1 antigen (MAC1) on phagocytes are both crucial for inducing tumour phagocytosis. Conversely, tumour cells rely on the expression of ‘don’t eat me’ signals, including CD47, programmed cell death 1 ligand 1 (PD- L1), β2-microglobulin (B2M) and other unidentified ligands that bind to leukocyte immunoglobulin-like receptor 2 (LILRB2) to inhibit their phagocytotic clearance by phagocytes; these molecules respectively bind to signal-regulatory protein α (SIRPα), programmed cell death 1 (PD-1) and leukocyte immunoglobulin-like receptor 1 (LILRB1) on phagocytes to inhibit tumor cell phagocytosis. Therapeutic antibodies targeting some of these receptor–ligand interactions have been investigated as potential immunotherapy approaches for multiple cancer types. MHC- I, major histocompatibility complex class I.

[0041] It was discovered and described in the present disclosure that a combination of Hu8F4 and anti-CD47 significantly increases phagocytosis of AML cells by macrophages. Hu8F4 (anti-PR1 / HLA-A2 humanized IgG1) binds to PR1 / HLA-A2 on target AML cells through the 8F4 Fab domain, and it binds to Fc receptors (FcR) on neighboring phagocytes such as macrophages through the Hu8F4 Fc domain, which provides an “eat me” signal in macrophages to mediate phagocytosis. The “don’t eat me” signal is prevented by anti-CD47 F(ab)’2 that block CD47 binding to SIRPα on phagocytes. Anti-CD47 F(ab)’2 lacks the Fc domain and cannot bind to FcR and therefore cannot mediate an “eat me” signal, which mimics the mechanism of action of Magrolimab (which is an IgG4 isotype and therefore alsoAttorney Docket No.: 090723-1472570 MDA24-017PCT does not bind to FcR). Significantly, therefore, Hu8F4 directs phagocytosis only against AML cells and not normal cells, because PR1 / HLA-A2 is differentially expressed on AML, which provides a potent “eat me” signal to phagocytes. It is envisioned that combining Hu8F4 or its variants with anti-CD47 antibodies increases phagocytosis of AML by blocking the “don’t eat me” signal, which mediates maximal phagocytosis of AML and elimination of AML in vitro and in preclinical models. It is also envisioned and described in the present disclosure that various anti-peptide / MHC-I TCR mimic antibodies, such as, but not limited, to those that bind to VLQELNVTV (SEQ ID NO:45) epitope, when bound by an HLA- A2, receptor, can be used in combination with “don’t eat me” blockade. Examples of such antibodies are described in International Patent Publications WO2014011489A2 abd WO2010065962A2, It is envisioned that such antibodies can block the “don’t eat me” interaction of leukocyte immunoglobulin like receptor B1( LILRB1) on the phagocyte by interacting with the peptide / MHC-I.

[0042] To examine the relationship between CD47 and Hu8F4 activity, both in vitro Antibody-dependent Cellular Phagocytosis (ADCP) assays and in vivo NSG mouse models were used. CD47 blockade was studied as a potential strategy to enhance Hu8F4 activity. To characterize the in vitro phagocytosis, an HLA-A2 transfected leukemia cell line, U937 (U937-A2), and an endogenously expressing HLA-A2 cell line THP1, were used as targets (T). NSG mouse bone marrow derived macrophages (BMDM) were used as effectors (E). At an initial E:T=1, Hu8F4 alone (10 µg / ml) was insufficient to prevent growth of U937-A2 and THP1. However, addition of neutralizing anti-CD47 F(ab)’2 antibody significantly slowed leukemia growth and eliminated both target cells by day 5. To further confirm the critical role of CD47 in Hu8F4-mediated ADCP, CRISPR / Cas9 CD47 KO U937-A2 and THP1 cells lines were created, confirming the role of CD47 in the 8F4-mediated ADCP. Complete phagocytosis of both CD47KO cell lines was only observed in the presence, but not the absence of Hu8F4. It was discovered that the presence of CD47 on leukemia cells can significantly limit the activity of Hu8F4 both in vitro and in vivo. Importantly, combining Hu8F4 with CD47 blockade significantly increased the Hu8F4 activity and together eliminated AML. Based on the discoveries described in the present disclosure, it was conceived that a therapy combining Hu8F4 or similar antibodies and anti-CD47 antibodies or other molecules that block the CD47-SIRPα axis (some suitable molecules are discussed in Yang et al. “The landscape overview of CD47‑based immunotherapy for hematological malignancies.” Biomarker Research 11:15 (2023), see, for example, Tables 1 and 2) can be used to treat patients with myeloid malignancies that express the HLA-A2 allele, includingAttorney Docket No.: 090723-1472570 MDA24-017PCT the patients with refractory and / or relapsed disease. Accordingly, therapeutic methods, compositions, and kits for treating or alleviating myeloid malignancies are conceived and envisioned. III. MYELOID MALIGNANCIES

[0043] Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells. These malignancies can be present in the bone marrow and peripheral blood. They can result from genetic and epigenetic alterations that perturb key processes such as self-renewal, proliferation and impaired differentiation.

[0044] Myeloid malignancies can be categorized as five types: (1) acute myeloid leukemia (AML); (2) myelodysplastic syndromes (MDS); (3) myeloproliferative neoplasms (MPN); (4) myelodysplastic and myeloproliferative (MDS / MPN) neoplasms; and (5) myeloid neoplasms associated with eosinophilia and abnormalities of growth factor receptors derived from platelets or fibroblasts.

[0045] According to the present disclosure, myeloid malignancies that express HLA-A2 allele include, for example, AML, MDS, and MPN. Compositions and methods according to the present disclosure can treat, alleviate, or otherwise be useful for myeloid malignancies, in particular those characterized by expression of HLA-A2 allele.

[0046] Leukemia cells overexpress the “don’t eat me” membrane protein CD47. CD47 on AML binds SIRPα on macrophages and neutrophils to inhibit phagocytosis. Blocking this interaction can allow antibodies to exert powerful phagocytic activity. CD47 may play in the anti-tumor activity of TCR-mimic antibodies. IV. ANTIBODIES

[0047] The present disclosure provides compositions and methods for treating or alleviating myeloid malignancies (for example in a subject having or suspected of having a myeloid malignancy, an animal model, an in vitro tissue culture model, and the like), such as, but not limited to, AML. Antibodies or antigen binding portions thereof that specifically or selectively bind antigen targets related to myeloid malignancies are provided herein. In certain aspects, antibodies as described herein are humanized antibodies. In embodiments of the present disclosure, antibodies (or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure) specific to PR1 / HLA-A2 tumor antigen complex are used. Examples of such antibodies are Hu8F4 and 8F4 antibodies, for instance, Hu8F4-1 and Hu8F4-2.8F4 antibodies, including humanizedAttorney Docket No.: 090723-1472570 MDA24-017PCT 8F4 (Hu8F4) antibodies, are described in U.S. Patent Nos.9,926,380 and 11,192,958. Hu8F4, a humanized monoclonal antibody that recognizes PR1 / HLA-A2 complex, is a first-in-class T cell receptor (TCR) - mimic antibody. The activity of Hu8F4 primarily depends on its binding to Fcγ receptors (FcγR) on effector cells. Antibody-dependent cellular phagocytosis (ADCP) is a principal mechanism of Hu8F4 anti-AML activity in vivo. In the embodiments of the present disclosure, antibodies (or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure) that bind to CD47 are also used. The non-limiting examples of such antibodies are a neutralizing anti-CD47 F(ab)’2 antibody used in the experiments described in the present disclosure, Magrolimab (also known as Hu5F9-G4) (Gilead Sciences), TJ011133 (I-Mab Biopharma Co), TTI-622 (Trillium Therapeutics Inc) or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure. The above can be collectively referred to as “antibodies according to the present disclosure.”

[0048] It is contemplated that antibody may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence, such as those in SEQ ID NOs: 3, 60, 5, 8, 9, 10 (heavy CDRs 1, 2 and 3; light CDRs 1 and 2, 3, respectively), and SEQ ID NO:16, which includes the heavy CDRs and framework regions 1, 2 and 3, which flank upstream of heavy CDRs 1, 2 and 3, respectively, and SEQ ID NOs: 19 or 20, which includes the light CDRs and framework regions 1, 2 and 3, which flank upstream of light CDRs 1, 2 and 3, respectively.

[0049] In some embodiments, the antibody comprises a heavy chain variable region comprising CDRs comprising amino acid sequences SEQ ID NOs: 3, 60 and 5, respectively, and a light chain variable region comprising CDRs comprising amino acid sequences SEQ ID NOs: 8, 9 and 10, respectively. In some embodiments, the antibody comprises a heavy chain variable region comprising CDRs consisting of amino acid sequences SEQ ID NOs: 3, 60 and 5, respectively, and a light chain variable region comprising CDRs consisting of amino acid sequences SEQ ID NOs: 8, 9 and 10, respectively.

[0050] In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16. In some embodiments, the antibody comprises a light chain comprising a light chain variable region at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:19. In some embodiments, the antibody comprises a light chain comprising a light chain variable region at least 90% identity (e.g.,Attorney Docket No.: 090723-1472570 MDA24-017PCT 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:20. In some embodiments, the antibody comprises a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16, and a light chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequences SEQ ID NOs: 19 or 20. In some embodiments, the antibody comprises a heavy chain variable region consisting of amino acid sequence SEQ ID NO:16, and a light chain variable region consisting of amino acid sequences SEQ ID NOs: 19 or 20.

[0051] In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16, and a light chain comprising a light chain variable region at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:19.

[0052] In some embodiments, the antibody comprises a heavy chain constant region that is a human gamma-1 heavy chain constant region and a light chain constant region that is a human kappa light chain constant region.

[0053] In some embodiments, the human gamma-1 heavy chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in amino acid sequence SEQ ID NO:38. In some embodiments, the human kappa light chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) residues 128-234 in amino acid sequence SEQ ID NO:42. In some embodiments, the human gamma-1 heavy chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in amino acid sequence SEQ ID NO:38, and the human kappa light chain constant region comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) residues 128-234 in amino acid sequence SEQ ID NO:42. In some embodiments, the human gamma-1 heavy chain constant region is residues 144-473 in amino acid sequence SEQ ID NO:38, and the human kappa light chain constant region is residues 128-234 in amino acid sequence SEQ ID NO:42.

[0054] In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%,Attorney Docket No.: 090723-1472570 MDA24-017PCT 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16 and a heavy chain constant region which is a human gamma-1 heavy chain constant region, and a light chain comprising a light chain variable region at least 90% identity to amino acid sequence SEQ ID NO:19 and a light chain constant region which is a human kappa light chain constant region.

[0055] In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:16 and the heavy chain constant region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 144-473 in the amino acid sequence SEQ ID NO:38, and a light chain comprising a light chain variable region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to amino acid sequence SEQ ID NO:19 and the light chain constant region comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to residues 128-234 in amino acid sequence SEQ ID NO: 42.

[0056] In some embodiments, the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to any one of SEQ ID NOs: 40 or 42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to of any one of SEQ ID NOs: 38 or 44. In some embodiments, the antibody can have a light chain sequence comprising at least 90% identity to SEQ ID NO:40 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38. In some embodiments, the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:38. In some embodiments, the antibody can have a light chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:42 and a heavy chain sequence comprising at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to SEQ ID NO:44.Attorney Docket No.: 090723-1472570 MDA24-017PCT TABLE 1. Exemplary Antibody SequencesAttorney Docket No.: 090723-1472570 MDA24-017PCTAttorney Docket No.: 090723-1472570 MDA24-017PCT

[0057] In some embodiments, the antibody is fused to a non-antibody peptide or polypeptide segment. In some embodiments, the antibody is linked to a diagnostic reagent or a therapeutic reagent. In some embodiments, the diagnostic reagent is a fluorophore, a chromophore, a dye, a radioisotope, a chemilluminescent molecule, a paramagnetic ion, or a spin-trapping reagent. In some embodiments, the therapeutic reagent is a cytokine, a chemotherapeutic, a radiotherapeutic, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule, or an immune co-stimulatory molecule. In some embodiments, the antibody is a bispecific antibody. For example, a bispecific antibody may have, in addition to binding affinity for PR1 / HLA-A2 complex, binding affinity for B cells (CD19, CD20), NK cells, phagocytes (CD16), or monocytes (CD14).

[0058] The design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables (Table 4). In particular embodiments, the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids. Codon preferences for various species of host cell are well known in the art. Codons preferred for use in humans, are well known to those of skill in the art (Wada et al., 1990). Codon preferences for other organisms also are well known to those of skill in the art (Wada et al., 1990, included herein in its entirety by reference). TABLE 2. Codon table. Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCUAttorney Docket No.: 090723-1472570 MDA24-017PCT Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0059] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0060] As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the embodiments of 4-1BB-specific antibodies or antigen binding fragments thereof described herein, for example, in the heavy chain variable region and / or light chain variable region, can occur that do not alter the nature or function of the antibodies or antigen binding fragments thereof. Such modifications include conservative amino acids substitutions, such that each recited sequence optionally contains one or more conservative amino acid substitutions. The list provided below identifies groups that contain amino acids that are conservative substitutions for one another; these groups are exemplary as other conservative substitutions are known to those of skill in the art: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0061] By way of example, when an aspartic acid at a specific residue is mentioned, also contemplated is a conservative substitution at the residue, for example, glutamic acid. Non- conservative substitutions, for example, substituting a proline with glycine or substituting a lysine with an asparagine, are also contemplated.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0062] In some instances, the affinity of 4-1BB-specific antibodies or antigen binding fragments thereof may be optimized through CRISPR or other site-directed mutagenesis methods to increase or decrease affinity as desired based on one or more of the known characteristics of the binding interaction with 4-1BB, the structure of either or both of the antibodies or antigen binding fragments thereof, or the 4-1BB protein.

[0063] Antibodies according to the present disclosure may also be engineered into various modalities, such as engineered chimeric antigen receptors (CAR), also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors, such as, for example CAR-T, CAR-NK, or CAR macrophage. CARs for CAR T, CAR NK and CAR macrophage have similar structures: the extracellular domain including the antigen binding domain and a spacer that is involved in engagement of target cells; a transmembrane domain that docks CAR to immune cells and is also involved in other functions of CAR, such as stability and interaction with other membrane proteins; and an intracellular signaling domain that is involved in signaling transduction and activation of immune cells.

[0064] As used herein, the following expressions - specifically binds to specific for, selectively binds and selective for a myeloid malignancy antigen or an epitope on a protein related to a myeloid malignancy - mean binding that is measurably different from a non- specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0065] An antibody, as used herein, can refer to an intact antibody (e.g., an intact immunoglobulin) and antibody fragment, for example, an antigen binding fragment, or a bispecific antibody. Antigen binding fragments can comprise at least one antigen binding domain. One example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. Antibodies and antigen binding fragments can be described by the antigen to which they specifically bind.

[0066] The VHand VLregions can be further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VHand VLgenerally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2Attorney Docket No.: 090723-1472570 MDA24-017PCT - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) CDR sequences on the heavy chain (VH) may be designated as CDRH1, 2, 3, while CDR sequences on the light chain (Vv) may be designated as CDRL1, 2, 3.

[0067] Provided herein are antibodies or antigen binding portions thereof that specifically bind to antigens related to myeloid malignancies. Such antibodies may be, for example, monoclonal antibodies (mAbs) or recombinant / chimeric antibodies (i.e., synthetic antibodies derived from synthetic nucleic acid constructs, such as viral vectors, that may also contain the sequences of the monoclonal antibodies disclosed herein).

[0068] In some instances, the affinity of myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof may be optimized through mutations to increase or decrease affinity as desired based on one or more of the known characteristics of the binding interaction with the cognant myeloid malignancy antigen, the structure of either or both of the antibodies or fragments thereof, or the myeloid malignancy antigen. In some instances, the mutations permit facile elution of purified antibodies or fragments thereof under desirable elution conditions during isolation and purification.

[0069] Methods of generating and screening for antibodies and antigen binding fragments are well-known in the art. Methods of further modifying antibodies for enhanced properties (e.g., enhanced affinity, chimerization, humanization) as well as generating antigen binding fragments, as described herein, are also well-known in the art.

[0070] The present disclosure also encompasses antibodies or fragments thereof that bind to the same epitope of myeloid malignancy antigens as the antibodies disclosed herein. Such antibodies can be identified using routine techniques known in the art, including, for example, competitive binding assays.

[0071] The term epitope, as used herein, means a component of an antigen capable of specific binding to an antibody or antigen binding fragment thereof. Such components optionally comprise one or more contiguous amino acid residues and / or one or more non- contiguous amino acid residues. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues thatAttorney Docket No.: 090723-1472570 MDA24-017PCT are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations.

[0072] The present disclosure also provides chimeric antibodies. The term chimeric antibody refers to an antibody in which a component of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0073] A human antibody is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources, genetically modified non-human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0074] In some embodiments, the antibody or antigen binding fragment thereof provided herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab′, F(ab′)2, Fc, Fd, Fd′, Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR- grafted, or an in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from either kappa or lambda light chains.

[0075] As used herein, the term monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are the same or substantially similar andAttorney Docket No.: 090723-1472570 MDA24-017PCT that bind the same epitope(s), except for variants that can normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target, for example, by affinity maturation, to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0076] Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 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 CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0077] In certain embodiments, antibodies and antibody compositions as used in the embodiments of the present disclosure are distinguishable from naturally occurring antibodies and compositions in one or more respects. Such distinguishable antibodies and compositions may be referred to as “synthetic,” or may be identified by the proviso that the antibody or composition “is not naturally occurring” or affirmatively as “non-naturally occurring.” As used herein the terms “corresponding antibody,” and “corresponding to” describes the relationship between (1) an antibody characterized by six specific CDR sequences of the antibodies according to the present disclosure and (2) a synthetic antibody comprising the same six CDR sequences. Synthetic antibodies of this disclosure may differ in structure from naturally occurring antibodies with the same CDRs. That is, synthetic antibodies identified by specified CDRs may be structurally different from antibodies comprising the specified CDRs. Possible differences for synthetic antibodies include variable region sequences that differ corresponding naturally occurring antibodies, different light chain sequences (i.e. lambda type instead of kappa type or vice versa), different isotypes, different allotypes, and differentAttorney Docket No.: 090723-1472570 MDA24-017PCT constant domain variants. These differences are discussed in more detail below. In some embodiments, the synthetic antibody is an engineered polypeptide, also referred to as a recombinant polypeptide, that is made using conventional protein and antibody engineering molecular biology, chemical, and biochemical methods.

[0078] In some embodiments, the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin producing human B cell, and further comprises a kappa or lambda light chain constant region. In some embodiments, the light chain constant region (kappa or lambda) is from the same type of light chain (i.e., kappa or lambda) as the light chain variable region that was derived from the immunoglobulin producing human B cell; as a non-limiting example, if an IgE- producing human B cell comprises a kappa light chain, then the antibody that is produced can comprise the light chain variable region from the IgE-producing B cell and further comprises a kappa light chain constant region.

[0079] In some embodiments, the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin-producing human B cell, and further comprises a heavy chain constant region having an IgG isotype (e.g., IgG4), an IgA isotype (e.g., IgA1), an IgM isotype, an IgD isotype, or that is derived from an IgG, IgA, IgM, or IgD isotype (e.g., is a modified IgG4 constant region). It will be appreciated by a person of ordinary skill in the art that the different heavy chain isotypes (IgA, IgD, IgE, IgG, and IgM) have different effector functions that are mediated by the heavy chain constant region, and that for certain uses it may be desirable to have an antibody that has the effector function of a particular isotype (e.g., IgG).

[0080] In some embodiments, the antibody comprises a native (i.e., wild-type) human IgG, IgA, IgM, or IgD constant region. In some embodiments, the antibody comprises a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, a native human IgG4 constant region, a native human IgA1 constant region, a native human IgA2 constant region, a native human IgM constant region, or a native human IgD constant region. In some embodiments, the antibody comprises a heavy chain constant region that comprises one or more modifications. It will be appreciated by a person of ordinary skill in the art that modifications such as amino acid substitutions can be made at one or more residues within the heavy chain constant region that modulate effector function. In some embodiments, the modification reduces effector function, e.g., results in a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on anAttorney Docket No.: 090723-1472570 MDA24-017PCT effector cell that mediates the effector function. In some embodiments, the modification (e.g., amino acid substitution) prevents in vivo Fab arm exchange, which can introduce undesirable effects and reduce the therapeutic efficacy of the antibody. See, e.g., Silva et al., J Biol Chem, 2015, 280:5462-5469.

[0081] In some embodiments, the antibody comprises a native (i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2 and comprises one or more modifications that modulate effector function. Ini some embodiments the antibody comprises a human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2. In some embodiments, the antibody comprises a native (i.e., wild-type) human IgM constant region, human IgD constant region, human IgG constant region that is derived from IgG1, IgG2, IgG3, or IgG4, or human IgA constant region that is derived from IgA1 or IgA2 and comprises one, two, three, four, five, six, seven, eight, nine, ten or more modifications (e.g., amino acid substitutions). In some embodiments, the constant region includes variations (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions) that reduce effector function.

[0082] Synthetic antibodies of this disclosure may comprise variations in heavy chain constant regions to change the properties of the synthetic antibody relative to the corresponding naturally occurring antibody. Exemplary changes include mutations to modulate antibody effector function (e.g., complement-based effector function or FcγR-based effector function), alter half-like, modulate co-engagement of antigen and FcγRs, introduce or remove glycosylation motifs (glyco-engineering). See Fonseca et al., 2018, “Boosting half- life and effector functions of therapeutic antibodies by Fc-engineering: An interaction- function review” Int J Biol Macromol.19:306-311; Wang et al., 2018, “IgG Fc engineering to modulate antibody effector functions” Protein Cell 2018, 9(1):63–73; Schlothauer, 2016, “Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions,” Protein Engineering, Design and Selection 29(10):457–466; Tam et al., 2017, “Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality” Antibodies 6(3):12, each incorporated herein by reference for all purposes.

[0083] In some embodiments, the heavy chain variable region and / or the light chain variable region of the antibody has an identical sequence to the heavy chain variable region and / or the light chain variable region encoded by the immunoglobulin producing single B cellAttorney Docket No.: 090723-1472570 MDA24-017PCT from the human subject having a myeloid malignancy. In some embodiments, the heavy chain variable region and / or the light chain variable region of the antibody comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0084] The heavy chain variable region sequence and / or light chain variable region sequence of an antibody described herein can be engineered to comprise one or more variations in the heavy chain variable region sequence and / or light chain variable region sequence. In some embodiments, the engineered variation(s) improves the binding affinity of the antibody for a myeloid malignancy. In some embodiments, the engineered variation(s) improves the cross-reactivity of the antibody for a second myeloid malignancy.

[0085] In some embodiments, the engineered variation is a variation in one or more CDRs, e.g., an amino acid substitution in a heavy chain CDR and / or a light chain CDR as described herein. In some embodiments, the engineered variation is a variation in one or more framework regions, e.g., an amino acid substitution in a heavy chain framework region and / or a light chain framework region. In some embodiments, the engineered variation is a reversion of a region of the heavy chain and / or light chain sequence to the inferred naïve sequence. Methods for determining an inferred naïve immunoglobulin sequence are described in the art. See, e.g., Magnani et al., PLoS Negl Trop Dis, 2017, 11:e0005655, doi:10.1371 / journal.pntd.0005655

[0086] In some embodiments, affinity maturation is used to engineer further mutations that enhance the binding affinity of the antibody for a myeloid malignancy or enhance the cross- reactivity of the antibody for a second myeloid malignancy or other non-myeloid related pathology. Methods for performing affinity maturation are known in the art. See, e.g., Renaut et al., Methods Mol Biol, 2012, 907:451-461.

[0087] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody toAttorney Docket No.: 090723-1472570 MDA24-017PCT distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0088] In some embodiments, an antigen binding fragment can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) J. Biol. Chem.274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is screened, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol 23(10):514-522.

[0089] Synthetic antibodies of this disclosure may differ from naturally occurring compositions in at least one or more of the following respects: (i) composition comprises antibodies that are purified, i.e., separated from tissue or cellular material with which they are associated in the human body, and optionally in an manufactured excipient or medium; and / or (ii) antibody compositions according to the present disclosure contain a single species of antibody (are monoclonal) such that all antibodies in the composition have the same structure and specificity. V. THERAPEUTICS THAT BLOCK THE CD47-SIRPα AXIS

[0090] Embodiments of the present disclosure are envisioned to use various therapeutics that block the CD47-SIRPα axis or, when appropriate, nucleic acid molecules encoding such therapeutics. A therapeutic that blocks the CD47-SIRPα axis may be or comprise any molecule that blocks the CD47-SIRPα axis. Non-limiting examples of such molecules include, but are not limited, antibodies described elsewhere in the present disclosure (such as a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622), or antigen binding fragments of such antibodies, or variants of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). Non-limiting examples of such molecules are small molecules, large molecules, peptides, proteins, etc. Some non-limiting examples ofAttorney Docket No.: 090723-1472570 MDA24-017PCT such molecules are shown in Table 3, which is a reproduction of Tables 1 and 2 from Yang et al. “The landscape overview of CD47‑based immunotherapy for hematological malignancies.” Biomarker Research 11:15 (2023). VI. THERAPEUTIC METHODS

[0091] As described herein, the present disclosure provides a method of treating a subject with a myeloid malignancy, such as AML, comprising administering to the subject a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure) and a therapeutically effective amount a molecule that blocks the CD47-SIRPα axis. Some examples of molecules that block the CD47-SIRPα axis are anti-CD47 antibodies or anti-CD172a antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). Some examples of molecules that block the CD47-SIRPα axis are shown in in Table 3. In some embodiments, the subject has or is determined to have a myeloid malignancy, such as AML.

[0092] The compositions described herein are useful in, inter alia, methods for treating a myeloid malignancy, such as AML, in a subject. As used herein, the term subject means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have a myeloid malignancy, such as AML. In some embodiments, the subject is diagnosed with a myeloid malignancy. In some embodiments, the subject is a human that is suspected of having a myeloid malignancy, for example, AML.

[0093] As used herein, administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and an antibody capable of blocking CD47-SIRPα axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, or a construct or constructs encoding same) into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneousAttorney Docket No.: 090723-1472570 MDA24-017PCT delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0094] The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavity, oral, intracranial injection, or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion. Techniques for preparing injectate or infusate delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those of skill in the art can readily determine the various parameters and conditions for producing antibody injectates or infusates without resort to undue experimentation.

[0095] Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No.20080241223; U.S. Patent Nos.5,501,856; 5,164,188; 4,863,457; and 3,710,795. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion- based systems, or electromechanical systems. In some embodiments, a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and a therapeutically effective amount of an antibody capable of blocking CD47-SIRPα axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure or Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, are delivered to a subject by way of local administration.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0096] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and the like.

[0097] Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a myeloid malignancy, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying or preventing metastasis. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a myeloid malignancy in a subject by administering an antibody as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0098] The principal symptoms of myeloid malignancy, such as AML, can include (without intending to be limiting) bone pain, nausea, constipation, loss of appetite, mental fogginess or confusion, fatigue, frequent infections, weight loss, weakness or numbness in the legs, excessive thirst, easily fractured or broken bones, anemia, leukopenia, thrombocytopenia, excessive urination, hypercalcemia, spinal cord compression, kidney dysfunction, hyperviscosity, and the like.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0099] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, and an antibody capable of blocking CD47-SIRPα axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof, that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of myeloid malignancy disease are ameliorated, or the likelihood of myeloid malignancy developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of an antibody or fragment thereof described herein, which dose is capable of treating a myeloid malignancy in a subject, can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole antibody may be required to treat a subject with a myeloid malignancy as compared to the dose of a fragment of an antibody (e.g., Fab’ antibody fragment) required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or extent of myeloid malignancy. For example, a subject that has had a previous myeloid malignancy may require administration of a different dosage than a subject who has not previously had a myeloid malignancy. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some instances, a therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0100] A pharmaceutical composition according to the present disclosure can include therapeutically effective amounts of antibodies according to the present disclosure. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the administered antibodies according to the present disclosure with one or more additional active agents, if more than one agent is used in or with the pharmaceutical composition. In certain aspects, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg.

[0101] Suitable human doses of any of the antibodies according to the present disclosure can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0102] Toxicity and therapeutic efficacy of antibodies according to the present disclosure can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. An antibody thereof that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0103] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the antibodies according to the present disclosure lies generally within a range of circulating concentrations of the the antibodies according to the present disclosure that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For the antibodies according to the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50(i.e., the concentration of the construct – e.g., antibody – which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may beAttorney Docket No.: 090723-1472570 MDA24-017PCT measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0104] In some embodiments, the antibodies according to the present disclosure can be administered to a subject simultaneously. That is, a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, is administered simultaneously with and an antibody capable of blocking CD47-SIRPα axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof. Alternatively, the antibodies according to the present disclosure can be administered separately in any order. That is, a therapeutically effective amount of a Hu8F4 or 8F4 antibody, or a corresponding antibody, or an antigen binding fragment thereof, is administered separately, before or after, a therapeutically effective amount of an antibody capable of blocking CD47-SIRPα axis, such as an anti-CD47 or an anti-CD172a antibody, including, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, TTI-622, or a corresponding antibody, or an antigen binding fragment thereof. Each or both of the antibodies according to the present disclosure can be administered in conjunction with other therapies for cancer (combination therapy). For example, each or both of the antibodies according to the present disclosure can be administered to a subject at the same time, prior to, or after, a second therapy. In some embodiments, each or both of the antibodies according to the present disclosure and the one or more additional active agents are administered at the same time. Optionally, the antibodies according to the present disclosure are administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the antibodies according to the present disclosure is administered second in time. Optionally, the each or both of the antibodies according to the present disclosure and the one or more additional agents are administered simultaneously in the same or different routes.

[0105] The antibodies according to the present disclosure can replace or augment a previously or currently administered therapy. For example, upon treating with a myeloid malignancy antigen-specific antibody or antigen binding fragment thereof, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lowerAttorney Docket No.: 090723-1472570 MDA24-017PCT levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the antibodies according to the present disclosure reach a level sufficient to provide a therapeutic effect.

[0106] Also provided are cancer treatment methods using a CAR comprising each or both of the antibodies according to the present disclosure. In some embodiments, these methods comprise using the CAR to redirect the specificity of an immune effector cell (e.g., a T cell) to target a cancer cell (e.g., a myeloid malignancy antigen-expressing cancer cell). Thus, provided herein are methods of stimulating an effector cell-mediated response (such as a T cell-mediated immune response) to a target cell population or tissue comprising cancer cells in a mammal, comprising the step of administering to the mammal an effector cell (such as a T cell) that expresses a CAR as described herein. In some embodiments, “stimulating” an immune cell refers to eliciting an effector cell-mediated response (such as a T cell-mediated immune response), which is different from activating an immune cell. CAR-expressing effector cells described herein can be infused to a subject in need of treatment (e.g., a cancer patient). In some embodiments, the infused cell is able to kill (or lead to the killing of) cancer cells in the subject. Formulations and methods for making CAR-expressing effector cells and using them in therapeutic methods are known in the art (see, e.g., Feins et al., 2019, Am. J. Hematol.94(S1):S3-S9).

[0107] Monitoring a subject (e.g., a human patient) for an improvement of myeloid malignancy, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in one or more symptoms of myeloid malignancy exhibited by the subject. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a myeloid malignancy described herein.

[0108] In some instances, each or both of the antibodies according to the present disclosure can be administered via virus-like particles. Virus-like particles (VLPs) comprise viralAttorney Docket No.: 090723-1472570 MDA24-017PCT protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0109] In some instances, each or both of the antibodies according to the present disclosure can be administered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0110] In some instances, each or both of the antibodies according to the present disclosure can be administered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006 / 110728.

[0111] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient cells that have been genetically engineered, using methods such as those described herein, to express and secrete the antibodies according to the present disclosure.

[0112] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient immune cells that express a CAR comprising each or both of the antibodies according to the present disclosure.

[0113] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding each or both of the antibodies according to the present disclosure.

[0114] There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.

[0115] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding expression of the nucleic acid molecule and / or adapter polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), whichAttorney Docket No.: 090723-1472570 MDA24-017PCT is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213-20 (1987); Massie et al., Mol. Cell. Biol.6:2872-83 (1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al., J. Virology 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host- restricted non-replicating vaccinia virus vectors. In some instances, the nucleic acid molecules encoding each or both of the antibodies according to the present disclosure can be delivered via virus-like particles.

[0116] Non-viral based delivery methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding the adapter polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0117] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., β-actin promoter or EF1α promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0118] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself.Attorney Docket No.: 090723-1472570 MDA24-017PCT They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0119] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EF1α promoter, and the retroviral long terminal repeat (LTR).

[0120] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0121] In certain embodiments, the effective amount of a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a each or both of the antibodies according to the present disclosure are being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0122] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the each or both of the antibodies according to the present disclosure in the formulation used. Such pharmacokinetic parameters are well known in the art, i.e., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol.58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci.84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol.24:103-108; the latest Remington's, supra). In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0123] In some cases, the dosage (of the active component) ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 20 mg / kg, of the patient’s body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or within the range of 0.1-20 mg / kg. In certain examples, each or both of the antibodies according to the present disclosure can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg once every other day at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In someAttorney Docket No.: 090723-1472570 MDA24-017PCT cases, the treatment comprises administering each or both of the antibodies according to the present disclosure according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen resumes. For example, a patient may be on a dosing regimen of each or both of the antibodies according to the present disclosure for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens each or both of the antibodies according to the present disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the each or both of the antibodies according to the present disclosure being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0124] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes, by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0125] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0126] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure in an ex vivo manner. In such instances, cells that have been removed from a subject may be exposed to a pharmaceutical composition comprising each or both of the antibodies according to the present disclosure, after which the cells are subsequently implanted back into the subject.

[0127] In some instances, the provided methods may include administering to the subject a each or both of the antibodies according to the present disclosure that is conjugated to aAttorney Docket No.: 090723-1472570 MDA24-017PCT therapeutic agent. The therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, or an immunosuppressive agent. Such therapeutic agents are described below.

[0128] In some instances, the provided methods may include administering the antibodies according to the present disclosure and a second form of cancer therapy to the subject. The second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies which may be administered as a second form of cancer therapy include, but are not limited to, rituximab (e.g., for treatment of B-cell lymphomas), trastuzumab (e.g., for treatment of breast cancer), and cetuximab (e.g., for treatment of lung cancer).

[0129] In some instances, each or both of the antibodies according to the present disclosure can be labeled, conjugated, or fused with a therapeutic agent or diagnostic agent (such as an imaging agent). The linkage can be covalent or noncovalent (e.g., ionic). Such antibodies and antibody fragments are referred to antibody-drug conjugates (ADC) or immunoconjugates. The antibody conjugates are useful for the local delivery of therapeutic agents, particularly cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated. Therapeutic agents include but are not limited to toxins, including but not limited to plant and bacterial toxins, small molecules, peptides, polypeptides and proteins. Genetically engineered fusion proteins, in which genes encoding for an antibody, or fragments thereof including the Fv region, or peptides can be fused to the genes encoding a toxin to deliver a toxin to the target cell are also provided. As used herein, a target cell or target cells are myeloid malignancy antigen- positive cells.

[0130] In some embodiments, each or both of the antibodies according to the present disclosure are conjugated to a moiety that specifically binds to an immune cell. In some embodiments, provided is a bispecific antibody comprising one or both of the antibodies according to the present disclosure or antigen binding fragment thereof as described herein and an antibody or antigen binding fragment thereof that specifically binds to an immune cell. In some embodiments, the bispecific antibody comprises an antibody according to the present disclosure and an antibody moiety that specifically binds to T cells. Such a moleculeAttorney Docket No.: 090723-1472570 MDA24-017PCT is referred to as a bispecific T cell engager and may induce T cell-mediated cytotoxicity of myeloid malignancy antigen-expressing cancer cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38). In some embodiments, the bispecific antibody comprises an antibody according to the present disclosure and an antibody moiety that specifically binds to natural killer cells (NK cells). Such a molecule is referred to as a NK cell engager and may induce NK cell-mediated cytotoxicity of myeloid malignancy antigen-expressing cancer cells (see, e.g., Demaria et al., 2021, European Journal of Immunology 51(8):1934-1942).

[0131] In certain aspects, each or both of the antibodies according to the present disclosure and CARs (or cells comprising CARs as described herein) according to the present disclosure can be administered as a co-therapy with other therapeutic agents. Other examples of therapeutic agents include chemotherapeutic agents, a radiotherapeutic agent, and immunotherapeutic agent, as well as combinations thereof. In this way, the antibody or peptide complex or CAR (or cell comprising a CAR) delivered to the subject can be multifunctional, in that it exerts one therapeutic effect by binding to the myeloid malignancy antigen protein and a second therapeutic effect by delivering a supplemental therapeutic agent.

[0132] The therapeutic agent can act extracellularly, for example by initiating or affecting an immune response, or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling. The therapeutic agent is optionally cleavable from an antibody according to the present disclosure. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.

[0133] In some instances, the therapeutic agent is a cytotoxic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof. Additional examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e. g., daunorubicinAttorney Docket No.: 090723-1472570 MDA24-017PCT (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Cytotoxic peptides such as auristatin (antineoplastic) peptides auristatin E (AE) and monomethylauristatin (MMAE), which are synthetic analogs of dolastatin, may also be conjugated to each or both of the antibodies according to the present disclosure. In some instances, each or both of the antibodies according to the present disclosure may be conjugated to a radioactive metal ion.

[0134] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm.), bortezomib (such as VELCADE®, Millenium Pharm.), fulvestrant (such as FASLODEX®, AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA®, Novartis), imatinib mesylate (such as GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (such as ELOXATIN®, Sanofi), 5-fluorouracil (5- FU), leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine (such as XELODA®, Roche), docetaxel (such as TAXOTERE®), and gefitinib (such as IRESSA®, Astrazeneca), AG1478, AG1571 (such as SU 5271; Sugen Inc.), alkylating agents such as thiotepa and cyclosphosphamide (such as CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ1Iand calicheamicin θ1I); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyneAttorney Docket No.: 090723-1472570 MDA24-017PCT antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (such as ADRIAMYCIN®, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Trametes Versicolor polysaccharide-K (Krestin, PSK) (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′, 2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (cytosine arabinoside, “Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (such as TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (a Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)), and doxetaxel (such as TAXOTERE®, Rhône-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitorAttorney Docket No.: 090723-1472570 MDA24-017PCT RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0135] Chemotherapeutic agents, as used herein, also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON®); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestanie, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) VEGF receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN-7®vaccine (plasmid / lipid complex containing the DNA sequences encoding HLA-B7 and ß2 microglobulin), LEUVECTIN®vaccine (plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID®vaccine (patient-specific naked DNA vaccine); IL-2 or aldesleukin (such as PROLEUKIN®); topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropin-releasing hormone antagonists (such as ABARELIX®); (x) anti-angiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0136] In some instances, the treatment methods provided herein may further comprise administering an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method. These treatments work by “taking the brakes off” the immune system (are immunosuppressive), allowing it to mount a stronger and more effective attack against cancer. Several different types of checkpoint inhibitors, targeting different checkpoints or “brakes” on immune cells, are currently in use. Exemplary immunosuppressive agents are PD-1 inhibitors (such as nivolumab and pembrolizumab), PD-L1 inhibitors (such as atezolizumab, durvalumab, and avelumab), and CTLA-4 inhibitors (such as ipilimumab). InAttorney Docket No.: 090723-1472570 MDA24-017PCT one example, the second form of cancer therapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor. In some instances, combinations of such inhibitors can be administered. In some instances, the PD-L1 inhibitor, the PD-1 inhibitor, and / or the CTLA4 inhibitor may be an inhibitory antibody that binds specifically to PD-L1, PD-1, or CTLA4, respectively.

[0137] In some instances, the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Systemic radiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells. VII. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0138] Compositions comprising one or both antibodies according to the present disclosure and pharmaceutically acceptable carrier are also provided. In some embodiments, the compositions comprise a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). In some embodiments, the compositions comprise any suitable molecule that blocks the CD47-SIRPα axis. Some examples of molecules that block the CD47-SIRPα axis are anti-CD47 or anti-CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variants of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). Some examples of molecules that block the CD47-SIRPα axis are shown in Table 3.

[0139] In some embodiments, the compositions comprise a nucleic acid encoding a Hu8F4 or 8F4 antibody, or an antigen binding fragment thereof, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). In some embodiments, the compositions comprise encoding any suitable molecule that blocks the CD47-SIRPα axis. Some examples of molecules that block the CD47-SIRPα axis are anti- CD47 or anti-CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, orAttorney Docket No.: 090723-1472570 MDA24-017PCT TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). Some examples of molecules that block the CD47-SIRPα axis are shown in Table 3. In the above compositions according to the embodiments of the present invention and comprising nucleic acids, one or more of the nucleic acids may be incorporated into one or more vectors. In the above compositions according to the embodiments of the present invention and comprising nucleic acids, one or more of the nucleic acids may be incorporated into one or more cells.

[0140] The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used in a subject with a myeloid malignancy, such as AML, that would benefit from any of the myeloid malignancy antigen-specific antibodies or antigen binding fragments thereof described herein.

[0141] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and / or I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbatesAttorney Docket No.: 090723-1472570 MDA24-017PCT such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Allen (2012) Remington – The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington – The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the antibodies according to the present disclosure.

[0142] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition comprising one or both antibodies of the present disclosure can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington – The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising one or both antibodies of the present disclosure can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some instances, the cryo-preservative may be sucrose or trehalose. In some instances, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0143] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalationAttorney Docket No.: 090723-1472570 MDA24-017PCT or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0144] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5- 6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5. In an embodiment, the pH is 7.2.

[0145] In certain embodiments when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising one or both antibodies of the present disclosure in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which one or both antibodies of the present disclosure is formulated as a sterile, isotonic solution and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0146] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, one or both antibodies of the present disclosure can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising one or both antibodies of the present disclosure can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in International Application Publication No. WO / 1994 / 020069, which describes pulmonary delivery of chemically modified proteins.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0147] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, one or both antibodies of the present disclosure that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of one or both antibodies of the present disclosure. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0148] In certain embodiments, a pharmaceutical composition can involve an effective quantity of one or both antibodies of the present disclosure in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0149] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations one or both antibodies of the present disclosure in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio- erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, International Application Publication No. WO / 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No.3,773,919; U.S. Patent No.5, 594,091; U.S. Patent No.8,383,153; U.S. Patent No.4,767,628; International Application Publication No. WO1998043615, Calo, E. et al. (2015) Eur. Polymer J 65:252-267 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al. (1993) Biopolymers 22:547-556), poly (2-hydroxyethyl-Attorney Docket No.: 090723-1472570 MDA24-017PCT methacrylate) (Langer et al. (1981) J Biomed Mater Res.15: 167-277; and Langer (1982) Chem Tech 12:98-105), ethylene vinyl acetate (Hsu and Langer (1985) J Biomed Materials Res 19(4):445-460) or poly-D(-)-3-hydroxybutyric acid (European Patent No. EP0133988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent No. EP 036,676; and U.S. Patent Nos.4,619,794 and 4,615,885).

[0150] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0151] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0152] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0153] In certain embodiments, the effective amount of a pharmaceutical composition comprising one or both antibodies of the present disclosure to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which the antibodies of the present disclosure are used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.Attorney Docket No.: 090723-1472570 MDA24-017PCT

[0154] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the one or both antibodies of the present disclosure in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0155] In certain embodiments, the route of administration of the pharmaceutical composition is in accordance with known methods, such as the following: orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0156] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0157] In certain embodiments, one or both antibodies of the present disclosure can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of theAttorney Docket No.: 090723-1472570 MDA24-017PCT cells by a subject’s immune system or by other detrimental factors from the surrounding tissues. VIII. KITS

[0158] The therapeutics according to the present disclosure are ideally suited for the preparation of kits, which can be used for carrying out the therapeutic methods according to the present disclosure. The kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.

[0159] In some instances, one of the containers may comprise an Hu8F4 or 8F4 antibody (or their fragments or variants, such as corresponding or synthetic antibodies, or other variations described in the present disclosure), or a nucleic acid encoding the foregoing. Another one of the containers may comprise a suitable molecule that blocks the CD47-SIRPα axis. Some examples of molecules that block the CD47-SIRPα axis are anti-CD47 or anti- CD172 antibodies, such as, but not limited to a neutralizing anti-CD47 F(ab)’2 antibody according to the Examples of the present disclosure, Magrolimab, TJ011133, or TTI-622, or antigen binding fragments of such anti-CD47 antibodies, or variant of any thereof, such as corresponding or synthetic antibodies (or other variations described in the present disclosure). Some examples of molecules that block the CD47-SIRPα axis are shown in Table 3. In some embodiments, another one of the containers may comprise a nucleic acid encoding. a suitable molecule that blocks the CD47-SIRPα axis. The kit may also have one or more containers containing pharmaceutical excipients.

[0160] A kit may include a pharmaceutical package for delivery to doctors and cancer patients. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit may include instructions for use (e.g., for administering according to a method as described herein).

[0161] In some embodiments, a kit comprises unit dose forms of antibodies according to the present disclosure. In some embodiments, a kit further comprises unit dose forms of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0162] In one embodiment, a kit comprises antibodies according to the present disclosure in defined, therapeutically effective dose in a single unit dosage forms or as separate unitAttorney Docket No.: 090723-1472570 MDA24-017PCT doses. The dose and form of the unit dose (e.g., tablet, capsule, immediate release, delayed release, etc.) can be any doses or forms as described herein. In one embodiment, the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months. In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits containing single or multi-chambered pre-filled syringes are included. In certain embodiments, kits containing one or more containers of a formulation described in this disclosure are included. EXAMPLES

[0163] The following examples are offered to illustrate, but not to limit the claimed invention. Example 1: Loss of CD47 expression delays the growth of AML.

[0164] The experiments were conducted that showed CD47 KO in THP1 AML cells slowing the growth of THP1 AML cells in both NSG and NSGS mouse models. NSG and NSGS mice were inoculated with THP1 WT cells or THP1 KO cells. BLI images were taken weekly. The data showing mean+ / - SEM of treatment groups (n=5 mice per group) is shown in FIG.1. Survival was calculated using Log-rank (Mantel-Cox) test and is illustrated in FIG. 2. Growth of CD47KO THP1 in NSG mice was delayed compared to parental THP1, median survival of untreated mice increased from 58 days for THP1 to 99 days for CD47 KO THP1 (p=0.0017, n=5). Example 2: Elimination of AML by Hu8F4 is dependent on CD47.

[0165] The experiments were conducted that showed Hu8F4 eliminating CD47 knock out (KO) THP1 AML cells in NSGS mice. NSGS mice were used to allow for faster growth of human myeloid cells in order facilitate THP1 cell growth in vivo. NSGS mice were injected with THP1-CD47 KO or THP1-WT cells (5x10^5) and treated with Hu8F4 antibody (Hu8F4- 2) at 1 mg / kg (three times per week for a total of 25 doses starting on day 2 (n=5 per group) (FIG.3). BLI was accessed weekly. Each data point shown in FIG.4 represents Total Flux Signal of one mouse at a timepoint. Survival was calculated using Log-rank (Mantel-Cox) test (FIG.5). Treatment with Hu8F4-2 for 7 weeks (1 mg / kg three times per week; started on day 2), significantly reduced THP1 leukemia growth and fully eliminated CD47 KO THP1 cells, confirming the role of CD47 in the activity of Hu8F4 antibody.Attorney Docket No.: 090723-1472570 MDA24-017PCT Example 3: Elimination of AML by Hu8F4 is dependent on CD47.

[0166] Repeated experiments were conducted that showed the combination of Hu8F4 (Hu8F4-2) and anti-CD47 F(ab’)2 eliminating AML. To test if CD47 blockade plays a role in Hu8F4 anti-AML activity in vivo, NSG mice were injected with with U937-A2 (5x10^3) and subsequently treated with Hu8F4-2 + / - anti-CD47 F(ab)’2 antibody at 1 mg / kg (three times per week for a total of 10 doses starting on day 5 (n=5 per group) (FIG.6). FIG.7 illustrates the summary of BLI data. The data shown are mean+ / - SEM of treatment groups (n=5).

[0167] The survival was calculated using Log-rank (Mantel-Cox) test (FIG.8). Control mice, treated with PBS, succumbed to leukemia by day 28. Both Hu8F4 and anti-CD47 F(ab)’2single-agent treatment slowed leukemia growth, while the combination of Hu8F4 and anti-CD47 F(ab)’2 completely prevented outgrowth of the aggressive U937-A2 cells, indicating that blocking CD47 significantly increased the activity of Hu8F4 in vivo. Interestingly, CD47 KO U937-A2 did not engraft in NSG mice as expected, (n=5), confirming that CD47 expression is critically important for cell line engraftment in NSG. HuCD47 is known to bind to NSG mouse SIRP ^ with high affinity providing protection to the human cells from phagocytosis by mouse macrophages. Example 4: Hu8F4 and anti-CD47 antibodies act synergistically to eliminate AML by phagocytosis.

[0168] The experiments were conducted that showed that Hu8F4-induced phagocytosis by mouse bone marrow-derived monocytes (BMDM) required CD47 blockade. Fresh bone marrow cells from NSG mouse (effectors) were labelled with red fluorescent linker PKH26 (Sigma, red) and incubated in presence of mouse M-CSF in 96 well plate for 7 - 10 days. GFP-transfected U937-A2 (FIG.9A) and THP1 (FIG.9B) target cells were treated with Hu8F4-2 + / - a-CD47 F(ab’)2 for 30 minutes and added to BMDM. To confirm a critical role of CD47 in H8F4-mediated ADCP, CD47 CRISPR / Cas 9 CD47 KO cells U937-A2 (FIG. 9C) and THP1 (FIG.9D) cells were incubated with and without Hu8F4 and a-CD47 F(ab’)2 antibodies. Fluorescent and bright field images were taken at various time points The bar show object area of target cells at 96 hours, analyzed and calculated by using Gen5 software. Each bar shows medium calculated object area of green (target) cells in 4 images, captured from corresponding cells using x4 objective by Cytation 3, error bar shows SEM. B-F.2way ANOVA comparison was conducted, adjusted p values: * p < 0.05; ** p < 0.01; ***p <0.001; ****p<0.0001Attorney Docket No.: 090723-1472570 MDA24-017PCT Example 5: Hu8F4 antibody anti-CD47 antibody synergize efficient phagocytosis of AML by human macrophages.

[0169] The experiments were conducted showing anti-CD47 blockade acting synergistically with Hu8F4 antibody (Hu8F4-2 was used in the experiments) to promote efficient phagocytosis of AML. The experimental results are illustrated in FIG.10. To test whether CD47 is also important in Hu8F4-mediated phagocytosis by human effector cells, human macrophages were generated from CD14+ monocytes from healthy donors. The macrophages were incubated with target cells, U937-A2 or THP1, labelled with CFSE, at E:T =1 in presence of antibodies (10 µg / ml ) for 3 hours. Cells were stained with anti-CD11b antibody and analyzed by flowcytometry. Phagocytosis was calculated as percent of phagocyted CD11b+ CFSE+ target cells out of total CFSE+ target cells. The bars in FIG.10 show mean + SEM of each treatment (n=4). Two-way ANOVA with Tukey multiple comparison was used. A-CD47 antibody F(ab’)2 increased ADCP, induced buy Hu8F4, but had no effect on control antibody Herceptin.. B-F.2way ANOVA comparison was conducted, adjusted p values: * p < 0.05; ** p < 0.01; ***p <0.001; ****p<0.0001

[0170] Fluorescently labeled human macrophages were incubated with CFSE-labelled target cells (U937-A2 or THP1), that had been pre-treated with Hu8F4 antibody or isotype control antibody (Herceptin), at E:T=10, with or without anti-CD47 F(ab)’2. After 3 hours, Hu8F4-mediated phagocytosis, defined as the mean percentage of double-positive target cells, increased from mean 9.7% to 12.3% (n=4, p=0.199) for U937-A2, and from 12.25% to 17.58% (n=4, p=0.005) for THP1 when anti-CD47 F(ab)’2 blocking antibody was added. Anti-CD47 F(ab)’2alone and in the presence of control with Herceptin did not induce phagocytosis.

[0171] It is understood that the examples and embodiments described in the present disclosure are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Patens, patent applications, and publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. Example 6: Hu8F4 combined with either anti-SIRP⍺ or anti-CD47 mAb blockage mediates synergistic ADCP activity against HLA-A2+ THP1 human AML cells.

[0172] The experiments were conducted showing that Hu8F4 combined with either anti- SIRP⍺ or anti-CD47 mAb blockage mediated synergistic ADCP activity against HLA-A2+Attorney Docket No.: 090723-1472570 MDA24-017PCT THP1 human AML cells. The experimental results are illustrated in FIG.11. Fresh bone marrow cells from NSG mouse (effectors) were labelled with red fluorescent linker PKH26 (Sigma) and incubated in presence of mouse M-CSF (50 ng / ml, BioLegend) in 96 well plate for 7 days. GFP-transfected THP1 target cells were treated with CD47- blocking antibodies + / - Hu8F4 antibodies for 30 minutes and added to BMDM at 1:1 ratio. Fluorescent images were taken at Day 7, the number of green fluorescent target cells weas analyzed by Gen5 software. (7 days time point shown). Bar graphs show medium number of target cells in images, captured from corresponding cells using x20 objective by Cytation 3, error bar shows SEM. One representative experiment out of three shown. The experiment showed H8F4- induced ADCP of target cells in presence of differentiated NSG mouse bone marrow-derived macrophages (BMDM) required inactivation of CD47.

[0173] The experiments were conducted demonstrating that blockage with either anti- CD47 or anti-SIRP⍺ showe synergistic ADCP activity with h8F4. The experimental results are illustrated in FIG.12. Target AML cells, transfected with GFP, were mixed 1:1 with NSG mouse macrophages, labelled with pkH26, in presence of antibodies. On Day 5 fluorescent images were assessed, and target AML cells were counted. The bar graphs in FIG.12 show medium number of target cells in images, captured from corresponding cells using x20 (for MV411) and x4 (for THP1) objectives (2way ANOVA comparison, adjusted p values: * p < 0.05; ** p < 0.01; ***p <0.001; ****p<0.0001). One representative experiment out of three is illustrated in FIG.12.

[0174] The experiments were conducted demonstrating that Anti-SIRPα (anti-CD172a) mediated anti-AML activity in vivo only in combination with Hu8F4. The experimental design is illustrated in FIG.13. The experimental results are illustrated in FIG.14. Adult female NSG mice were injected intravenously with 1 million THP-1 Luc / GFP leukemia cells via tail vein. Starting at 7 days post-inoculation, groups of mice (n=5 per group) were treated with Hu8F4 at 1 mg / kg, anti-SIRPα (anti-CD172a) at 4 mg / kg or 20 mg / kg, or a combination of Hu8F4 and anti-CD172a, administered three times per week for a total of 10 doses. In vivo bioluminescence imaging was performed on the day of inoculation and weekly thereafter to monitor leukemia burden. The total signal in the region of interest (ROI), measured in photons per second, was quantified using Living Image software. The same ROI was applied to all sequential images captured during a single imaging session for each mouse. The leukemia burden was plotted against the number of days post-inoculation.

[0175] The experiments described above demonstrated that blocking CD47-SIRPa interaction with either F(ab’)2 anti-CD47 or with Magrolimab showed synergistic activityAttorney Docket No.: 090723-1472570 MDA24-017PCT when combined with h8F4 mAb. Also, blocking the CD47-SIRPa interaction with anti-mouse SIRPa showed synergistic activity in combination with h8F4 mAb.

[0176] It is understood that the examples and embodiments described in the present disclosure are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Patens, patent applications, and publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

[0177] Disclosed are materials, compositions, and ingredients that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.7$% / (^^ 3104488046378919tsit nsitsitsitsitsitsitsits; itslile;tslile;tslileononononon n n n n c1n c1nn c1gg g g gogogo o ode d odeie ode data a a a a agagagantmnagat ganaantantant t t t t tanan n n n nmgilrtm on omrprtm onmgilro747 a a a a a4747 7 7 7 7 7 7 arghttiba ai 7rghttiba ai 7rghttibD C D C D4C D4C D4 4 4 4 4C D C D C D C D C D CoraPed h 4niD CoraPed h 4niD CoraPe idhni- -ra -c - rnn s s ua dthL ehaiT -Ia ecP o Tnin eoca-a(h gomrse ei ur dtditmgec C ic cns e loa ccs gLo etiul ra oloeliaccniOCLLT-o ditL B htpnoi ei ocinBeHCoemi S h iapoBtSteuenne u s iciB,.o nevcB S do ded usniL rc ba eic hBnI nevc ecamtubaC)oG mmIepM- Ini j nnI s anI se eclMy tL gindeLi T-X si kAiGeKoCaiJMaZ oiBdt n laoLaHcit nnInI sci4C9J5T0;-i34 cS73n91e1- 18 G-2 3015 6 101 –918719F 13- 1- 02 7- 0022GD01- 3JT I1BI K5AuH MR1- CHSLTZA -BX3- HIBIba9 m 5ilb b0r-ic 2 a ba a apmimi mil30140S 0n0-ozlp lafor561021790 22eC matu ga9-LGMIeLeL giLMI-R1- -T 0- 3- MHSLZAB XHIBI^^215 9 7 1663202 32 1 12259 1111 1 9R0202 2 12 2020R202da u u pe ed ga lu ec aed ga inec1 ad ga lusin 4m a oDsa odo flrp nC / sin sin sinlu ecstay otc mn bb-soh 0 a olert274gocy otFc mn odea0 a ga e dty otn bo onieC;C;dtmagois αPo o o ber mnac m9 algga tortoh274tngi roh174 or rp ottsnain e1m 1re ni uddrfcRI gS,agagaolrtntntngocFte n pDDn sgi pDDamalotipDDnot0l o mtiniip niotiF-ni e a a a natsiroiatmCCum Tcyl nnmCC7A -Be ;rmm gotmIaoyl ngga-Be ;r 4 rgotDghota bCrPe idhmC ynl nC;u prmec1- uldgoat urecto E; brdi biα ei-BegotmIercBmnaerrpotca hPni RtkIoc 7Srp ol47b D47umnC D4C DmmoCmIagga- c ponI nc - ip-psciB oi eBo nI oioi t-g umCy sci B BesoecTindtLMJuSgoty lue gocol co pndt cndatrenO iu o pnoOLOL haepnouCah oghnzhacrI ehsicxn eOnouCenoTuCm mommrng et Til m m uilCmIaahh nSaHoiBdtLGeTx XELmrAmIamrhmIal hirTcnI- eR74 ma6 U0N 0r37037A1e0 41;-1RU841 11h-1- 2tMOP1 04- 71-I3-A; 1-0-0- 26U4XLM M -IOMICGSNA01AMIMITT6t0p1371ee30 0 4 0 4ca1011gurm- 6M -T 21- 81- 11pr-0- 2M M6- DaNMIMJGSG-TLoXvEMIIMITT^^RTC / / sagba lr -t s tng c gayh bionaeni uenao Fattnte hnimm mgil soserogroiat 7 numsiaTc 4n 7 mesnear h ryTsattiA Dmmo 4A CmIaggaDNaC Drgeigota;r a bfpErPedoth iphniscituepsarc ei enhnIaTxrp mtnmuoilelCiC irTcnIpEe maNre2210h0t6-I-xOTTRR2 1 ge2urma6 0-I 0-xD NTTRR^^reggrnnieg g ti gnnitniet reg itiel gnnit52mhtwA(mpa h onoto3380222- yht r snasrecaio-ll tcnan no nn0o / o / y- a dn- imwyskohstndersotocpe-c-eit 2Ns’Denimode yrom-derono s’nimovB a C vi k h cntcut as tcNl k ha giln gda ddpeipa 02Lphtig pa a dmy mpa arleg pamna omtalrfHylaPererDeCvitiHR tN / R i swo dpomvr HyLda fielrol sroohlpefRecR- dBom HyL-il-i b ;8;48ba raxputi a 4G-ba14X1miozr;mil 9FmiLXLxumbaba pat5uxuA Atirelm melHtir)-l-nijcnnc ol- co n Ie d nI nc oiaitn scveae sOnOBb T(dteev igSlicXLy Ag XoLy Agah LoM,.oolyt G;n- cIeToC nnI oirBoF ceniIcS81814 9 9 221 4 038 1 513 4 3 3010396753 3 390 0 03 2T T0 0C CTCTCTC N N N N N 4 8483314X1118 G-9LX 181FALA0J- TI5BI uH ^^n - oitm / aclyLadyersoe yrtsi di aeizw msdrote ya cderomoiidaltle mL noCsBpacslarf ocog éSor n ns tchdd eitav parpI eDC-Thp LDeRey nRMuFnnaySapd aaler fermyll;o12ba1co1t262o6-I 6r-ITTmiTx-I 1uT 22T 62-I6-IP T.1tir.2TTTTscisciscscrtoutitits eu u unm ou parmeuparmeupeamu papill SireThTcilnleI irThTcilnlreI irThTcilnl IirreThc TnIre818 8 3 b51m 356338060u6N66 9 326028 50203TC TCTCT0CTC N N N N N e maN g121 1 262 2 2ur-I6-I6-I6-IDTTTTTTTT^^

Claims

Attorney Docket No.: 090723-1472570 MDA24-017PCT WHAT IS CLAIMED IS:

1. A method of treating a myeloid malignancy in a subject, comprising: (a) administering to the subject a therapeutically effective amount of a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) administering to the subject a therapeutically effective amount of (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

2. The method of claim 1, wherein the second antibody is an anti-CD47 or an anti- CD172a antibody.

3. The method of claim 1, wherein (b)(2) comprises one or more molecules shown in Table 3.

4. The method of claim 1 or 2, wherein the second antibody is a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab, TJ011133, or TTI-622.

5. The method of any one of claims 1 to 4, wherein the myeloid malignancy is characterized by expression of HLA-A2 allele.

6. The method of any one of claims 1 to 5, wherein the myeloid malignancy is AML, MDS, or MPN.

7. The method of any one of claims 1 to 6, wherein the myeloid malignancy is a refractive myeloid malignancy.Attorney Docket No.: 090723-1472570 MDA24-017PCT 8. The method of any one of claims 1 to 7, wherein the patient has relapsed.

9. The method of any one of claims 1 to 8, wherein steps (a) and (b) are performed simultaneously.

10. The method of any one of claims 1 to 8, wherein steps (a) and (b) are not performed simultaneously.

11. The method of any one of claims 1 to 10, further comprising administering at an additional anti-cancer therapy to the patient.

12. The method of claim 11, wherein the additional anti-cancer therapy is a chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy.

13. A pharmaceutical composition, comprising: (a) a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

14. The pharmaceutical composition of claim 13, wherein the second antibody is an anti- CD47 or an anti-CD172a antibody.

15. The pharmaceutical composition of claim 13, wherein (b)(2) comprises one or more molecules shown in Table 3.Attorney Docket No.: 090723-1472570 MDA24-017PCT 16. The pharmaceutical composition of claim 13 or 14, wherein the second antibody is a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab, TJ011133, or TTI-622.

17. The pharmaceutical composition of any one of claims 13 to 16, wherein (a) is the antigen binding fragment of the first antibody.

18. The pharmaceutical composition of any one of claims 13 to 17, wherein (b)(1) is the antigen binding fragment of the second antibody.

19. The pharmaceutical composition of claim 17 or 18, wherein the antigen binding fragment of the first antibody and / or the antigen binding fragment of the second antibody is a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.

20. A pharmaceutical composition, comprising: (a) a nucleic acid encoding a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and, (b) a nucleic acid encoding (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

21. The pharmaceutical composition of claim 20, wherein the second antibody is an anti- CD47 or an anti-CD172a antibody.

22. The pharmaceutical composition of claim 20, wherein (b)(2) comprises one or more molecules shown in Table 3.Attorney Docket No.: 090723-1472570 MDA24-017PCT 23. The pharmaceutical composition of claim 20 or 21, wherein the second antibody is a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab, TJ011133, or TTI-622.

24. The pharmaceutical composition of any one of claims 20 to 23, wherein (a) is the antigen binding fragment of the first antibody.

25. The pharmaceutical composition of any one of claims 20 to 24, wherein (b)(1) is the antigen binding fragment of the second antibody.

26. The pharmaceutical composition of claim 24 or 25, wherein the antigen binding fragment of the first antibody and / or the antigen binding fragment of the second antibody is a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody.

27. The pharmaceutical composition of any one of claims 20 to 26, wherein (a) and (b) are incorporated in one or more vectors.

28. The pharmaceutical composition of any one of claims 20 to 26, wherein (a) and (b) are incorporated in one or more cells.

29. A method of treating a myeloid malignancy in a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 1 to 28.

30. The method of claim 29, wherein the myeloid malignancy is characterized by expression of HLA-A2 allele.

31. The method of claim 29 or 30, wherein the myeloid malignacy is AML, MDS, or MPN.

32. The method of any one of claims 29 to 31, wherein the myeloid malignancy is a refractive myeloid malignancy.

33. The method of any one of claims 29 to 32, wherein the patient has relapsed.Attorney Docket No.: 090723-1472570 MDA24-017PCT 34. A kit comprising: (a) a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is Hu8F4 or 8F4 antibody; and (b) (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

35. The kit of claim 34, wherein the second antibody is an anti-CD47 or an anti-CD172a antibody.

36. The kit of claim 34, further comprising one or more of: (c) a pharmaceutical package; (d) a chemotherapeutic agent; (e) a cytotoxic agent; (f) a radiotherapeutic agent, or (g) an immunotherapeutic agent.

37. The kit of any one of claims 34 to 36, wherein the second antibody is a neutralizing anti-CD47 F(ab)’2 antibody, Magrolimab, TJ011133, or TTI-622.

38. The kit of any one of claims 34 to 37, wherein (b)(2) comprises one or more molecules shown in Table 3.Attorney Docket No.: 090723-1472570 MDA24-017PCT 39. A method of treating a myeloid malignancy in a subject, comprising: (a) administering to the subject a therapeutically effective amount of a first antibody, an antibody corresponding to the first antibody, a variant of the first antibody or the antibody corresponding to the first antibody, or an antigen binding fragment of the first antibody or the antibody corresponding to the first antibody, wherein the first antibody is capable of specifically binding to SEQ ID NO:45; and (b) administering to the subject a therapeutically effective amount of (1) a second antibody, an antibody corresponding to the second antibody, a variant of the second antibody or the antibody corresponding to the second antibody, or an antigen binding fragment of the second antibody or the antibody corresponding to the second antibody, wherein the second antibody is an antibody capable of blocking CD47-SIRPα axis, or (2) a therapeutic capable of blocking CD47-SIRPα axis.

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