Compositions and methods for inducing phagocytosis

Abstract

Compositions and methods that are useful for inducing phagocytosis of a cancer cell and / or increasing killing of a cancer cell (e.g., by an immune cell) by contacting the cancer cell with an antibody capable of selectively binding to a CD43 protein expressed on the surface of cancer cells. In various embodiments, the CD43 protein is not expressed on the surface of a healthy cell.

Description

[0001] Atorney Docket No.: 167741-053601 / PCT

[0002] Electronic Deposit Date: October 27, 2025

[0003] COMPOSITIONS AND METHODS FOR INDUCING PHAGOCYTOSIS

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 712,752, filed October 28, 2024, the entire contents of which are incorporated herein by reference.

[0006] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grants No. CA242457, CA009172, and HL116324 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] During the past few decades, research has provided breakthroughs that have enhanced our understanding of the mechanisms and pathways that regulate the immune system’s response to cancer. However, despite these advances, obstacles still exist for the field of cancer immunotherapy. These include, among other things, the inability to predict treatment efficacy and patient response; the need for additional biomarkers; the development of resistance to cancer immunotherapies; the lack of clinical study designs that are optimized to determine efficacy; and high treatment costs. Thus, there is a present need for improved immunotherapies.

[0010] SUMMARY

[0011] As described below, the present disclosure features compositions and methods that are useful for inducing phagocytosis and / or increasing killing (e.g., by an immune cell) of a cancer cell by contacting the cancer cell with an antibody capable of selectively binding to a CD43 protein expressed on the surface of cancer cells. In various embodiments, the CD43 protein is not expressed on the surface of a healthy cell.

[0012] In one aspect, the disclosure provides a method for increasing phagocytosis and / or killing of a cancer cell by an immune cell. The method involves contacting the cancer cell with an antibody that selectively binds to a CD43 protein expressed on the surface of the cancer cell.

[0013] In another aspect, the disclosure provides a method for increasing cancer cell phagocytosis and / or killing by immune cells in a subject in need thereof. The method involves Atorney Docket No.: 167741-053601 / PCT

[0014] Electronic Deposit Date: October 27, 2025 administering to the subject an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell, thereby increasing phagocytosis of the cancer cell. The cancer cell is characterized as expressing CD43.

[0015] In another aspect, the disclosure provides a method for treating a CD43-positive cancer in a selected subject. The method involves administering to the subject an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell, thereby treating cancer in the subject. The subject is selected as having a cancer expressing CD43.

[0016] In another aspect, the disclosure provides a kit containing an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell and instructions for using the antibody in the method of any aspect of the disclosure, or embodiments thereof.

[0017] In another aspect, the disclosure provides a method for identification of genes modulating phagocytosis of a cell by a macrophage. The method involves a) editing a cell to knock-out expression of a gene and yield an edited cell. The method also involves b) co-culturing the cell with macrophages to yield a co-culture. The method further involves c) using nucleotide sequencing to determine whether the edited cell is enriched or depleted in a macrophage fraction of the co-culture. Enrichment or depletion of the edited cell in the macrophage fraction relative to a reference cell identifies the gene as modulating phagocytosis.

[0018] In any aspect of the disclosure, or embodiments thereof, the cancer cell is a myeloma cell. In any aspect of the disclosure, or embodiments thereof, the cancer cell is a lymphoma cell. In any aspect of the disclosure, or embodiments thereof, the cancer cell expresses CD43. In any aspect of the disclosure, or embodiments thereof, the cancer cell is a leukemia cell.

[0019] In any aspect of the disclosure, or embodiments thereof, the phagocytosis is macrophage- mediated phagocytosis.

[0020] In any aspect of the disclosure, or embodiments thereof, the antibody selectively binds a glycoform of CD43 expressed on the surface of the cancer cell. In any aspect of the disclosure, or embodiments thereof, the antibody is a VHH antibody.

[0021] In any aspect of the disclosure, or embodiments thereof, the nucleotide sequencing is single-cell sequencing.

[0022] In any aspect of the disclosure, or embodiments thereof, expression of the gene is knocked out using CRISPR. In any aspect of the disclosure, or embodiments thereof, the method involves contacting the cell with a library of single guide RNAs (sgRNAs), where each sgRNA targets a unique gene for knock-out. In any aspect of the disclosure, or embodiments thereof, the cells express a polynucleotide programmable endonuclease. Atorney Docket No.: 167741-053601 / PCT

[0023] Electronic Deposit Date: October 27, 2025

[0024] In any aspect of the disclosure, or embodiments thereof, the cells are cancer cells. In any aspect of the disclosure, or embodiments thereof, the cancer cells are leukemia cells.

[0025] In any aspect of the disclosure, or embodiments thereof, a) further involves selecting for an edited cell before co-culturing the edited cell with macrophages.

[0026] In any aspect of the disclosure, or embodiments thereof, the immune cell is a natural killer (NK) cell or a T cell. In any aspect of the disclosure, or embodiments thereof, the immune cell is a macrophage.

[0027] Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the embodiments of the disclosure will be apparent from the detailed description, and from the claims.

[0028] Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0030] By “agent” is meant a small molecule compound, polypeptide, nucleic acid molecule, or a functional fragment or portion thereof. In various embodiments, an agent is an anti-CD43 antibody or an antigen binding fragment thereof.

[0031] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0032] By “alteration” is meant a change in the structure, expression levels or activity of a polynucleotide or polypeptide as detected by standard art known methods such as those described herein. The alteration can be an increase or a decrease. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels.

[0033] By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0034] As used herein, the term “antibody (Ab)” refers to an immunoglobulin molecule that specifically recognizes, binds to, or is immunologically reactive with, a particular antigen. Antibodies include polyclonal, monoclonal, genetically and molecularly engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), nanobodies (also called single chain antibodies (e.g., scFv antibodies), camelid species antibodies, or variable heavy domain of heavy chain (VHH) antibodies) and antigen-binding fragments of such antibodies, including e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. As is appreciated by one having skill in the art, the basic form of an antibody molecule is Y-shaped, with two, identical antigen-binding sites, one at the tip of each arm of the Y. Unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as, for example, without limitation, Fab, Fab', and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (see, Wahl et al., J. Nucl. Med. 24:316, 1983; incorporated herein by reference). An F(ab')2 fragment can be split into two Fab' fragments using mild reduction conditions.

[0035] Antibodies, antibody binding fragments, antibody binding domains, or antibody binding portions, particularly, functional antibody fragments, domains, or portions, can be synthetically or recombinantly produced. As would be understood by the skilled practitioner in the art, antibody binding fragments, domains, or portions are regions of antibody molecules that bind to antigens. In an embodiment, the antibody is a nanobody (also called a single chain antibody (e.g., scFv antibody), camelid species antibody, or variable heavy domain of heavy chain (VHH) antibody), or an antigen-binding fragment, domain, or portion of the nanobody. Methods and procedures for producing antibodies, antigen-binding fragments, domains, or portions of antibodies, and nanobodies are well known in the art and include, without limitation, phageantibody technology (Knappik et al., J. Mol. BioL, 296:57-86, 2000), technologies involving screening of B-cell derived DNA libraries, e.g., as described in WO 91 / 17271 and WO 92 / 01047, ribosome display technology (see, e.g., Rouet, et al, “Next-Generation Sequencing of Antibody Display Repertoires,” Frontiers in Immunology, volume 9, article 118 (2018), doi: Atorney Docket No.: 167741-053601 / PCT

[0036] Electronic Deposit Date: October 27, 2025

[0037] 10.3389 / ftmmu.2018.00118, the disclosure of which is incorporated herein by reference in its entirety for all purposes), and yeast display technology. Briefly, such methods involve the production of phage libraries in which phage members of the library display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. The phage displaying antibodies are selected by affinity enrichment for binding to a selected protein. Antibodies can also be produced using trioma methodology (e.g., Oestberg et al., Hybridoma 2:361-367, 1983; U.S. Patent Nos. 4,634,664; 4,634,666). Antibodies can also be isolated and purified from any cell that produces and expresses the antibodies, including host cells that have been transfected or transformed with antibody-encoding expression constructs. The host cells can be cultured under conditions in which the antibodies are expressed. For isolation and purification, an antibody can be separated from other cellular components, such as certain proteins, carbohydrates, or lipids, using methods known in the art. Without limitation, such methods include size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. The purity of an antibody preparation can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis. A preparation of purified antibodies can contain more than one type of antibody. Alternatively, an antibody can be produced using synthetic chemical procedures to synthesize its amino acid sequence, such as by direct peptide synthesis using solidphase techniques (e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963; Roberge et al., Science, 269:202-204, 1995). Protein synthesis can be performed using manual or automation techniques. In some cases, fragments of antibodies can be separately synthesized and combined using chemical methods to produce a full-length molecule.

[0038] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, Fab’, F(ab')2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and Cm 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 Cm domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining Atorney Docket No.: 167741-053601 / PCT

[0039] Electronic Deposit Date: October 27, 2025 region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art. In some embodiments, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, scFab, Fv, rlgG, and scFv fragments) of a biparatopic antibody, which are joined by a synthetic linker, are provided.

[0040] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, Fab’, F(ab')2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and Cm 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 Cm domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or Atorney Docket No.: 167741-053601 / PCT

[0041] Electronic Deposit Date: October 27, 2025 chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art. In some embodiments, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, scFab, Fv, rlgG, and scFv fragments) of a biparatopic antibody, which are joined by a synthetic linker, are provided.

[0042] By “anti-cluster of differentiation 43 (CD43) antibody” is meant a polypeptide capable of specifically binding a CD43 polypeptide, or a fragment thereof. In an embodiment, the CD43 antibody or antigen-binding fragment thereof specifically binds a post-translationally modified form of CD43, such as a CD43 polypeptide that has been glycosylated (e.g., sialylated). In another embodiment, an anti-CD43 antibody selectively binds a CD43 glycoform expressed on the surface of a tumor cell.

[0043] By “anti-cluster of differentiation 43 (CD43) antibody polynucleotide” is meant a polynucleotide encoding an anti-CD43 antibody.

[0044] By “cluster of differentiation 43 (CD43) polypeptide” or “sialophorin (SPN) polypeptide” is meant a CD43 protein having at least 85% amino acid sequence identity to GenBank® Accession No. CAA36294.1, which is provided below, and capable of modulating phagocytosis of at least a portion of a cell. >CAA36294.1 sialophorin [Homo sapiens] MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSA LPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSH TVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATD SLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGML PVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVG GSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAV DAPAPDEPEGGDGAAP

[0045] By “cluster of differentiation 43 (CD43) polynucleotide” or “sialophorin (SPN) polynucleotide” is meant a nucleic acid molecule encoding a CD43 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD43 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD43 expression. An exemplary CD43 gene sequence is provided at Ensemble Accession No. ENSG00000197471. An exemplary CD43 nucleotide sequence from Homo sapiens is provided below (GenBank® Accession No.: X52075.1): >X52075.1 : 1528-2730 Human gene for sialophorin (CD43) Atorney Docket No.: 167741-053601 / PCT

[0046] Electronic Deposit Date: October 27, 2025

[0047] ATGGCCACGCTTCTCCTTCTCCTTGGGGTGCTGGTGGTAAGCCCAGACGCTCTGGGGAGCACAA CAGCAGTGCAGACACCCACCTCCGGAGAGCCTTTGGTCTCTACTAGCGAGCCCCTGAGCTCAAA GATGTACACCACTTCAATAACAAGTGACCCTAAGGCCGACAGCACTGGGGACCAGACCTCAGCC CTACCTCCCTCAACTTCCATCAATGAGGGATCCCCTCTTTGGACTTCCATTGGTGCCAGCACTG GTTCCCCTTTACCTGAGCCAACAACCTACCAGGAAGTTTCCATCAAGATGTCATCAGTGCCCCA GGAAACCCCTCATGCAACCAGTCATCCTGCTGTTCCCATAACAGCAAACTCTCTAGGATCCCAC ACCGTGACAGGTGGAACCATAACAACGAACTCTCCAGAAACCTCCAGTAGGACCAGTGGAGCCC CTGTTACCACGGCAGCTAGCTCTCTGGAGACCTCCAGAGGCACCTCTGGACCCCCTCTTACCAT GGCAACTGTCTCTCTGGAGACTTCCAAAGGCACCTCTGGACCCCCTGTTACCATGGCAACTGAC TCTCTGGAGACCTCCACTGGGACCACTGGACCCCCTGTTACCATGACAACTGGCTCTCTGGAGC CCTCCAGCGGGGCCAGTGGACCCCAGGTCTCTAGCGTAAAACTATCTACAATGATGTCTCCAAC GACCTCCACCAACGCAAGCACTGTGCCCTTCCGGAACCCAGATGAGAACTCACGAGGCATGCTG CCAGTGGCTGTGCTTGTGGCCCTGCTGGCGGTCATAGTCCTCGTGGCTCTGCTCCTGCTGTGGC GCCGGCGGCAGAAGCGGCGGACTGGGGCCCTCGTGCTGAGCAGAGGCGGCAAGCGTAACGGGGT GGTGGACGCCTGGGCTGGGCCAGCCCAGGTCCCTGAGGAGGGGGCCGTGACAGTGACCGTGGGA GGGTCCGGGGGCGACAAGGGCTCTGGGTTCCCCGATGGGGAGGGGTCTAGCCGTCGGCCCACGC TCACCACTTTCTTTGGCAGACGGAAGTCTCGCCAGGGCTCCCTGGCGATGGAGGAGCTGAAGTC TGGGTCAGGCCCCAGCCTCAAAGGGGAGGAGGAGCCACTGGTGGCCAGTGAGGATGGGGCTGTG GACGCCCCAGCTCCTGATGAGCCCGAAGGGGGAGACGGGGCTGCCCCTTAA

[0048] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments.

[0049] As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616, 1977 and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; by Chothia et al., (J Mol. Biol. 196:901-917, 1987), and by MacCallum et al., (J. Mol. Biol. 262'.'l32-'l 45 , 1996) where the definitions include overlapping or subsets of amino acid residues when compared against each Atorney Docket No.: 167741-053601 / PCT

[0050] Electronic Deposit Date: October 27, 2025 other. In certain embodiments, the term “CDR” is a CDR as defined by Kabat based on sequence comparisons. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. In various aspects and embodiments, antibodies comprising modifications in these hybrid hypervariable positions are provided. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a beta-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see, Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.

[0051] By “core 1 synthetase, glycoprotein-n-acetylgalactosamine 3-beta-galactosyltransferase, 1 (C1GALT1) polypeptide” is meant a protein having glycosyltransferase activity with at least 85% identity to GENBANK™ Accession No. AAF81981.1, which is provided below, or a functional fragment thereof.

[0052] >AAF81981.1 corel UDP-galactose:N-acetylgalactosamine-alpha-R beta 1,3- galactosyltransferase [Homo sapiens] MASKSWLNFLTFLCGSAIGFLLCSQLFSILLGEKVDTQPNVLHNDPHARHSDDNGQNHLEGQMN FNADSSQHKDENTDIAENLYQKVRILCWVMTGPQNLEKKAKHVKATWAQRCNKVLFMSSEENKD FPAVGLKTKEGRDQLYWKTIKAFQYVHEHYLEDADWFLKADDDTYVILDNLRWLLSKYDPEEPI YFGRRFKPYVKQGYMSGGAGYVLSKEALKRFVDAFKTDKCTHSSSIEDLALGRCMEIMNVEAGD SRDTIGKETFHPFVPEHHLIKGYLPRTFWYWNYNYYPPVEGPGCCSDLAVSFHYVDSTTMYELE YLVYHLRPYGYLYRYQPTLPERILKEISQANKNEDTKVKLGNP

[0053] By “core 1 synthetase, glycoprotein-n-acetylgalactosamine 3-beta-galactosyltransferase, 1 (C1GALT1) polynucleotide” is meant a polynucleotide encoding a C1GALT1 polypeptide. Atorney Docket No.: 167741-053601 / PCT

[0054] Electronic Deposit Date: October 27, 2025

[0055] Exemplary C1GALT1 polynucleotide sequences are provided at GENBANK™ Accession No. AF155582.1, which is provided below, and at ENSEMBL™ Accession No. ENSG00000106392. >AF155582.1 :63-1154 Homo sapiens corel UE)P-galactose:N-acetylgalactosamine-alpha-R beta 1,3 -galactosyltransferase (C1GALT1) mRNA, complete cds ATGGCCTCTAAATCCTGGCTGAATTTTTTAACCTTCCTCTGTGGATCAGCAATAGGATTTCTTT

[0056] TATGTTCTCAGCTATTTAGTATTTTGTTGGGAGAAAAGGTTGACACCCAGCCTAATGTTCTTCA TAATGATCCTCATGCAAGGCATTCAGATGATAATGGACAGAATCATCTAGAAGGACAAATGAAC TTCAATGCAGATTCTAGCCAACATAAAGATGAGAACACAGACATTGCTGAAAACCTCTATCAGA AAGTTAGAATTCTTTGCTGGGTTATGACCGGCCCTCAAAACCTAGAGAAAAAGGCCAAACACGT CAAAGCTACTTGGGCCCAGCGTTGTAACAAAGTGTTGTTTATGAGTTCAGAAGAAAATAAAGAC TTCCCTGCTGTGGGACTGAAAACCAAAGAAGGCAGAGATCAACTATACTGGAAAACAATTAAAG CTTTTCAGTATGTTCATGAACATTATTTAGAAGATGCTGATTGGTTTTTGAAAGCAGATGATGA CACGTATGTCATACTAGACAATTTGAGGTGGCTTCTTTCAAAATACGACCCTGAAGAACCCATT TACTTTGGGAGAAGATTTAAGCCTTATGTAAAGCAGGGCTACATGAGTGGAGGAGCAGGATATG TACTAAGCAAAGAAGCCTTGAAAAGATTTGTTGATGCATTTAAAACAGACAAGTGTACACATAG TTCCTCCATTGAAGACTTAGCACTGGGGAGATGCATGGAAATTATGAATGTAGAAGCAGGAGAT TCCAGAGATACCATTGGAAAAGAAACTTTTCATCCCTTTGTGCCAGAACACCATTTAATTAAAG GTTATCTACCTAGAACGTTTTGGTACTGGAATTACAACTATTATCCTCCTGTAGAGGGTCCTGG TTGCTGCTCTGATCTTGCAGTTTCTTTTCACTATGTTGATTCTACAACCATGTATGAGTTAGAA TACCTCGTTTATCATCTTCGTCCATATGGTTATTTATACAGATATCAACCTACCTTACCTGAAC GTATACTAAAGGAAATTAGTCAAGCAAACAAAAATGAAGATACAAAAGTGAAGTTAGGAAATCC TTGA

[0057] By “ClGALTl-speciftc chaperone 1 (C1GALT1C1) polypeptide” is meant a protein having chaperone activity with at least 85% identity to GENBANK™ Accession No. AAH1 1930.1, which is provided below, or a functional fragment thereof.

[0058] >AAH11930.1 ClGALTl-speciftc chaperone 1 [Homo sapiens]

[0059] MLSESSSFLKGVMLGSIFCALITMLGHIRIGHGNRMHHHEHHHLQAPNKEDILKISEDERMELS KSFRVYCI ILVKPKDVSLWAAVKETWTKHCDKAEFFSSENVKVFESINMDTNDMWLMMRKAYKY AFDKYRDQYNWFFLARPTTFAI IENLKYFLLKKDPSQPFYLGHTIKSGDLEYVGMEGGIVLSVE SMKRLNSLLNIPEKCPEQGGMIWKISEDKQLAVCLKYAGVFAENAEDADGKDVFNTKSVGLSIK EAMTYHPNQVVEGCCSDMAVTFNGLTPNQMHVMMYGVYRLRAFGHIFNDALVFLPPNGSDND

[0060] By “ClGALTl-speciftc chaperone 1 (C1GALT1C1) polynucleotide” is meant a polynucleotide encoding a C1GALT1C1 polypeptide. Exemplary C1GALT1C1 polynucleotide sequences are provided at GENBANK™ Accession No. BC011930.2, which is provided below, and at ENSEMBL™ Accession No. ENSG00000171155. Atorney Docket No.: 167741-053601 / PCT

[0061] Electronic Deposit Date: October 27, 2025

[0062] >BC011930.2:59-1015 Homo sapiens Cl GALT 1 -specific chaperone 1, mRNA (cDNA clone MGC: 19947 IMAGE:3355639), complete cds

[0063] ATGCTTTCTGAAAGCAGCTCCTTTTTGAAGGGTGTGATGCTTGGAAGCATTTTCTGTGC TTTGATCACTATGCTAGGACACATTAGGATTGGTCATGGAAATAGAATGCACCACCATGAGCAT CATCACCTACAAGCTCCTAACAAAGAAGATATCTTGAAAATTTCAGAGGATGAGCGCATGGAGC TCAGTAAGAGCTTTCGAGTATACTGTATTATCCTTGTAAAACCCAAAGATGTGAGTCTTTGGGC TGCAGTAAAGGAGACTTGGACCAAACACTGTGACAAAGCAGAGTTCTTCAGTTCTGAAAATGTT AAAGTGTTTGAGTCAATTAATATGGACACAAATGACATGTGGTTAATGATGAGAAAAGCTTACA AATACGCCTTTGATAAGTATAGAGACCAATACAACTGGTTCTTCCTTGCACGCCCCACTACGTT TGCTATCATTGAAAACCTAAAGTATTTTTTGTTAAAAAAGGATCCATCACAGCCTTTCTATCTA GGCCACACTATAAAATCTGGAGACCTTGAATATGTGGGTATGGAAGGAGGAATTGTCTTAAGTG TAGAATCAATGAAAAGACTTAACAGCCTTCTCAATATCCCAGAAAAGTGTCCTGAACAGGGAGG GATGATTTGGAAGATATCTGAAGATAAACAGCTAGCAGTTTGCCTGAAATATGCTGGAGTATTT GCAGAAAATGCAGAAGATGCTGATGGAAAAGATGTATTTAATACCAAATCTGTTGGGCTTTCTA TTAAAGAGGCAATGACTTATCACCCCAACCAGGTAGTAGAAGGCTGTTGTTCAGATATGGCTGT TACTTTTAATGGACTGACTCCAAATCAGATGCATGTGATGATGTATGGGGTATACCGCCTTAGG GCATTTGGGCATATTTTCAATGATGCATTGGTTTTCTTACCTCCAAATGGTTCTGACAATGACT GA

[0064] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In some embodiments, the analyte is an antigen, epitope, or fragment thereof. In one embodiment, the term “detect” refers to detecting antibody binding to an agent of interest.

[0065] By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0066] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasias, such as a leukemia. In some embodiments, the neoplasia is a lymphoma or a myeloma. The cells of a neoplasia may express CD43.

[0067] By “effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the Atorney Docket No.: 167741-053601 / PCT

[0068] Electronic Deposit Date: October 27, 2025 subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

[0069] By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. In embodiments, portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0070] As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), singlechain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.

[0071] As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., Cm, Cm, Cm), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell (e.g., yeast) that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 1998 / 46645; WO 1998 / 50433; WO 1998 / 24893; WO 1998 / 16654; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; incorporated by reference herein. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0072] As used herein, the term “humanized” antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FR regions may also be those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370 to Queen et al.; EP239400; PCT publication WO 91 / 09967; U.S. Pat. No. 5,225,539; EP592106; and EP519596; incorporated herein by reference.

[0073] “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0074] By “immune cell” is meant a cell of the immune system capable of generating an immune response. Non-limiting examples of immune cells include B cells, dendritic cells, eosinophils, macrophage, mast cells, monocytes, natural killer (NK) cells, neutrophils, plasma cells, and T cells (e.g., cytotoxic T cells, regulatory T cells, or helper T cells). In some embodiments, the immune cell is a NK cell or a T cell.

[0075] By “increase” is meant to alter positively relative to a reference. An increase may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0076] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from an original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are Atorney Docket No.: 167741-053601 / PCT

[0077] Electronic Deposit Date: October 27, 2025 typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0078] By “isolated polynucleotide” is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0079] By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, and or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0080] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a developmental state, condition, disease, or disorder.

[0081] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0082] By “neoplasia” is meant a disease or disorder characterized by excess proliferation or reduced apoptosis. In embodiments, a neoplasia is a cancer or tumor. Illustrative neoplasms include breast cancer, esophageal cancer, head-and-neck cancer, pancreatic cancer, skin cancer, colorectal cancer, hepatocellular cancer, bladder cancer, bile duct cancer, luminal and nonluminal bladder cancer, basal bladder cancer, muscle-invasive bladder cancer, and non-muscle- invasive bladder cancer, pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic Atorney Docket No.: 167741-053601 / PCT

[0083] Electronic Deposit Date: October 27, 2025 leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin’s disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, liver cancer, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In embodiments, the neoplasia may be colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD), stomach cancer, and uterine corpus endometrial carcinoma (UCEC). In embodiments, the neoplasia may be a liquid tumor such as, for example, leukemia or lymphoma. In embodiments, the cancer is a colon, kidney, lung, pancreatic, renal (e.g., renal cell carcinoma or clear renal cell carcinoma), or skin cancer (e.g., a melanoma).

[0084] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing, a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0085] By “polynucleotide” or “nucleic acid molecule” is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced stability in the presence of nucleases.

[0086] By “polypeptide” or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post- translational modification is glycosylation or phosphorylation. In various embodiments, Atorney Docket No.: 167741-053601 / PCT

[0087] Electronic Deposit Date: October 27, 2025 conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some aspects the disclosure embraces sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.

[0088] By “reduce” is meant to alter negatively relative to a reference. A reduction may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0089] By “reference” is meant a standard or control condition. In some embodiments, a reference is a healthy control cell. In some instances, a reference is a cell that has not been contacted with an anti-CD43 antibody. A reference may be a CD43 glycoform that is or is not detectably expressed on the surface of a target cell.

[0090] A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer thereabout or therebetween.

[0091] As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an Atorney Docket No.: 167741-053601 / PCT

[0092] Electronic Deposit Date: October 27, 2025 antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene gly col-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019, Flo et al., (Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51 :6363, 1991); and Takkinen et al., (Protein Engineering 4 :837 , 1991). The VL and VH domains of a scFv molecule can be derived from one or more antibody molecules. It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules of some aspects and embodiments herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, in one embodiment, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and / or framework residues). Alternatively, or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art-recognized techniques. scFv fragments are described, for example, in WO 2011 / 084714; incorporated herein by reference.

[0093] By “selectively binds” is meant a compound or antibody that binds to one form of a protein and exhibits reduced binding or fails to detectably bind to another form of a protein. In various embodiments, an antibody that selectively binds one CD43 glycoform or glycoepitope does not bind to an alternative CD43 glycoform or glycoepitope. In some embodiments, an antibody is capable of binding to a CD43 glycoform expressed on the surface of a cancer cell and exhibits reduced or undetectable binding to a CD43 glycoform expressed on the surface of a corresponding control cell.

[0094] By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the disclosure. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0095] Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule.

[0096] By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0097] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, of at least about 37° C, or of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0098] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C. In a preferred Atorney Docket No.: 167741-053601 / PCT

[0099] Electronic Deposit Date: October 27, 2025 embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0100] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least about 90%, 95% or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison. In various embodiments, a polypeptide or polynucleotide suitable for use in compositions or methods of the disclosure comprises an amino acid or polynucleotide sequence having about or at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a sequence provided herein.

[0101] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST™, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST™ program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0102] By “subject” is meant an animal. The animal can be a mammal. The mammal can be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.

[0103] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0104] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0105] As used herein, the term “VH” refers to the variable region or domain of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region or domain of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab. The VH and VL regions or domains form antigen binding sites that recognize and bind to an antigen, e.g., antigenic determinants or epitopes of the antigen. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.

[0106] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0107] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art. In some cases, a range of normal tolerance in the art is within 1 or 2 standard deviations of the mean. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0108] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0109] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. Atorney Docket No.: 167741-053601 / PCT

[0110] Electronic Deposit Date: October 27, 2025

[0111] BRIEF DESCRIPTION OF THE DRAWINGS

[0112] FIGs. 1A-1L provide schematic diagrams, scatter plots, flow cytometry plots, and bar plots showing how genome-wide CRISPR screens were used to identify key pathways that regulate antibody-dependent cellular phagocytosis and antibody-independent phagocytosis of human leukemia. FIG 1A provides a schematic diagram of antibody-independent cellular phagocytosis screen design. The screen was a genome-wide CRISPR screen facilitating the identification of human macrophage: human leukemia inhibitory checkpoints. FIG IB provides a scatter plot of log fold change of enrichment or depletion of genes in the leukemia fraction of MV41 1 (x-axis) or M0LM13 (y-axis) co-cultured with macrophages versus leukemia alone. The circle size indicates average -loglO (p-value) across both cell lines. FIG. 1C provides a scatter plot of log fold change of enrichment or depletion of genes in the macrophage fraction of MV41 1 (x-axis) or M0LM13 (y-axis) co-cultured with macrophages versus leukemia alone. The circle size indicates average -loglO (p-value) across both cell lines. FIG ID provides a schematic diagram of competitive co-culture experiment of human leukemia cells with human or mouse macrophages. FIG. IE provides a flow cytometry plot of leukemia cells with and without CD47 knockout cultured alone or with human macrophages stimulated with LPS. FIG. IE also provides barplots showing quantification of the percent of total cells phagocytosed and log2 normalized ratio of knockout cells versus control. The data of the bar plots of FIG. IE show the mean ± s.e.m. of four technical replicates and are representative of two different experiments. FIG. IF provides a flow cytometry plot of human leukemia cells co-cultured with LPS stimulated mouse macrophages. FIG. IF also provides barplots quantifying the percent of phagocytosed cells and ratio of knockout to control remaining after macrophage co-culture. The data of the bar plots of FIG. IF show the mean ± s.e.m. of four technical replicates and are representative of two different experiments. FIG. 1G provides a schematic diagram of a coculture experiment where human leukemia were treated with anti-CD47 (MIAP410) antibody prior to co-culture with human macrophages pre-treated with or without Fc receptor blocking antibodies. FIG. 1G also provides a barplot showing the percent of phagocytosed cells in the presence or absence of macrophage Fc receptor blockade. The data of the bar plot of FIG. 1G show the mean ± s.e.m. of four technical replicates and are representative of two different experiments. FIG. 1H provides a schematic diagram of antibody-dependent cellular phagocytosis screen design. FIG. II provides a scatter plot of log fold change of enrichment or depletion of genes in the leukemia fraction of anti-CD47 treated MV411 (x-axis) or M0LM13 (y-axis) co-cultured with macrophages versus leukemia alone. The circle size indicates average Atorney Docket No.: 167741-053601 / PCT

[0113] Electronic Deposit Date: October 27, 2025 loglO (p-value) across both cell lines. FIG. 1J provides a schematic diagram and two bar plots showing the competitive co-culture experiment of control or knockout leukemia cells co-cultured with human macrophages. Log2 normalized ratio of the knockout to control and percent of phagocytosed leukemia are plotted. The data in FIGs. 1E-1G and 1 J were analyzed by unpaired, two-sided Student’s / -test, * p<0.05, ** p<0.01, *** p<0.001. FIGs. IK and IL provide scatter plots showing an alternative representation of the data shown in FIGs. IB and 1C, respectively.

[0114] FIGs. 2A-2D provide a plot, schematic diagrams, a scatter plot, and bar plots showing that integrated analysis of genome-scale antibody-dependent and antibody-independent cellular phagocytosis screens revealed O-glycan pathway genes as key regulators of macrophage phagocytosis. FIG. 2A provides a plot showing genes ranked by the average of the difference between the log fold change of enrichment or depletion of the leukemia fraction and macrophage fraction. The circle size is scaled -loglO (p-value). The top 15 enriched genes and top 5 depleted genes are listed and arranged by statistical significance. FIG. 2B provides a schematic diagram and bar plots of competitive co-culture experiment of PTPN6 knockout leukemia. The percent of phagocytosed cells and log2 ratio of knockout to control are shown. The data of the bar plots represent mean ± s.e.m. of four technical replicates and are representative of two independent experiments. FIG. 2C provides a scatter plot of the difference between the log fold change of enrichment or depletion in the leukemia fraction and macrophage fraction in the antibody-dependent cellular phagocytosis (ADCP) screen (x-axis) and antibody-independent cellular phagocytosis (AICP) screen (y-axis). The circle size is the average -loglO(p-value). FIG. 2D provides a schematic diagram showing the O-glycosylation pathway highlighting top gene knockouts that enhanced antibody-independent cellular phagocytosis (AICP) and antibodydependent cellular phagocytosis (ADCP). The data in FIG. 2B were analyzed by unpaired, two- sided Student’s / -test, ** p< 0.01

[0115] FIGs. 3A-3G provide schematic diagrams, flow cytometry charts, bar graphs and step graphs showing how the O-linked glycosylation pathway inhibited human macrophage phagocytosis of leukemia cells through terminal sialic acid residues. FIG. 3A provides a schematic diagram of competitive phagocytosis assay, representative flow cytometry plots, and bar graphs of relative phagocytosis of C7GA 77-deficient, C / Gd / / / 7C / -deficient, or control versus control leukemia cells (MV411 and M0LM13). FIG. 3B provides a schematic diagram of competitive phagocytosis assay, representative flow cytometry plots, and bar graphs of relative phagocytosis of SLC39A9-deficient, S / XA5A 2-dedcien or control versus control sgRNA leukemia cells (MV411 and M0LM13) FIG. 3C provides a schematic diagram of antibody- Atorney Docket No.: 167741-053601 / PCT

[0116] Electronic Deposit Date: October 27, 2025 dependent cellular phagocytosis (ADCP) strategy: control or CAM / / / ' / -deficient MV411 cells were engineered to ectopically express mouse CD8a. FIG. 3D provides a schematic diagram of an in vivo ADCP model: control or C / GM / / / ' / -deficient were intravenously engrafted into sublethally irradiated NOD SCID IL2rg- / ~ mice. Mice bearing (7 GM / / / ' / -deficient or control MV41 1 cells were intraperitoneally injected with systemic anti-CD8a antibodies starting on day +5. FIG. 3E provides a step graph and bar plots of survival of mice challenged with C1GALT1- defi cient or control MV411 leukemia cells. FIG. 3F provides a schematic diagram of competitive phagocytosis assay experimental design and bar graphs of relative phagocytosis of C1GALT1, C1GALT1C1, or control sgRNA versus control sgRNA leukemia cells pre-treated with varying doses of V. cholerae sialidase prior to phagocytosis assays. Both knockout and control cells were pre-treated with sialidase prior to co-culture. FIG. 3G provides a schematic diagram of competitive phagocytosis assay and bar graphs of relative phagocytosis of C1GALT1- deficient, C / GM / / / 7C / -deficient, or control MV411 leukemia cells versus control cells pretreated with V. cholerae sialidase prior to phagocytosis assays. Only control cells (but not knockout cells) were pre-treated with sialidase prior to co-culture. For FIGs. 3A-3B and 3F-3G, genetically modified or sialidase-treated leukemia cells were co-cultured with or without fFNy- stimulated macrophages for 18-24 hours. The bar graphs indicate log (fold change) of the ratio of knockout cells relative to control after co-culture with macrophages. The data of the bar graphs of FIGs. 3A, 3B, and 3E represent mean ± s.d. of four technical replicates and are representative of 3-5 independent experiments For FIG. 3E, data represent three independent survival experiments.

[0117] FIGs. 4A-4J provide scatter plots, schematic diagrams, flow cytometry plots, bar graphs, step graphs, flow plots, and Western blots showing how sialylated CD43 was found to be the major downstream effector of the O-linked glycosylation pathway and was sufficient to inhibit macrophage phagocytosis of human leukemia. FIG. 4A provides a scatter plot of functional impact in CRISPR antibody-independent cellular phagocytosis (AICP) screens (x-axis) vs. relative gene expression (y-axis) of all known human cell surface proteins. The size of each dot is scaled to reflect the % of potential O-linked glycosylation sites. FIG. 4B provides a schematic diagram of competitive phagocytosis assay, representative flow cytometry plots, and bar graphs of relative phagocytosis of SPN (CD43) -deficient or control sgRNA versus control leukemia cells (MV411 and M0LM13) in coculture assays with fFNy-stimulated macrophages. FIG. 4C provides representative flow cytometry plots and bar graphs of relative phagocytosis of SPN (CD 43) -deficient or control leukemias overexpressing CD8a versus control MV411 leukemia cells overexpressing CD8a in coculture assays with varying doses of anti-CD8a opsonizing Atorney Docket No.: 167741-053601 / PCT

[0118] Electronic Deposit Date: October 27, 2025 antibodies. FIG. 4D provides representative flow cytometry plots and bar graphs of relative phagocytosis of control leukemias overexpressing luciferase, C1GALT1, or CD43 versus control MV41 1 leukemia cells overexpressing luciferase in coculture assays with IFNy-stimulated macrophages. FIG. 4E provides a schematic diagram and a step graph of survival of mice challenged with SPN (CD43) -deficient or control MV411 leukemia cells overexpressing CD8a. Mice were intraperitoneally injected with systemic anti-CD8a antibodies starting on day +5. FIG. 4F provides representative flow plots for surface expression of sialylated CD43 in control, C1GALT1 -deficient, C1GALT1C1 -deficient, or ( 7773-deficient MV4 I I leukemias. FIG. 4G provides representative Western blots for sialylated CD43 levels in MV411 leukemia cells treated without or with V. cholerae sialidase. FIG. 4H provides representative Western blots for total CD43 levels in MV411 leukemia cells treated without or with V. cholerae sialidase FIG. 41 provides representative flow cytometry plots and bar graphs of relative phagocytosis of Cl GALT 1 -deficient leukemias overexpressing luciferase, C1GALT1, or CD43 versus control MV41 1 leukemia cells overexpressing luciferase in coculture assays with IFNy-stimulated macrophages. FIG. 4J provides bar graphs of relative phagocytosis of CD43 -deficient or dual C1GALT1- and CD43 -deficient leukemias after co-culture with IFNy-stimulated macrophages. The FIGs. 4B-4D and 4H bar graphs indicate log (fold change) of the ratio of knockout cells relative to control after co-culture with IFNy-stimulated macrophages. The data of the bar graphs of FIGs. 4B, 4C, 4D, and 4 J represent mean ± s.d. of four technical replicates and are representative of 3-5 independent experiments.

[0119] FIGs. 5A-5K provide UMAP, dot plot summaries, Western blots, flow cytometry plots, a violin plot, and bar graphs showing how anti-CD43 antibodies enhanced human macrophage phagocytosis of leukemia cell lines and primary patient samples. FIGs. 5A and 5B provide uniform manifold approximation and projection (UMAP) of single cell RNA-sequencing (scRNA- seq) profiles of bone marrow aspirate cells taken from healthy patients or acute myelogenous leukemia (AML) patients. Expression of SPN / CD43 is shown on the right panel if FIG. 5B. FIG. 5C provides a dot plot summary of z-score scaled expression of CD43 across normal and malignant cell subtypes. The size of each dot is scaled to reflect the absolute % of cells in which SPN transcripts were detected. FIG. 5D provides representative Western blots for total CD43 expression in human leukemia cell lines (MV411, M0LM13), primary AML blasts (AML 01 - AML07), and normal immune cell subsets (bulk peripheral blood mononuclear cells (PBMCs), monocytes). FIG. 5E provides flow cytometry plots for sialylated CD43 expression on fresh blasts from AML patients (AML01 - AML06). FIG. 5F provides a schematic diagram of phagocytosis experiments with anti-CD43 antibodies and bar graphs of absolute phagocytosis Atorney Docket No.: 167741-053601 / PCT

[0120] Electronic Deposit Date: October 27, 2025 of human leukemia cell lines (MV411, M0LM13, HEL) after addition of various doses of anti- CD43 antibodies to macrophage co-culture assays. FIG. 5G provides a schematic diagram of phagocytosis assays of patient-derived AML blasts and bar graphs of absolute phagocytosis of primary AML cells after addition of varying doses of anti-CD43 antibodies to macrophage coculture assays. FIG. 5H provides a schematic diagram of phagocytosis assays with FcR blockade prior to addition of either anti-CD47 or anti-CD43 antibodies and bar graphs of absolute phagocytosis of MV411 leukemias with or without Fc blockade prior to addition of anti-CD47 or anti-CD43 antibodies. FIG. 51 provides a schematic diagram of phagocytosis assays with AML patient- derived macrophages and bar graphs of relative phagocytosis of control or CD43- deficient leukemias versus control MV411 leukemias. FIG. 5 J provides a schematic diagram of phagocytosis assays with AML patient-derived macrophages and bar plots of absolute phagocytosis of MV411 leukemias with or without anti-CD43 antibodies. In FIGs. 5F-5J, the data represent mean ± s.d. of four technical replicates and are representative of 3-5 independent experiments. FIG. 5K provides a violin plot showing an alternative representation of the data presented in FIG. 5J.

[0121] FIGs. 6A-6D provide flow cytometry plots, live cell microscopy images, and bar graphs showing the establishment of a human macrophage in vitro co-culture assay. FIG. 6A provides a flow cytometry gating strategy to quantify and assess phagocytosis. Macrophages are CD1 1b- FITC positive and leukemia cells express red fluorescent protein (RFP) or blue fluorescent protein (BFP). The gating strategy is shown. FIG. 6B provides a live cell microscopy images of PHRODO™-stained leukemia cells co-cultured with macrophages that are unstimulated, stimulated with fFNy, or following leukemia treatment with Anti-CD47. FIG. 6C provides bar graphs of the quantification of phagocytosis with macrophage colony-stimulating factor (M- CSF) differentiated macrophages following treatment with various cytokines. FIG. 6D provides bar graphs of assessment of percent of phagocytosis seen with M-CSF or granulocytemacrophage colony-stimulating factor (GM-CSF) differentiated macrophages stimulated with IFNy at a range of effector to target ratios. The data in FIG. 6C were analyzed by two-way ANOVA, and the data in FIG. 6D were analyzed with unpaired, two-sided Student’s t-test, * p<0.05, ** p<0.01, *** pO.OOl.

[0122] FIGs. 7A-7F provide line plots, Peason correlations, and heatmaps relating to the genetic screen quality metrics for antibody independent cellular phagocytosis screens. FIGs. 7A-7B provide plots of the abundance of sgRNAs in leukemic cells in the indicated conditions compared to a Gaussian distribution of sgRNAs in MV411 and M0LM13. FIGs. 7C-7D provide net replicate Pearson correlations of each individual replicate versus pre-input Atorney Docket No.: 167741-053601 / PCT

[0123] Electronic Deposit Date: October 27, 2025 representing the initial distribution of the sgRNA library in MV411 and M0LM13. FIGs. 7E- 7F provide Pearson correlation heatmaps of replicates in the leukemia fraction and macrophage fraction versus one another in the MV411 and M0LM13 antibody independent cellular phagocytosis screens, respectively.

[0124] FIGs. 8A-8D provide scatter plots and volcano plots of hits that modulated antibody independent cellular phagocytosis. FIG. 8A provides a scatter plot of log fold change (LFC) of enrichment or depletion of sgRNAs in the leukemia fraction versus leukemia only (y-axis) and macrophage fraction versus leukemia only (x-axis) for MV411. FIG. 8B provides a scatter plot of log fold change (LFC) values of enrichment or depletion of sgRNAs in the leukemia fraction versus leukemia only (y-axis) versus macrophage fraction versus leukemia only (x-axis). The circle sizes for points in FIGs. 8A-8B are scaled by average log 10 p-value. FIG. 8C provides a volcano plot of LFC of MV411 leukemia fraction or MV411 macrophage fraction (x-axis) versus the average -loglO(p-value) (y-axis). FIG. 8D provides a volcano plot of LFC of MV411 leukemia fraction or macrophage fraction (x-axis) versus the average -loglO(p-value) (y-axis).

[0125] FIGs 9A-9D provide bar graphs and flow cytometry plots of showing the impact of targeting CD47 with antibodies or genetic deletion on macrophage phagocytosis. FIG. 9A provides a bar graph of the percent of leukemia cells phagocytosed following treatment with isotype control or varying doses of anti-CD47 (MIAP410) antibody. The data of FIG. 9A show the mean ± s.e.m. of four technical replicates and are representative of two different experiments. FIG. 9B provides a low cytometry plot of CD47 expression in MV411 Cas9 cells transduced with sgRNAs targeting CD47 or a control locus. FIG. 9C provides the flow cytometry plot of CD47 expression in MV411 Cas9 cells transduced with sgRNAs targeting CD47 or a control locus. FIG. 9D provides a bar graph of the percent of leukemia cells phagocytosed following treatment with SIRPa-Fc or Anti-CD47 (MIAP410) antibody. The data of FIGs. 9A and 9D show the mean ± s.e.m. of four technical replicates and are representative of two different experiments. The data in FIG. 9A were analyzed by two-way ANOVA, *** p<0.001.

[0126] FIGs. 10A-10F provide line plots, Pearson correlations, and heatmaps of the genetic screen quality metrics for antibody dependent cellular phagocytosis screens. FIGs. 10A and 10B provide plots of the abundance of sgRNAs in leukemic cells in the indicated conditions compared to a Gaussian distribution of sgRNAs in MV411 and M0LM13 antibody-dependent cellular phagocytosis screens. FIGs. 10C and 10D provide net replicate Pearson correlations of each individual replicate versus pre-input representing the initial distribution of the sgRNA library in MV411 and M0LM13. FIGs. 10E and 10F provide Pearson correlation heatmaps of Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 all replicates in the leukemia fraction and macrophage fraction versus one another in the MV411 and M0LM13 antibody independent cellular phagocytosis screens, respectively.

[0127] FIGs. 11A-11B provide volcano plots of hits that modulated antibody dependent cellular phagocytosis. FIG. 11A provides a volcano plot of LFC of MV411 leukemia fraction or MV411 macrophage fraction (x-axis) versus the average -loglO(p-value) (y-axis). FIG. 11B provides a volcano plot of log-fold chang (LFC) of MV411 leukemia fraction or macrophage fraction (x- axis) versus the average -log 10 (p-value) (y-axis).

[0128] FIGs. 12A-12D provide bar graphs, flow cytometry plots, and Western blots relating to the validation of genetic factors that modulated antibody-dependent cellular phagocytosis. FIG. 12A provides a bar graph showing the ratio of phagocytosis of MV411 leukemia cells with MHC-I loss versus control leukemia in co-culture. The leukemia cells were treated with either anti-isotype or anti-CD33. The percent of leukemia cells is quantified in each condition. FIG. 12B provides the flow cytometry plot of macrophages following editing with Cas9-RNP with sgRNAs targeting AAVS1, LILRB1, or LILRB2. The expression of LILRB1 or LILRB2 is shown. The percent of macrophages positive for LILRB1 or LILRB2 expression is quantified. FIG. 12C provides bar graphs showing quantification of the ratio of phagocytosis of MV411 leukemia with MHC-I loss versus control leukemia in co-culture with sgAAVSl, sgLILRBl, or sgLILRB2 edited macrophages. The percent of leukemia cells phagocytosed is quantified in each macrophage background. FIG. 12D provides Western blot images for PTPN6 in control MV41 1 leukemia or in leukemic cells transduced with sgRNAs targeting PTPN6. The data in FIGs. 12A and 12C were analyzed with unpaired, two-sided Student’s t-test, * p<0.05, ** p<0.01, *** pO.OOl.

[0129] FIGs. 13A-13J provide flow plots, growth curves, bar graphs, Western blots, flow cytometry plots, step graphs, and schematic diagrams showing that loss of O-glycosylation and cell surface sialylation enhanced phagocytosis in vitro and in vivo. FIG. 13A provides a flow plot of VVA, PNA, and SIGLEC 7-Fc staining of control, Cl GALT 1 -deficient, or C1GALT1C1- deficient MV411 leukemias. FIG. 13B provides INCUCYTE®-based growth curves for control, C1GALT1 -deficient, or C1GALT1 Cl -deficient MV411 leukemias. FIG. 13C provides a bar graph of relative antibody-dependent cellular phagocytosis of control, C1GALT1 -deficient, or C1GALT1 Cl -deficient MV411 leukemias by unstimulated macrophages after addition of anti- CD47 antibodies. FIG. 13D provides representative Western blots for MGAT1 expression after editing MV411 cells with various MGAT1 sgRNAs. FIG. 13E provides bar graphs of relative and absolute phagocytosis of control or MGAT1 -deficient leukemias versus control leukemias after co-culture with fFNy-stimulated macrophages. FIG. 13F provides representative flow Atorney Docket No.: 167741-053601 / PCT

[0130] Electronic Deposit Date: October 27, 2025 cytometry plots of mouse CD8a overexpression in MV411 leukemias. FIG. 13G provides bar graphs of relative phagocytosis of control leukemias overexpressing mouse CD8a versus control leukemias overexpressing luciferase in the presence of various doses of anti-CD8a antibodies. FIG. 13H provides bar graphs of relative phagocytosis of control, C1GALT1 -deficient, or CD47- deficient leukemias cells overexpressing mouse CD8a relative to control leukemias overexpressing mouse CD8a in the presence of varying doses of CD8a antibodies, FIG. 131 provides a step graph of survival of mice challenged with MV411 overexpressing mouse CD8a leukemias treated with isotype or anti-CD8a antibodies. FIG. 13J provides a schematic diagram and bar graph relating to sialic acid biosynthesis and relative phagocytosis of control, CMAS- deficient, or SLC35A / -deficient leukemias versus control MV411 leukemias after co-culture with IFNy-stimulated macrophages. The data in FIGs. 13C-13D, 13G-13H, and 13J were analyzed with unpaired, two-sided Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001.

[0131] FIGs. 14A-14C provide growth curves, flow cytometry plots, and bar graphs showing that CD43 loss was epistatic with C1GALT1C1 loss. FIG. 14A provides INCUCYTE®-based growth curves for control or CD43 -deficient MV411 leukemias. FIG. 14B provides representative flow cytometry plots of sialylated CD43 expression after treatment of leukemias cells with varying doses of V. cholerae sialidase. FIG 14C provides bar graphs of relative phagocytosis of CD43 -deficient or dual C1GALT1C1- and CD43 -deficient leukemias after coculture with IFNy-stimulated macrophages. The data in FIG. 14C were analyzed with unpaired, two-sided Student’s t-test, ** p<0.01, n.s. not significant.

[0132] FIG. 15A-15G provide heatmaps, flow cytometry plots, schematic diagrams, bar graphs, and plots showing that loss or inhibition of SIGLEC7 and 9 did not modify phagocytosis of CD43 or C1GALT1C1 deficient leukemia cells. FIG. 15A provides a heatmap of RNA transcript levels of SIGLEC family members taken from bulk RNA-sequencing of unstimulated or IFNy-stimulated human macrophages. FIG. 15B provides representative flow cytometry plots depicting SIGLEC family member expression in unstimulated or IFNy-stimulated macrophages. FIG. 15C provides flow plots of SIGLEC 7-Fc or SIGLEC 9-Fc staining on control or CD43- deficient MV411 leukemias. FIG. 15D provides representative flow cytometry plots, a schematic diagram, and a bar graph for SIGLEC 7 and SIGLEC 9 expression after genetic deletion of both SIGLEC 7 and SIGLEC 9 in human macrophages and relative phagocytosis of control, C1GALT1 -deficient, or CD43 -deficient leukemias by control or SIGLEC 7 / 9-defi cient macrophages. FIG. 15E provides a schematic diagram and a bar graph showing relative phagocytosis of control, C1GALT1 -deficient, or CD43 -deficient leukemias by IFNy-stimulated macrophages after addition of isotype or anti-SIGLEC 7 / anti-SIGLEC 9 neutralizing antibodies. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0133] FIG. 15F provides a bar graph of absolute phagocytosis of control, C1GALT1 -deficient, or CD43 -deficient leukemias after addition of anti-SIGLEC 7 / anti-SIGLEC 9 neutralizing antibodies. FIG 15G provides a plot of cell binding avidity measured using a Z-MOVI™ avidity analyzer of IFNy-stimulated macrophages co-cultured with control or CD43 -deficient MV411 leukemias (n=3). The data in FIGs. 15D-15E were analyzed by unpaired, two-sided Student’s t- test, ** p< 0.01. The data in FIG. 15G are representative of three independent Z-MOVI™ avidity analyzer chips.

[0134] FIGs. 16A-16D provide UMAP, dot plots, fluorescence microscopy images, bar graphs, and schematic diagrams showing the expression O-glycan pathway genes and the impact of anti- CD43 targeting antibodies on phagocytosis of leukemia cells. FIG. 16A provides a uniform manifold approximation and projection (UMAP) and dot plot of the expression of C1GALT1, C1GALT1C1, SLC39A9, and SLC35A2 across a UMAP projection of scRNA-seq profiles of bone marrow aspirate cells taken from healthy patients or acute myelogenous leukemia (AML) patients. FIG. 16B provides representative fluorescence microscopy images of human macrophages co-cultured with PHRODO™-labeled human leukemia cells in the presence or absence of anti-CD43 antibody (clone MEM59). FIG. 16C provides a bar graph of the absolute phagocytosis of MV411 leukemia cells by unstimulated human macrophages after addition of various concentrations of anti-CD43 (clone 10G7). FIG. 16D provides a schematic diagram and bar graph of the absolute phagocytosis of MV411 leukemia cells by IFNy-stimulated macrophages after addition of anti-CD43 antibodies in the presence or absence of Fc blockade. The data in FIGs. 16C and 16D were analyzed by unpaired, two-sided Student’s t-test, *p<0.05, ** p< 0.01.

[0135] FIGs. 17A-17C provide schematic diagrams and bar graphs showing that genetic deletion of the O-glycan pathway or CD43 in acute myeloid leukemia (AML) cells increased human macrophage phagocytosis. FIG. 17A provides a schematic diagram showing the O- glycan pathway. FIG. 17B provides a schematic diagram describing the experiment undertaken to evaluate enrichment or depletion of cells co-cultured in the presence of control cells and human macrophages when the cells were edited to knock out expression of C1GALT1, C1GALT1C1, or CD43. FIG. 17C provides bar graphs showing log2 enrichment values for control cells and edited cells in an experiment carried out as described in FIG. 17B. In FIGs. 17A-17C, “sgCtrl” indicates cells edited using a CRISPR / Cas9 system containing a control guide RNA that did not target a polynucleotide encoding C1GALT1, C1GALT1C1, or CD43, “sgKO” indicates cells edited using a CRISPR / Cas9 system containing a guide RNA targeting a Atorney Docket No.: 167741-053601 / PCT

[0136] Electronic Deposit Date: October 27, 2025 polynucleotide encoding C1GALT1, C1GALT1C1, or CD43 to knock out expression of the encoded polypeptide, and “S / T” indicates a serine (S) or a threonine (T) amino acid.

[0137] FIG. 18 provides a series of flow cytometry histograms demonstrating that a SIGLEC-1 Fc, which is a fusion protein containing the extracellular domain of SIGLEC-1 and the Fc domain of an antibody, recognizes SLC35A2 generated ligands but not CD43. In FIG. 18, “sgCh2-l” indicates control cells contacted with a CRISPR / Cas9 system containing a control guide RNA that did not target C1GALT1 or CD43 for editing, “sgClGALTl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding C1GALT1, “sgCD43” indicates cells contacted with a CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding CD43, “MV411” indicates a leukemia cell line established from the blast cells from a 10-y ear-old male with biphenotypic B-myelomonocytic leukemia, “M0LM13” indicates an acute myeloid leukemia (AML) cell line established from the peripheral blood of a 20-year-old man with acute myeloid leukemia, and “+ sialidase” indicates cells contacted with sialidase.

[0138] FIG. 19 provides a series of flow cytometry histograms demonstrating that a SIGLEC-7 recognizes O-glycans but CD43 loss does not eliminate SIGLEC-7 binding. In FIG. 18, “sgCh2- 1” indicates control cells contacted with a CRISPR / Cas9 system containing a control guide RNA that did not target C1GALT1, C1GALT1C1, or CD43 for editing, “sgClGALTl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding C1GALT1, “sgClGALTICl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding C1GALT1C1, “sgCD43” indicates cells contacted with a CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding CD43, “MV411” indicates a leukemia cell line established from the blast cells from a 10-y ear-old male with biphenotypic B-myelomonocytic leukemia, “MOLM-13” indicates an acute myeloid leukemia (AML) cell line established from the peripheral blood of a 20-year-old man with acute myeloid leukemia, and “+ sia” indicates cells contacted with sialidase.

[0139] FIG. 20 provides a series of flow cytometry histograms demonstrating that a SIGLEC-9 recognizes ligands other than CD43. In FIG. 18, “sgCh2-l” indicates control cells contacted with a CRISPR / Cas9 system containing a control guide RNA that did not target C1GALT1, C1GALT1C1, or CD43 for editing, “sgClGALTl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide Atorney Docket No.: 167741-053601 / PCT

[0140] Electronic Deposit Date: October 27, 2025 encoding C1GALT1, “sgClGALTlCl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding C1GALT1C1, “sgCD43” indicates cells contacted with a CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding CD43, “MV411” indicates a leukemia cell line established from the blast cells from a 10-year-old male with biphenotypic B-myelomonocytic leukemia, “MOLM-13” indicates an acute myeloid leukemia (AML) cell line established from the peripheral blood of a 20-year-old man with acute myeloid leukemia, and “+ sia” indicates cells contacted with sialidase.

[0141] FIG. 21 provides a bar graph demonstrating that a SIGLEC1 blockade did not eliminate the preference for macrophage to phagocytose C1GALT1 or CD43 knockout MV411 cells. In FIG. 21, “sgCtrl” indicates control cells contacted with a CRISPR / Cas9 system containing a control guide RNA that did not target C1GALT1 or CD43 for editing, “sgClGALTl” indicates cells contacted with CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding C1GALT1, “sgCD43” indicates cells contacted with a CRISPR / Cas9 system containing a guide RNA directing Cas9 to introduce a knockout edit to a polynucleotide encoding CD43, and “anti-SIGLECl” indicates a monoclonal antibody capable of binding to SIGLEC1.

[0142] FIGs. 22A to 22C provide a schematic diagram and bar graphs showing that genetic deletion of CD43 or antibody-mediated blockade of CD43 enhanced NK cell cytotoxicity in vitro. FIG. 22A provides a schematic diagram of the experiment conducted. FIG. 22B provides a bar graph showing that genetic deletion of CD43 led to increased killing of leukemia cells by natural killer (NK) cells compared to unedited (control) leukemia cells at effector-to-target (E:T) ratios of 1 :4 and 1 :2. FIG. 22B provides a bar graph showing that contacting of cells with an anti-CD43 antibody led to increased killing of leukemia cells by natural killer (NK) cells compared to cells contacted with an isotype control antibody at an effector-to-target ratio (E:T) of 1 :4 or 1 :2.

[0143] FIGs. 23A and 23B provide a schematic diagram and a bar graph demonstrating that CD43 inhibited human T cell cytotoxicity in vitro. FIG. 23A provides a schematic diagram of the experiment conducted. The cells evaluated in the experiment either expressed an anti-CD3 antibody and CD80 on their surface (+anti-CD3 / 80 ORF) or did not. In FIG. 23A “TCR” indicates a T cell receptor and “OKT3” refers to a monoclonal anti-CD3 antibody antigenbinding domain. FIG. 23B provides a bar graph showing that CD43 knockout leukemia cells showed higher rates of killing by T cells than non-edited leukemia cells when co-cultured with the T cells at an effector-to-target ratio (E:T) of 1 :4 or 1 :2. Atorney Docket No.: 167741-053601 / PCT

[0144] Electronic Deposit Date: October 27, 2025

[0145] FIGs. 24A-24D provide a schematic diagram, flow cytometry hystograms, a bar graph, and a plot demonstrating that CD43 inhibited human T cell cytotoxicity in vivo. FIG. 24A provides a schematic diagram of the experiment conducted. Unedited leukemia cells were administered to the mice (NSG mice) as a control. FIG. 24B provides a flow cytometry histogram showing levels of CD43 expression on the surface of MV411 leukemia cells, CD8+ T cells, and CD4+ T cells administered to the mice. FIG. 24C provides a bar graph showing an alternative presentation of the data shown in FIG. 24B. In FIG. 24C, “MFI” indicates mean fluorescent intensity. FIG. 24D provides a plot showing the percent survival of mice exposed to the indicated conditions. In FIG. 24D, “+T cells” indicates mice administered T cells, “no T cells” indicates mice not administered T cells, “CD43 KO leukemia” indicates leukemia cells edited using a CRISPR / Cas9 system to knock out expression of CD43, and “ctrl leukemia” indicates unedited leukemia cells.

[0146] DETAILED DESCRIPTION

[0147] The disclosure features compositions and methods that are useful for inducing phagocytosis or increasing killing of a cancer cell (e.g., by an immune cell) by contacting the cancer cell with an antibody capable of selectively binding to a CD43 protein expressed on the surface of cancer cells. In various embodiments, the CD43 protein is not expressed on the surface of a healthy cell.

[0148] The present disclosure is based, at least in part, upon the discovery disclosed in the Examples provided herein that inhibiting, in whole or in part, O-linked glycosylation (e.g., through knockout or inhibition of C1GALT1, C1GALT1C1, or CD43) led to a striking increase in phagocytosis by macrophages of cancer cells (e.g., human leukemic cells), and that contacting a cancer cell with an anti-CD43 antibody (blockade of CD43) lead to an increased phagocytosis of cancer cells by macrophages. The present disclosure is also based, at least in part, upon the development of a CRISPR screen to identify genes encoding polypeptides modulating phagocytosis of cells by macrophage. The present disclosure is also based, at least in part, upon the discovery detailed herein that O-linked glycosylation and CD43 suppressed natural killer (NK) cell and T cell cytotoxicity, thereby indicating that CD43 may be a pan-immune regulator of anti-leukemic immunity.

[0149] The present disclosure is based, at least in part, upon results from genome-scale CRISPR knockout screens carried out in human leukemia cells co-cultured with human monocyte-derived macrophages in the presence or absence of antibody opsonization to systematically identify regulators of phagocytosis. This approach allowed for the cataloging and validation of the genes Atorney Docket No.: 167741-053601 / PCT

[0150] Electronic Deposit Date: October 27, 2025 that impact human macrophage-mediated antibody-independent cellular phagocytosis (AICP) and antibody-dependent cellular phagocytosis (ADCP). As there are notable differences in receptor expression, activation, and function between mouse and human macrophages, healthy human donor-derived monocytes differentiated into macrophages were used to perform unbiased genome- scale screens. AML was evaluated because it is refractory to T-cell directed immune checkpoint blockade and has a tumor microenvironment that is rich in myeloid cells. Surprisingly, it was found that whereas the classic “don’t eat me” signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were found to be strong negative regulators of phagocytosis. In acute myelogenous leukemia (AML), the cell surface O-linked glycoprotein CD43 was a major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. It was found that CD47 was only a weak regulator of human macrophage phagocytosis, whereas the O-linked sialoglycoprotein CD43 was a potent inhibitor of human macrophage AICP and ADCP. The experiments described in the Examples of the present disclosure highlight the importance of using human platforms to identify immune checkpoints, and nominate CD43 as a glyco-immune regulator of human macrophage phagocytosis.

[0151] Macrophages and Phagocytosis

[0152] Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

[0153] Phagocytosis is a core function of macrophages that promotes tumor clearance and enhances adaptive immunity through antigen processing and cross-presentation to T lymphocytes. Macrophages phagocytose tumor cells through Fc-receptor mediated recognition of antibody-opsonized targets (antibody-dependent cellular phagocytosis, ADCP) and / or antibody-independent phagocytosis (AICP), which involves the integration of pro-phagocytic “eat me” signals and anti-phagocytic “don’t eat me” signals. Preclinical studies have identified CD47 on tumor cells as a major “don’t eat me” signal that transmits inhibitory signals to macrophages to restrain phagocytosis. However, recent phase III clinical trials of CD47- neutralizing antibodies for the treatment of MDS and AML have shown limited clinical efficacy. The mechanism explaining these clinical results is unknown, and highlights the need for the discovery of new therapeutic targets with potential to activate the phagocytosis of cancer cells. Atorney Docket No.: 167741-053601 / PCT

[0154] Electronic Deposit Date: October 27, 2025

[0155] CD43

[0156] The O-linked glycosylation and sialylation pathways are important negative regulators of human macrophage phagocytosis (FIG. 2D). For example, C1GALT1 (T synthetase) is required for the formation of O-linked glycosylation modifications (Core 1 O-glycan / T antigen), C1GALT1C1 (COSMC) is a molecular chaperone of C1GALT1, and CD43 is a surface glycoprotein that undergoes O-linked glycosylation and sialyation. The gene encoding CD43 is called SPN. Loss of C1GALT1 / C1GALT1C1 expression in a cell results in reduced terminal sialylation of cell surface proteins.

[0157] CD43 is a heavily O-glycosylated surface glycoprotein with 93 extracellular serine / threonine residues. The core O-linked glycosylation structures are terminally sialylated. Several glycoforms of CD43 exist and are variably expressed by different cell types. Several glycoforms of CD43 exist and are variably expressed by different cell types.

[0158] CD43 is differentially glycosylated in leukemia cells compared to normal hematopoietic cells. Myeloid leukemias contain different glycoforms of CD43 than healthy thymocytes or T cells. CD43 glycoforms vary depending on the hematopoietic lineage (myeloid vs. lymphoid). For example, myeloid cells express CD43 proteins with increased glycosylation compared to healthy cells.

[0159] Anti-CD43 Antibodies

[0160] Anti-CD43 antibodies, which bind to cancer cells, thereby targeting them for phagocytosis and / or killing by immune cells, are useful for the treatment of neoplasias. A number of anti-CD43 antibodies are available to the skilled practitioner. Non-limiting examples of anti-CD43 antibodies include those disclosed in International Patent Application Publication No. PCT / KR2016 / 011428, European Patent Application No. 06716320, Patent Application Publications No. CA2989551, CA2652703A, and CA3143891A, and International Patent Application Publications No. WO2021113853, WO2017065493, and WO2018115485. Anti- CD43 antibodies capable of selectively binding to a CD43 protein expressed on the surface of cancer cells include as non-limiting examples, those disclosed in Gillissen, et al. “Patient-derived antibody recognizes a unique CD43 epitope expressed on all acute myelogenous leukemia (AML) cells and has antileukemia activity in mice,” Blood Advances, 1 : 1551-1564 (2017). Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0161] Methods of Treatment

[0162] The methods and compositions provided herein (e.g., anti-CD43 antibodies) are useful for treating a disease (e.g., a neoplasia) in a subject in need thereof, increasing phagocytosis of a cell (e.g., by macrophage), and / or increasing killing of a cell (e.g., by a NK cell and / or a T cell). In embodiments, a subject is administered, for example, an anti-CD43 antibody capable of selectively binding to CD43 proteins expressed on the surface of cancer cells or other target cells. The methods provided herein include methods for the treatment of a neoplasia.

[0163] Generally, the methods provided herein include administering a therapeutically effective amount of an agent (e.g., an anti-CD43 antibody) as provided herein, to a subject who is in need thereof.

[0164] An effective amount of an agent can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of an agent of the disclosure (i.e., an effective dosage) may depend on the therapeutic compounds or agents selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic agents provided herein can include a single treatment or a series of treatments.

[0165] Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, 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. Agents which exhibit high therapeutic indices may be advantageous to use for administration to a patient in some embodiments. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0166] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents may lie within a range of circulating concentrations 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 any agent used in the method of the present disclosure, the therapeutically effective Atorney Docket No.: 167741-053601 / PCT

[0167] Electronic Deposit Date: October 27, 2025 dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0168] Dosages and desired drug concentrations of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration (e.g., oral administration, intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, intracranial, intraspinal, subcutaneous, intraarticular, intrasy novi al, intrathecal, topical, or inhalation routes) is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles described in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0169] For in vivo administration of any of the agents of the present disclosure, normal dosage amounts may vary from about 10 ng / kg up to about 100 mg / kg of an individual's and / or subject's body weight or more per day, depending upon the route of administration. In some embodiments, the dose amount is about 1 mg / kg / day to 10 mg / kg / day. For repeated administrations over several days or longer, depending on the severity of the disease, disorder, or condition to be treated, the treatment is sustained until a desired suppression of symptoms is achieved.

[0170] An effective amount of an agent of the instant disclosure may vary, e.g., from about 0.001 mg / kg to about 1000 mg / kg or more in one or more dose administrations for one or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose varies from about 0.001 mg / kg to about 1000 mg / kg, from about 0.01 mg / kg to about 750 mg / kg, from about 0.1 mg / kg to about 500 mg / kg, from about 1.0 mg / kg to about 250 mg / kg, and from about 10.0 mg / kg to about 150 mg / kg.

[0171] An exemplary dosing regimen may include administering an initial dose of an agent of the disclosure of about 200 pg / kg, followed by a weekly maintenance dose of about 100 pg / kg every other week. Other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is contemplated herein. In certain embodiments, dosing Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 ranging from about 3 pg / kg to about 2 mg / kg (such as about 3 pg / kg, about 10 pg / kg, about 30 pg / kg. about 100 pg / kg, about 300 pg / kg, about 1 mg / kg. or about 2 mg / kg) may be used. In certain embodiments, dosing frequency is three times per day, twice per day, once per day. once every other day. once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. Progress of the therapy is easily monitored by conventional techniques and assays. The dosing regimen, including the agent(s) administered, can vary over time independently of the dose used.

[0172] Methods for characterizing the efficacy of a treatment for a neoplasia are well known in the art (e.g., computerized tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), position emission tomography (PET) scan, ultrasound X-ray, biopsy, etc.).

[0173] Screens

[0174] The present disclosure provides methods for identification of genes modulating phagocytosis of cells by macrophage (see, e.g., FIG. 1A). In embodiments, the methods involve contacting a population of cells (e.g., a population of cancer cells) expressing a polynucleotide programmable endonuclease with a library of guide RNAs targeting genes for knockout to yield edited cells. The methods further involve selecting for edited cells (e.g., using flow cytometry or other methods familiar to one of skill in the art) and subsequently co-culturing the edited cells with stimulated macrophages. The methods in various embodiments further involve identifying edited cells phagocytosed by the macrophage (e.g., through single-cell sequencing). Identification of those edited cells enriched in macrophage of the co-culture, genes that modulate phagocytosis of the cells may be identified (e.g., if cells edited to knock out expression of a particular gene are enriched in the macrophage fraction of the co-culture, the gene is indicated as an inhibitory checkpoint of phagocytosis of the cells).

[0175] Generation and Screening of Antibodies that Bind to CD43

[0176] Antibodies, including recombinantly produced antibodies, that selectively bind to an antigen (e.g., a CD43 protein expressed on the surface of a cancer cell) are provided and described herein.

[0177] Methods for generating antibodies against a protein or peptide of interest are known and practiced by the skilled practitioner. When animals are immunized with antigens they respond by generating a polyclonal antibody response comprised of many individual monoclonal antibody Atorney Docket No.: 167741-053601 / PCT

[0178] Electronic Deposit Date: October 27, 2025 specificities. It is the sum of these individual specificities that make polyclonal antibodies useful in so many different contexts. Individual monoclonal antibodies were originally isolated by immortalizing individual B cells using hybridoma technology (Kohler and Milstein, Nature 256, 495, 2011), in which B cells from an immunized animal are fused with a myeloma cell. With the advent of molecular biology, in vitro methods to generate antibodies against proteins of interest, such as CD43, have been developed.

[0179] The terms “antigen of interest” or “target protein” are used herein interchangeably and refer generally to the agent recognized and specifically bound by an antibody. In an embodiment, such an antigen of interest or target protein is a CD43 polypeptide (e.g., a particular glycoform of CD43), or an antigenic and / or immunogenic portion thereof.

[0180] An antibody is a polypeptide chain-containing molecular structure with a specific shape that specifically binds an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. In one embodiment, an antibody molecule is an immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD). Antibodies from a variety of sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, or fowl are considered “antibodies.” Numerous antibody coding sequences have been described; and others may be raised by methods well-known by a skilled practitioner.

[0181] For example, antibodies or antigen binding fragments may be produced by genetic engineering. Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to a wild-type nucleic acid sequence. Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in some aspects and embodiments herein are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions obtained from antibody producing cells of one species with the constant Atorney Docket No.: 167741-053601 / PCT

[0182] Electronic Deposit Date: October 27, 2025 light and heavy chain regions from another. Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art and may be achieved by standard means (as described, e.g., in U.S. Pat. No. 5,624,659, incorporated fully herein by reference).

[0183] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody and fitting them to the structure of the human antibody chains. Although apparently complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated fully herein by reference.

[0184] In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab')2, or other fragments) may be synthesized. “Fragment,” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques. For instance, “Fv” immunoglobulins for use in some aspects and embodiments herein may be produced by synthesizing a variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities.

[0185] Immunoglobulins may be modified post-translationally, e.g., to add chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of some aspects and embodiments herein.

[0186] Screening of libraries for CD43-binding polypeptides or peptides

[0187] Methods for high throughput screening of polypeptide (e.g., antibody or antigen-binding antibody fragment) libraries for molecules capable of selectively binding to a CD43 protein include, without limitation, display techniques including phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate ligands that bind biologically relevant molecules has been reviewed, e.g., in Felici et al. (Biotechnol. Annual Rev. 1 : 149-183, 1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), and Hoogenboom et al. (Immunotechnology 4: 1-20, 1998). Several randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind different targets, e.g., cell surface receptors or DNA (reviewed by Kay (Per sped. Drug Discovery Des . 2, 251-268, 1995), Kay et al., (Mol. Divers. 1 : 139-140, 1996)). Proteins and Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 multimeric proteins have been successfully phage-displayed as functional molecules (see. e.g., EP 0349578A, EP 4527839A, EP 0589877A; Chiswell and McCafferty (Trends Biotechnol. 10, 80-84 1992)). In addition, functional antibody fragments (e.g. Fab, single-chain Fv [scFv]) have been expressed as reported by McCafferty et al. (Nature 348: 552-554, 1990), Barbas et al. (Proc. Natl. Acad Set. USA 88:7978-7982, 1991), and Clackson et al. (Nature 352:624-628, 1991). These references are hereby incorporated by reference in their entirety.

[0188] In addition to generating CD43 -binding polypeptides of some aspects and embodiments herein, in vitro display techniques, which are known and practiced in the art, also provide methods for improving the affinity of an anti-CD43 -binding polypeptide, antibody, or antigenbinding fragments thereof. For instance, rather than screening libraries of antibodies and fragments thereof containing completely randomized hypervariable regions, narrower libraries of antibodies and antigen-binding fragments thereof that feature targeted mutations at specific sites within hypervariable regions can be screened. This can be accomplished, for example, by assembling libraries of polynucleotides encoding antibodies or antigen-binding fragments thereof that encode random mutations only at particular sites within hypervariable regions. These polynucleotides can then be expressed in, e.g., filamentous phage, bacterial cells, yeast cells, mammalian cells, or in vitro using, e.g., ribosome display, mRNA display, or cDNA display techniques in order to screen for antibodies or antigen-binding fragments thereof that specifically bind to a CD43 polypeptide or peptide (and epitopes thereof) with improved binding affinity. Yeast display, for instance, is well-suited for affinity maturation, and has been used previously to improve the affinity of a single-chain antibody to a KD of 48 fM (Boder et al. (Proc Natl Acad Sci USA 9TA I ., 2000)).

[0189] Additional in vitro techniques that can be used for the generation and affinity maturation of CD43-binding polypeptides, antibodies, and antigen-binding fragments thereof (e.g., singlechain polypeptides, antibodies, and antigen-binding fragments thereof) of some aspects and embodiments herein include the screening of combinatorial libraries of antibodies or antigenbinding fragments thereof for functional molecules capable of specifically binding to peptides derived from the CD43 polypeptide. Combinatorial antibody libraries can be obtained, e.g., by expression of polynucleotides encoding randomized hypervariable regions of an antibody or antigen-binding fragment thereof in a eukaryotic or prokaryotic cell. This can be achieved, e.g., using gene expression techniques described herein or known in the art. Heterogeneous mixtures of antibodies can be purified, e.g., by Protein A or Protein G selection, sizing column chromatography), centrifugation, differential solubility, and / or by any other standard technique for the purification of proteins. Libraries of combinatorial libraries thus obtained can be Atorney Docket No.: 167741-053601 / PCT

[0190] Electronic Deposit Date: October 27, 2025 screened, e.g., by incubating a heterogeneous mixture of these antibodies with a peptide derived from the CD43 polypeptide that has been immobilized to a surface for a period of time sufficient to allow antibody-antigen binding. Non-binding antibodies or fragments thereof can be removed by washing the surface with an appropriate buffer (e.g., a solution buffered at physiological pH (approximately 7.4) and containing physiological salt concentrations and ionic strength, and optionally containing a detergent, such as TWEEN-20®). Antibodies that remain bound can subsequently be detected, e.g., using an ELISA-based detection protocol (see, e.g., U.S. Patent No. 4,661,445; incorporated herein by reference).

[0191] Additional techniques for screening combinatorial libraries of polypeptides (e.g., antibodies, and antigen-binding fragments thereof) for those that specifically bind to CD43 polypeptide-derived peptides include the screening of one-bead-one-compound libraries of antibody fragments. Antibody fragments can be chemically synthesized on a solid bead (e.g., using established split-and-pool solid phase peptide synthesis protocols) composed of a hydrophilic, water-swellable material such that each bead displays a single antibody fragment. Heterogeneous bead mixtures can then be incubated with a CD43 polypeptide-derived peptide that is optionally labeled with a detectable moiety (e.g., a fluorescent dye) or that is conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HA tag) that can later be detected by treatment with a complementary tag (e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody, respectively). Beads containing antibody portions or fragments that specifically bind to a CD43 polypeptide-derived peptide can be identified by analyzing the fluorescent properties of the beads following incubation with a fluorescently-labeled antigen or complementary tag (e.g., by confocal fluorescent microscopy or by fluorescence-activated bead sorting; see, e.g., Muller et al. (J. Biol. Chem., 16500-16505, 1996); incorporated herein by reference). Beads containing antibody fragments that specifically bind to CD43 polypeptide-derived peptides can thus be separated from those that do not contain high-affinity antibody fragments. The sequence of an antibody fragment that specifically binds to a CD43 polypeptide-derived peptide can be determined by techniques known in the art, including, e.g., Edman degradation, tandem mass spectrometry, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gel electrophoresis, among others (see, e.g., WO 2004 / 062553; incorporated herein by reference).

[0192] Further methods for screening libraries of antibodies (e.g., VHH antibodies) for those capable of selectively binding to a target antigen also include those described in International Patent Application No. PCT / US24 / 34097, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Atorney Docket No.: 167741-053601 / PCT

[0193] Electronic Deposit Date: October 27, 2025

[0194] Methods of Identifying Antibodies and Ligands

[0195] Methods for high throughput screening of antibody, antibody fragment, and ligand libraries for molecules capable of binding the CD43 polypeptide or peptide can be used to identify antibodies suitable for the uses as described herein. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate ligands that bind biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1 : 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1- 20, 1998, the disclosures of each of which are incorporated herein by reference as they pertain to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind cell surface antigens as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Afo / . Divers. 1 : 139-140, 1996, the disclosures of each of which are incorporated herein by reference as they pertain to the discovery of antigenbinding molecules. Proteins, such as multimeric proteins, have been successfully phage- displayed as functional molecules (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as they pertain to the use of in vitro display techniques for the discovery of antigen-binding molecules). In addition, functional antibody fragments, such as Fab and scFv fragments, have been expressed in in vitro display formats (see, for example, McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they pertain to in vitro display platforms for the discovery of antigen-binding molecules). These techniques, among others, can be used to identify and improve the affinity of antibodies that bind to the CD43 polypeptide or peptide.

[0196] Expression of Anti-CD43 Antibodies

[0197] Mammalian cells can be transfected with a polynucleotide(s) encoding an antibody of some aspects and embodiments herein, which are expressed as recombinant polypeptides. In one embodiment, a mammalian cell is co-transfected with polynucleotides encoding the heavy and light chains of an anti-CD43 polypeptide antibody, which are expressed in the cell and assembled as the anti-CD43 antibody. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0198] It is possible to express antibodies or antigen-binding fragments thereof in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of polypeptides or antigen-binding fragments thereof is performed in eukaryotic cells, e.g., mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody. Exemplary, nonlimiting mammalian host cells for expressing the recombinant antibodies or antigen-binding fragments thereof of some aspects and embodiments herein include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, HEK293T cells, SP2 / 0, NIH3T3, and BaF3 cells. Additional, nonlimiting cell types that may be useful for the expression of antibodies and fragments thereof include bacterial cells, such as BL-21(DE3) E. coli cells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expression of antibodies include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete medium according to established procedures known in the art. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody protein in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.

[0199] Polypeptides (e.g., antibodies or antigen-binding fragments thereof) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Also included in some aspects and embodiments herein are methods in which the above procedure is varied according to established protocols known in the art. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-CD43 antibody of some aspects and embodiments herein in order to produce an antigen-binding fragment of the antibody.

[0200] Once a CD43-binding polypeptide (e.g., an anti-CD43 polypeptide antibody or an antigen-binding fragment thereof) of some aspects and embodiments herein has been produced by recombinant expression, it can be purified by any method known in the art, such as a method useful for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, affinity for antigen (e.g., a CD43 polypeptide or peptide) after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, an CD43- Atorney Docket No.: 167741-053601 / PCT

[0201] Electronic Deposit Date: October 27, 2025 binding polypeptide (e.g., an anti-CD43 antibody of some aspects and embodiments described herein ) or an antigen-binding portion or fragment thereof, can be fused to heterologous polypeptide sequences as known in the art, for example, to facilitate purification, e.g., a histidine tag, a detectable / detectably labeled marker, and the like.

[0202] Once isolated, an anti-CD43 antibody, or antigen-binding portion or fragment thereof can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a Superdex.TM. 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0203] Pharmaceutical Compositions

[0204] Provided also are pharmaceutical compositions for use in treating a neoplasia, increasing killing of a cell (e.g., by an immune cell), and / or increasing phagocytosis of a cell. In an embodiment, the compositions include an anti-CD43 antibody capable of selectively binding to a CD43 protein expressed on the surface of a cancer cell, as described herein, and an acceptable carrier, excipient, or diluent.

[0205] The agents of the disclosure (e.g., an anti-CD43 antibody) may be contained in any appropriate amount in any suitable carrier substance and are generally present in an amount of 0.01-95% by weight of the total weight of the composition. The pharmaceutical composition may be provided in a form that is suitable for a parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route, such that the agent, such as a vector described herein, is systemically delivered.

[0206] The pharmaceutical compositions of the present disclosure can be prepared in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005). In general, the immune cell, or population thereof is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, Atorney Docket No.: 167741-053601 / PCT

[0207] Electronic Deposit Date: October 27, 2025 dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

[0208] Some nonlimiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

[0209] Pharmaceutical compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.

[0210] Pharmaceutical compositions can also contain one or more osmotic modulating agents, / .< ., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and / or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art Atorney Docket No.: 167741-053601 / PCT

[0211] Electronic Deposit Date: October 27, 2025 may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and / or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.

[0212] The skilled artisan may determine the number of cells and amount of optional additives, vehicles, and / or carriers in compositions and to be administered in methods of the present disclosure. Typically, additives are present in an amount of 0.001 to 50 % (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt%, about 0.0001 to about 1 wt%, about 0.0001 to about 0.05 wt% or about 0.001 to about 20 wt%, about 0.01 to about 10 wt%, or about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it may be advantageous to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent such as a mouse); and, the dosage of the composition(s), concentration of components therein, and the timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein, and the time for sequential administrations can be ascertained without undue experimentation.

[0213] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. The pharmaceutical composition may be administered systemically.

[0214] The pharmaceutical composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation. Apart from the agent (e.g., CAR T cells, immune checkpoint blockade (ICB), or other chemotherapeutic agent), the composition may include suitable parenterally acceptable carriers and / or excipients. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and / or dispersing, agents. Atorney Docket No.: 167741-053601 / PCT

[0215] Electronic Deposit Date: October 27, 2025

[0216] In some embodiments, the pharmaceutical composition are formulated for intravenous delivery. As noted above, the compositions according to the described embodiments may be in a form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be employed include water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3 -butanediol, Ringer's solution, isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p- hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10- 60% w / w of propylene glycol or the like.

[0217] The pharmaceutical composition described herein can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; / .< ., carrier, or vehicle.

[0218] Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as chickens, ducks, geese, and / or turkeys.

[0219] Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0220] In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and / or conditions. Atorney Docket No.: 167741-053601 / PCT

[0221] Electronic Deposit Date: October 27, 2025

[0222] Genome Editing

[0223] In various embodiments, immune cells of the present disclosure are modified using genome editing. Immune cells can be modified by knocking out (e.g., by deletion) a target gene(s). Gene editing tools provide the ability to manipulate the DNA sequence of a cell (e.g., to delete a target gene) at a specific chromosomal locus, without introducing mutations at other sites of the genome. This technology effectively enables the researcher to manipulate the genome of a subject’s cells in vitro, ex vivo, or in vivo.

[0224] In one embodiment, gene editing involves targeting an endonuclease (an enzyme that causes DNA breaks internally within a DNA molecule) to a specific site of the genome and thereby triggering formation of a chromosomal double strand break (DSB) at the chosen site. If, concomitant with the introduction of the chromosome breaks, a donor DNA molecule may be introduced (for example, by plasmid or oligonucleotide introduction), interactions between the broken chromosome and the introduced DNA can occur, especially if the two sequences share homology. In this instance, a process termed “gene targeting” can occur, in which the DNA ends of the chromosome invade homologous sequences of the donor DNA by homologous recombination (HR). By using the donor plasmid sequence as a template for HR, a seamless repair of the chromosomal DSB can be accomplished. In some embodiments, no donor DNA molecule is introduced and the double-stranded break is repaired by the error-prone non- homologous end joining NHEJ pathway leading to knock-out or deletion of the target gene (e.g., through the introduction of indels or nonsense mutations). In some embodiments, an endonuclease(s) can be targeted to at least two distinct chosen sites located within a gene sequence so that chromosomal double strand breaks at the distinct sites leads to excision and deletion of a nucleotide sequence flanked by the two distinct sites.

[0225] Current genome editing tools use the induction of double strand breaks (DSBs) to enhance gene manipulation of cells, including the deletion or knockout of genes. Such methods include zinc finger nucleases (ZFNs; described for example in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, and U.S. Pat. Publ. Nos. 20030232410 and US2009020314, which are incorporated herein by reference), Transcription Activator-Like Effector Nucleases (TALENs; described for example in U.S. Patent Nos. 8,440,431, 8,440,432, 8,450,471, 8,586,363, and 8,697,853, and U.S. Pat. Publ. Nos. 20110145940, 20120178131, 20120178169, 20120214228, 20130122581, 20140335592, and 20140335618, which are incorporated herein by reference), and the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system (described for example in U.S. Atorney Docket No.: 167741-053601 / PCT

[0226] Electronic Deposit Date: October 27, 2025

[0227] Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,871,445, 8,889,356, 8,906,616, 8,932,814, 8,945,839, 8,993,233, and 8,999,641, and U.S. Pat. Publ. Nos. 20140170753, 20140227787, 20140179006, 20140189896, 20140273231, 20140242664, 20140273232, 20150184139, 20150203872, 20150031134, 20150079681, 20150232882, and 20150247150, which are incorporated herein by reference). For example, ZFN DNA sequence recognition capabilities and specificity can be unpredictable. Similarly, TALENs and CRISPR / Cas9 cleave not only at the desired site, but often at other “off-target” sites, as well. These methods have significant issues connected with off-target double-stranded break induction and the potential for deleterious mutations, including indels, genomic rearrangements, and chromosomal rearrangements, associated with these off-target effects. ZFNs and TALENs entail use of modular sequencespecific DNA binding proteins to generate specificity for ~18 bp sequences in the genome.

[0228] RNA-guided nucleases-mediated genome editing, based on Type 2 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR Associated) systems, offers a valuable approach to alter the genome. In brief, Cas9, a nuclease guided by single-guide RNA (sgRNA), binds to a targeted genomic locus next to the protospacer adjacent motif (PAM) and generates a double-strand break (DSB). The DSB is then repaired either by non-homologous end joining (NHEJ), which leads to insertion / deletion (indel) mutations, or by homology-directed repair (HDR), which requires an exogenous template and can generate a precise modification at a target locus (Mali et al., Science. 2013 Feb 15;339(6121): 823-6). Genetic manipulation using engineered nucleases has been demonstrated in tissue culture cells and rodent models of rare diseases.

[0229] CRISPR has been used in a wide range of organisms including baker’s yeast (5. cerevisiae), zebra fish, nematodes (C. elegans), plants, mice, and several other organisms. Additionally, CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. Libraries of tens of thousands of guide RNAs are now available.

[0230] Since 2012, the CRISPR / Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates. By inserting a plasmid containing Cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.

[0231] CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 prokaryote's genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.

[0232] CRISPR-associated (Cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR / Cas systems. Particular combinations of Cas genes and repeat structures have been used to define 8 CRISPR subtypes (E. coh, Y. pest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR / Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

[0233] Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (about 30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype E. coif) proteins (called CasA-E in E. colt) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacerrepeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in / ■ / coli requires Cascade and Cas3, but not Casl and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. See also U.S. Patent Publication 2014 / 0068797, which is incorporated by reference in its entirety.

[0234] In another embodiment, gene editing may involve the use of a base editor or PRIME editing to alter a gene such that expression of the gene is disrupted and / or activity of a polypeptide encoded by the gene is reduced or eliminated in a cell. Methods for base editing and prime editing are well known in the art and described, for example, in Kantor, et al., “CRISPR- Cas9 DNA Base-Editing and Prime Editing,” IntJMol Sci, 21 :6240 (2020). Atorney Docket No.: 167741-053601 / PCT

[0235] Electronic Deposit Date: October 27, 2025

[0236] Cas9 and Casl2

[0237] Cas9 and Casl2 are polynucleotide programmable endonucleases, enzymes specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. Researchers have demonstrated that they could disable one or both sites while preserving a programmable endonuclease’s ability to home located its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA into a “single-guide RNA” molecule that, mixed with Cas9, could find and cut the correct DNA targets. It has been proposed that such synthetic guide RNAs might be able to be used for gene editing (Jinek et al., Science. 2012 Aug 17;337(6096):816-21). In various embodiments, the Cas9 nuclease may be substituted with any polynucleotide capable of binding a guide RNA and being specifically directed thereby to bind a target nucleotide sequence (i.e., a polynucleotide programmable endonuclease) (see, e.g., Yamagata, SynBio, 1 :65-76 (2023)).

[0238] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR / Cas- mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR / Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Delivery of Cas9 or Casl2 DNA sequences also is contemplated. guide RNA (gRNA)

[0239] As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to a targeting sequence (i.e., a “spacer”) within the gRNA (alternatively referred to as a single guide RNA (sgRNA)) to bind to a target sequence (e.g., to create a double strand break). In some embodiments, a spacer of a gRNA is about 18, 19, 20, 21, or 22 nucleotides in length. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type 21 promoter U6). Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a portion (i.e., the “spacer”) which targets a ~20 protospacer nucleotides immediately preceding the protospacer-adjacent motif (PAM) sequence NGG; gRNAs do not contain a PAM sequence. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0240] Combination Therapies

[0241] Compositions and methods of the disclosure may be used in combination with any conventional therapy known in the art. In one embodiment, an anti-CD43 antibody of the disclosure that selectively targets a neoplastic cell may be used in combination with any anti- neoplastic therapy known in the art. Exemplary anti -neoplastic therapies include, for example, chemotherapy, cryotherapy, hormone therapy, immunotherapy (e.g., administration of CAR-T cells or other immune effector cells to a subject in need thereof), radiotherapy, and surgery. Non-limiting examples of chemotherapeutics include abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS184476, 2,3,4,5,6-pentafluoro-N- (3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N- methyl-L-valyl-L-proly- 1-Lproline-t-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'-deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU), cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin), etoposide, 5 -fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6.sup.th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers.

[0242] Characterizing Cells

[0243] In various aspects, the methods of the disclosure involve characterizing a neoplasia and / or cell(s). In some instances, the characterization of a neoplasia involves determining whether or not the neoplasia expresses CD43. Such characterization and measurements can be carried out using methods familiar to one of skill in the art, which include, but are not limited to, those described herein.

[0244] Gene expression levels can be detected using biomarkers (e.g., CD43 polynucleotides or CD43 polypeptides, or fragments thereof). In some cases, a biomarker is a polynucleotide (e.g., mRNA, a portion of a genome, and / or a gene). The biomarkers of this disclosure can be detected by any suitable method. The methods described herein can be used individually or in Atorney Docket No.: 167741-053601 / PCT

[0245] Electronic Deposit Date: October 27, 2025 combination for a more accurate detection of the biomarkers (e.g., biochip in combination with mass spectrometry, immunoassay in combination with mass spectrometry, and the like).

[0246] Detection paradigms that can be employed in the disclosure include, but are not limited to, optical methods, electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).

[0247] These and additional methods are describe below.

[0248] Detection by sequencing and / or probes

[0249] In particular embodiments, the biomarkers of the disclosure are measured by a sequencing- and / or probe-based technique (e.g., RNA-seq).

[0250] RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling. In embodiments, to mitigate sequence-dependent bias resulting from amplification complications to allow truly digital RNA-Seq, a set of barcode sequences can be used to ensure that every cDNA molecule prepared from an mRNA sample is uniquely labeled by random attachment of barcode sequences to both ends (see, e.g., Shiroguchi K, et al. Proc Natl Acad Sci USA. 2012 Jan. 24;109(4): 1347-52). After PCR, paired-end deep sequencing can be applied to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance can be measured based on the number of unique barcode sequences observed for a given cDNA sequence. The barcodes may be optimized to be unambiguously identifiable. This method is a representative example of how to quantify a whole transcriptome from a sample.

[0251] Detecting a target polynucleotide sequence or fragment thereof associated with a biomarker that hybridizes to a probe sequence may involve sequencing, FACS, qPCR, RT-PCR, a genotyping array, and / or a NanoString assay (see, e.g., Malkov, et al. “Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System”, BMC Research Notes, 2: Article No: 80 (2009)), or any of various other techniques known to one of skill in the art. Various detection methods may be used and are described as follows.

[0252] Preparation of a library for sequencing may involve an amplification step. Amplification may involve thermocycling or isothermal amplification (such as through the methods RPA or LAMP). Cross-linking may involve overlap-extension PCR or use of ligase to associate multiple Atorney Docket No.: 167741-053601 / PCT

[0253] Electronic Deposit Date: October 27, 2025 amplification products with each other. Amplification can refer to any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR. In particular, the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT-PCR) in order to quantify the expression level of a biomarker.

[0254] Detection of the expression level of a biomarker can be conducted in real time in an amplification assay (e.g., qPCR). In one aspect, the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art. DNA-binding dyes suitable for this application include, as non-limiting examples, SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.

[0255] Other fluorescent labels such as sequence specific probes can be employed in the amplification reaction to facilitate the detection and quantification of the amplified products. Probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are taught, for example, in U.S. Pat. No. 5,210,015.

[0256] Sequencing may be performed on any high-throughput platform. Methods of sequencing oligonucleotides and nucleic acids are well known in the art (see, e.g., WO93 / 23564, WO98 / 28440 and WO98 / 13523; U.S. Pat. App. Pub. No. 2019 / 0078232; U.S. Pat. Nos. 5,525,464; 5,202,231; 5,695,940; 4,971,903; 5,902,723; 5,795,782; 5,547,839 and 5,403,708; Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977); Drmanac et al., Genomics 4:114 (1989); Koster et al., Nature Biotechnology 14: 1123 (1996); Hyman, Anal. Biochem. 174:423 (1988); Rosenthal, International Patent Application Publication No. WO1993021340A1; Metzker et al., Nucl. Acids Res. 22:4259 (1994); Jones, Biotechniques 22:938 (1997); Ronaghi et al., Anal. Biochem. 242:84 (1996); Ronaghi et al., Science 281 :363 (1998); Nyren et al., Anal. Biochem. 151 :504 (1985); Canard and Arzumanov, Gene 11 : 1 (1994); Dyatkina and Atorney Docket No.: 167741-053601 / PCT

[0257] Electronic Deposit Date: October 27, 2025

[0258] Arzumanov, Nucleic Acids Symp Ser 18: 117 (1987); Johnson et al., Anal. Biochem. 136: 192 (1984); and Eigen and Rigler, Proc. Natl. Acad. Sci. USA 91(13):5740 (1994), all of which are expressly incorporated by reference).

[0259] The sequencing of a polynucleotide can be carried out using any suitable commercially available sequencing technology. In embodiments, the sequencing of a polynucleotide is carried out using a chain termination method of DNA sequencing (e.g., Sanger sequencing). In some embodiments, commercially available sequencing technology is a next-generation sequencing technology, including as non-limiting examples combinatorial probe anchor synthesis (cPAS), DNA nanoball sequencing, droplet-based or digital microfluidics, heliscope single molecule sequencing, nanopore sequencing (e.g., Oxford Nanopore technologies), GeneGap sequencing, massively parallel signature sequencing (MPSS), microfluidic Sanger sequencing, microscopybased techniques (e.g., transmission electronic microscopy DNA sequencing), RNA polymerase (RNAP) sequencing, single-molecule real-time (SMRT) sequencing, SOLiD sequencing, ion semiconductor sequencing, polony sequencing, Pyrosequencing (454), sequencing by hybridization, sequencing by synthesis (e.g., Illumina™ sequencing), sequencing with mass spectrometry, and tunneling currents DNA sequencing.

[0260] In embodiments, levels of biomarkers in a sample are quantified using targeted sequencing. Methods for targeted sequencing are well known in the art (see, e.g., Rehm, “Disease-targeted sequencing: a cornerstone in the clinic”, Nature Reviews Genetics, 14:295-300 (2013)).

[0261] In embodiments, a probe comprises a molecular identifier, such as a fluorescent or chemiluminescent label, a radioactive isotope label, an enzymatic ligand, or the like. The molecular identifier can be a fluorescent label or an enzyme tag, such as digoxigenin, P- galactosidase, urease, alkaline phosphatase or peroxidase, avidin / biotin complex.

[0262] Methods used to detect or quantify binding of a probe to a target biomarker will typically depend upon the molecular identifier. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. Enzymatic labels can be detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and colorimetric labels can be detected by visualizing a colored label.

[0263] Specific non-limiting examples of molecular identifiers include radioisotopes, such as 32P, 14C, 1251, 3H, and 1311, fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, P-galactosidase, P-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin, and ruthenium. In the case Atorney Docket No.: 167741-053601 / PCT

[0264] Electronic Deposit Date: October 27, 2025 where biotin is employed as a molecular identifier, streptavidin bound to an enzyme (e.g., peroxidase) may further be added to facilitate detection of the biotin.

[0265] Examples of fluorescent molecular identifiers include, but are not limited to, Atto dyes, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS); 4-amino- N-[3-vinyl sulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-naphthyl)mal eimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5'5"-dibromopyrogallol- sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansylchloride); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives; eosin, eosin isothiocyanate, erythrosin and derivatives; erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N' tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine

[0266] A fluorescent molecular identifier may be a fluorescent protein, such as blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, red fluorescent protein, yellow fluorescent protein or any photoconvertible protein. Colorimetric molecular identifiers, bioluminescent molecular identifiers and / or chemiluminescent molecular identifiers may be used in embodiments of the disclosure. Atorney Docket No.: 167741-053601 / PCT

[0267] Electronic Deposit Date: October 27, 2025

[0268] Detection of a molecular identifier may involve detecting energy transfer between molecules in a hybridization complex by perturbation analysis, quenching, or electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes. The fluorescent molecular identifier may be a perylene or a terrylen. In the alternative, the fluorescent molecular identifier may be a fluorescent bar code.

[0269] The molecular identifier may be light sensitive, wherein the label is light-activated and / or light cleaves the one or more linkers to release the molecular cargo. The light-activated molecular cargo may be a major light-harvesting complex (LHCII). In another embodiment, the fluorescent molecular label may induce free radical formation.

[0270] In an advantageous embodiment, agents may be uniquely labeled in a dynamic manner (see, e.g., international patent application serial no. PCT / US2013 / 61182 filed Sep. 23, 2012). The unique labels are, at least in part, nucleic acid in nature, and may be generated by sequentially attaching two or more detectable oligonucleotide tags to each other and each unique label may be associated with a separate agent. A detectable oligonucleotide tag may be an oligonucleotide that may be detected by sequencing of its nucleotide sequence and / or by detecting non-nucleic acid detectable moieties to which it may be attached.

[0271] In embodiments, the molecular identifier is a microparticle, including, as non-limiting examples, quantum dots (Empodocles, et al., Nature 399: 126-130, 1999), or gold nanoparticles (Reichert et al., Anal. Chem. 72:6025-6029, 2000).

[0272] Detection by Immunoassay

[0273] In particular embodiments, the biomarkers of the disclosure are measured by immunoassay. Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.

[0274] This disclosure contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence- based immunoassays (e.g., flow cytometry), and chemiluminescence. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR). Atorney Docket No.: 167741-053601 / PCT

[0275] Electronic Deposit Date: October 27, 2025

[0276] Immunoassays can be carried out on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection. A single marker may be detected at a time or a multiplex format may be used. Multiplex immunoanalysis may involve planar microarrays (protein chips) and beadbased microarrays (suspension arrays).

[0277] In a SELDI-based immunoassay, a biospecific capture reagent for the biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry.

[0278] Detection by Biochip

[0279] In embodiments, a sample is analyzed by means of a biochip (also known as a microarray). The polypeptides and nucleic acid molecules of the disclosure are useful as hybridizable array elements in a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

[0280] The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14: 1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93: 10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289: 1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.

[0281] Detection by Protein Biochip

[0282] In embodiments, a sample is analyzed by means of a protein biochip (also known as a protein microarray). Such biochips are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a biomarker, or a fragment thereof. In embodiments, a protein biochip of the disclosure binds a biomarker present in a Atorney Docket No.: 167741-053601 / PCT

[0283] Electronic Deposit Date: October 27, 2025 sample and detects an alteration in the level of the biomarker. Typically, a protein biochip features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer- based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the disclosure) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).

[0284] In embodiments, the protein biochip is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of nonspecific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.

[0285] Many protein biochips are described in the art. These include, for example, protein biochips produced by CIPHERGEN BIOSYSTEMS™ (Fremont, CA), Zyomyx (Hayward, CA), Packard BioScience Company (Meriden, CT), Phylos (Lexington, MA), Invitrogen (Carlsbad, CA), Biacore (Uppsala, Sweden) and Procognia (Berkshire, UK). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent Nos. 6,225,047; 6,537,749; 6,329,209; and 5,242,828; PCT International Publication Nos. WO 00 / 56934; WO 03 / 048768; and WO 99 / 51773.

[0286] Detection by Nucleic Acid Biochip

[0287] In aspects of the disclosure, a sample is analyzed by means of a nucleic acid biochip (also known as a nucleic acid microarray). To produce a nucleic acid biochip, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an inkjet application apparatus, as described in PCT application WO 1995 / 025116 Al (Baldeschweiler, et al.). Alternatively, a gridded array may be used to arrange and link cDNA Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.

[0288] A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, e.g., as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are well known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the biochip.

[0289] Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions include, as non-limiting examples, temperatures of at least about 30 °C, of at least about 37 °C, or of at least about 42 °C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In an embodiment, hybridization will occur at 30 °C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In embodiments, hybridization will occur at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In other embodiments, hybridization will occur at 42 °C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0290] The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency Atorney Docket No.: 167741-053601 / PCT

[0291] Electronic Deposit Date: October 27, 2025 can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, of at least about 42 °C, or of at least about 68 °C. In embodiments, wash steps will occur at 25 °C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In other embodiments, wash steps will occur at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

[0292] Detection system for measuring the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences are well known in the art. For example, simultaneous detection is described in Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997. In embodiments, a scanner is used to determine the levels and patterns of fluorescence.

[0293] Detection by Mass Spectrometry

[0294] In embodiments, the biomarkers of this disclosure are detected by mass spectrometry (MS). Mass spectrometry is a well-known tool for analyzing chemical compounds that employs a mass spectrometer to detect gas phase ions. Mass spectrometers are well known in the art and include, but are not limited to, time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The method may be performed in an automated (Villanueva, et al., Nature Protocols (2006) l(2):880-891) or semiautomated format. This can be accomplished, for example with the mass spectrometer operably linked to a liquid chromatography device (LC-MS / MS or LC-MS) or gas chromatography device (GC-MS or GC-MS / MS). Methods for performing mass spectrometry are well known and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; US Patent No. 5,800,979 and the references disclosed therein.

[0295] Laser Desorption / Ionization

[0296] In embodiments, the mass spectrometer is a laser desorption / ionization mass spectrometer. In laser desorption / ionization mass spectrometry, the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from Atorney Docket No.: 167741-053601 / PCT

[0297] Electronic Deposit Date: October 27, 2025 an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer. The analysis of proteins by LDI can take the form of MALDI or of SELDI. The analysis of proteins by LDI can take the form of MALDI or of SELDI.

[0298] Laser desorption / ionization in a single time of flight instrument typically is performed in linear extraction mode. Tandem mass spectrometers can employ orthogonal extraction modes.

[0299] Matrix-assisted Laser Desorption / ionization (MALDI) and Electrospray Ionization (ESI)

[0300] In embodiments, the mass spectrometric technique for use in the disclosure is matrix- assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI). In related embodiments, the procedure is MALDI with time of flight (TOF) analysis, known as MALDL TOF MS. This involves forming a matrix on a membrane with an agent that absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV or IR laser light into the vapor phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass / charge (m / z) reading which is very accurate and sensitive. MALDI spectrometers are well known in the art and are commercially available from, for example, PERCEPTIVE BIOSYSTEMS™ (Framingham, Mass., USA).

[0301] Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, in embodiments, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.

[0302] MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the disclosure to produce an array of spots on a collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using an server (e.g., ExPASy) to generate the data in a form suitable for computers.

[0303] Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on a collection membrane. These include, but are not limited to, the use of delayed ion extraction, energy reflectors, ion-trap modules, and the like. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 ion-trap, TOF, single quadrupole, multi-quadrupole mass spectrometers, and the like. The use of such devices (other than a single quadrupole) allows MS-MS or MSnanalysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.

[0304] Capillary infusion may be employed to introduce the biomarker to a desired mass spectrometer implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques including, but not limited to, gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with mass spectrometry. One variation of the technique is the coupling of high-performance liquid chromatography (HPLC) to a mass spectrometer for integrated sample separation / and mass spectrometer analysis.

[0305] Quadrupole mass analyzers may also be employed as needed to practice the various aspects and embodiments of the disclosure. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some embodiments. It offers high resolution and the ability of tandem mass spectrometry experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.

[0306] Surface-enhanced laser desorption / ionization (SELDI)

[0307] In embodiments, the mass spectrometric technique for use in the various aspects or embodiments of the disclosure is “Surface Enhanced Laser Desorption and Ionization” or “SELDI,” as described, for example, in U.S. Patents No. 5,719,060 and No. 6,225,047, both to Hutchens and Yip. This refers to a method of desorption / ionization gas phase ion spectrometry (e.g., mass spectrometry) in which an analyte (here, one or more of the biomarkers) is captured on the surface of a SELDI mass spectrometry probe.

[0308] SELDI has also been called “affinity capture mass spectrometry.” It also is called “Surface-Enhanced Affinity Capture” or “SEAC”. This version involves the use of probes that have a material on the probe surface that captures analytes through a non-covalent affinity interaction (adsorption) between the material and the analyte. The material is variously called an “adsorbent,” a “capture reagent,” an “affinity reagent” or a “binding moiety.” Such probes can be referred to as “affinity capture probes” and as having an “adsorbent surface.” The capture reagent can be any material capable of binding an analyte. The capture reagent is attached to the probe surface by physisorption or chemisorption. In certain embodiments the probes have the Atorney Docket No.: 167741-053601 / PCT

[0309] Electronic Deposit Date: October 27, 2025 capture reagent already attached to the surface. In other embodiments, the probes are preactivated and include a reactive moiety that is capable of binding the capture reagent, e.g., through a reaction forming a covalent or coordinate covalent bond. Epoxide and acyl-imidizole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors. Nitrilotriacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact non-covalently with histidine containing peptides. Adsorbents are generally classified as chromatographic adsorbents and biospecific adsorbents.

[0310] “Chromatographic adsorbent” refers to an adsorbent material typically used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators (e.g., nitrilotriacetic acid or iminodiacetic acid), immobilized metal chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple biomolecules (e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g., hydrophobic attraction / electrostatic repulsion adsorbents).

[0311] A biospecific adsorbent is an adsorbent comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). In certain instances, the biospecific adsorbent can be a macromolecular structure such as a multi protein complex, a biological membrane or a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic acids. Biospecific adsorbents typically have higher specificity for a target analyte than chromatographic adsorbents. Further examples of adsorbents for use in SELDI can be found in U.S. Patent No. 6,225,047. A “bioselective adsorbent” refers to an adsorbent that binds to an analyte with an affinity of at least 10'8M.

[0312] Protein biochips produced by CIPHERGEN™ comprise surfaces having chromatographic or biospecific adsorbents attached thereto at addressable locations. CIPHERGEN™’ s PROTEINCHIP® arrays include NP20 (hydrophilic); H4 and H50 (hydrophobic); SAX-2, Q-10 and (anion exchange); WCX-2 and CM- 10 (cation exchange); IMAC-3, IMAC-30 and IMAC-50 (metal chelate); and PS-10, PS-20 (reactive surface with acylimidazole, epoxide) and PG-20 (protein G coupled through acyl-imidazole). Hydrophobic ProteinChip arrays have isopropyl or nonylphenoxy-poly(ethylene glycol)methacrylate functionalities. Anion exchange ProteinChip arrays have quaternary ammonium functionalities. Cation exchange ProteinChip arrays have carboxylate functionalities. Immobilized metal chelate ProteinChip arrays have nitrilotriacetic acid functionalities (IMAC 3 and IMAC 30) or O- methacryloyl-N,N-bis-carboxymethyl tyrosine functionalities (IMAC 50) that adsorb transition Atorney Docket No.: 167741-053601 / PCT

[0313] Electronic Deposit Date: October 27, 2025 metal ions, such as copper, nickel, zinc, and gallium, by chelation. Preactivated ProteinChip arrays have acyl-imidazole or epoxide functional groups that can react with groups on proteins for covalent binding.

[0314] Such biochips are further described in: U.S. Patent No. 6,579,719 (Hutchens and Yip, “Retentate Chromatography,” June 17, 2003); U.S. Patent 6,897,072 (Rich et al., “Probes for a Gas Phase Ion Spectrometer,” May 24, 2005); U.S. Patent No. 6,555,813 (Beecher et al., “Sample Holder with Hydrophobic Coating for Gas Phase Mass Spectrometer,” April 29, 2003); U.S. Patent Publication No. U.S. 2003 -0032043 Al (Pohl and Papanu, “Latex Based Adsorbent Chip,” July 16, 2002); and PCT International Publication No. WO 03 / 040700 (Um et al., “Hydrophobic Surface Chip,” May 15, 2003); U.S. Patent Application Publication No. US 2003 / -0218130 Al (Boschetti et al., “Biochips With Surfaces Coated With Polysaccharide- Based Hydrogels,” April 14, 2003) and U.S. Patent 7,045,366 (Huang et al., “Photocrosslinked Hydrogel Blend Surface Coatings” May 16, 2006).

[0315] In general, a probe with an adsorbent surface is contacted with the sample for a period of time sufficient to allow the biomarker or biomarkers that may be present in the sample to bind to the adsorbent. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; preferably, aqueous solutions are employed. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature. Unless the probe has both SEAC and SEND properties (as described herein), an energy absorbing molecule then is applied to the substrate with the bound biomarkers.

[0316] In yet another method, one can capture the biomarkers with a solid-phase bound immuno-adsorbent that has antibodies that bind the biomarkers. After washing the adsorbent to remove unbound material, the biomarkers are eluted from the solid phase and detected by applying to a SELDI biochip that binds the biomarkers and analyzing by SELDI.

[0317] The biomarkers bound to the substrates are detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer. The biomarkers are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a biomarker typically will involve detection of signal intensity. Thus, both the quantity and mass of the biomarker can be determined. Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0318] Computer Systems

[0319] The present disclosure also relates to a computer system involved in carrying out the methods of the disclosure.

[0320] A computer system (or digital device) may be used to receive, transmit, display and / or store results, analyze the results, and / or produce a report of the results and analysis. A computer system may be understood as a logical apparatus that can read instructions from media (e.g. software) and / or network port (e.g. from the internet), which can optionally be connected to a server having fixed media. A computer system may comprise one or more of a CPU, disk drives, input devices such as keyboard and / or mouse, and a display (e.g. a monitor). Data communication, such as transmission of instructions or reports, can be achieved through a communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and / or receiving data. For example, the communication medium can be a network connection, a wireless connection, or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections (or any other suitable means for transmitting information, including but not limited to mailing a physical report, such as a print-out) for reception and / or for review by a receiver. The receiver can be but is not limited to an individual, or electronic system (e.g. one or more computers, and / or one or more servers).

[0321] In some embodiments, the computer system may comprise one or more processors. Processors may be associated with one or more controllers, calculation units, and / or other units of a computer system, or implanted in firmware as desired. If implemented in software, the routines may be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other suitable storage medium. Likewise, this software may be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the internet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc. The various steps may be implemented as various blocks, operations, tools, modules, and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and / or software. When implemented in hardware, some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc. Atorney Docket No.: 167741-053601 / PCT

[0322] Electronic Deposit Date: October 27, 2025

[0323] A client-server, relational database architecture can be used in embodiments of the disclosure. A client-server architecture is a network architecture in which each computer or process on the network is either a client or a server. Server computers are typically powerful computers dedicated to managing disk drives (file servers), printers (print servers), or network traffic (network servers). Client computers include PCs (personal computers) or workstations on which users run applications, as well as example output devices as disclosed herein. Client computers rely on server computers for resources, such as files, devices, and even processing power. In some embodiments of the disclosure, the server computer handles all of the database functionality. The client computer can have software that handles all the front-end data management and can also receive data input from users.

[0324] A machine readable medium which may comprise computer-executable code may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0325] The subject computer-executable code can be executed on any suitable device which may comprise a processor, including a server, a PC, or a mobile device such as a smartphone or tablet. Any controller or computer optionally includes a monitor, which can be a cathode ray tube (“CRT”) display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display, etc.), or others. Computer circuitry is often placed in a box, which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The Atorney Docket No.: 167741-053601 / PCT

[0326] Electronic Deposit Date: October 27, 2025 box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard, mouse, or touch-sensitive screen, optionally provide for input from a user. The computer can include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.

[0327] A computer can transform data into various formats for display. A graphical presentation of the results of a calculation can be displayed on a monitor, display, or other visualizable medium (e.g., a printout). In some embodiments, data or the results of a calculation may be presented in an auditory form.

[0328] In aspects, software used to analyze the data can include code that applies an algorithm to the analysis of the results.

[0329] Types of Samples

[0330] This disclosure provides methods involving the characterization whether or not cells within a sample express CD43. In one embodiment, the samples are biological samples generally derived from a human subject, such as from a bodily fluid (such as ascites, blood, plasma, pleural fluid, serum, cerebrospinal fluid, phlegm, saliva, stool, urine, semen, prostate fluid, breast milk, or tears, or tissue sample (e.g. a tissue sample obtained by biopsy). In a further embodiment, the samples are biological samples derived from an animal, such as a bodily fluid (such as blood, cerebrospinal fluid, phlegm, saliva, or urine) or tissue sample (e.g. a tissue sample obtained by biopsy). In still another embodiment, the samples are biological samples from in vitro sources (such as cell culture medium). Cell free (cfDNA) attached to a substrate may be first suspended in a liquid medium, such as a buffer or a water, and then subject to sequencing and / or analysis. In yet another embodiment, the sample contains DNA within a cell, which may be extracted, sequenced and subject to the same analysis. In some instances, the sample is a biopsy (e.g., a needle biopsy) or a section.

[0331] Kits

[0332] The disclosure also provides kits for use in increasing phagocytosis of a cell, increasing killing of a cell (e.g., by an immune cell), and / or treating a neoplasia. Kits of the present disclosure may include one or more containers comprising an agent (e.g., an anti-CD43 antibody) for increasing phagocytosis and / or killing of a cell (e.g., for treatment of a neoplasia). In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, these instructions comprise a description of Atorney Docket No.: 167741-053601 / PCT

[0333] Electronic Deposit Date: October 27, 2025 use of the agent (e.g., an anti-CD43 antibody) of the kit for treatment of a neoplasia, increasing killing of a cell (e.g., by an immune cell), and / or increasing phagocytosis of a cell.

[0334] Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for practicing any of the methods described herein.

[0335] Components of the kits of this disclosure may be disposed within suitable packaging. Suitable packaging includes, but is not limited to, containers such as vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0336] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for specific embodiments will be discussed in the sections that follow.

[0337] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

[0338] EXAMPLES

[0339] Example 1: Development of a human macrophage co-culture assay to determine regulators of phagocytosis

[0340] To establish a scalable and sensitive phagocytosis assay for unbiased genetic screens, monocytes were isolated from healthy donors and an evaluation was performed of the mode of monocyte differentiation (M-CSF vs GM-CSF), macrophage stimulation (no stimulation, IL-6, Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0341] IFNy, TNFa, or LPS), and effector to target ratios on the phagocytosis of two human leukemia cell lines, MV411 and M0LM13. Phagocytosis was quantified with flow cytometry to measure the absolute number of remaining leukemia cells after 18-24 hours of co-culture compared to leukemia cells grown in the absence of macrophages (FIG. 6A). As a complementary approach, an evaluation was performed of phagocytosis of PHRODO™ fluorescent dye-labeled leukemia cells with live-cell imaging, which allowed for the identification of macrophages that had recently ingested leukemia cells (FIG. 6B).

[0342] Baseline phagocytosis of leukemia cells by unstimulated human macrophages was low (FIGs. 6B-6C). IFNy stimulation had the most potent effect on macrophage phagocytosis, enhancing absolute phagocytosis by 4-6 fold (FIGs. 6B-6C). Macrophage colony-stimulating factor (M-CSF) differentiated macrophages exhibited increased phagocytosis compared to granulocyte-macrophage colony-stimulating factor (GM-CSF) differentiated macrophages at all effectortarget ratios (FIG. 6D) and this mode of differentiation was selected for further phagocytosis assays.

[0343] Example 2: Genome-scale CRISPR screens revealed pathways that regulated antibodyindependent tumor phagocytosis

[0344] To define the tumor-intrinsic factors that regulated antibody-independent cellular phagocytosis (AICP), pooled genome-scale CRISPR knockout screens were performed by coculturing CRISPR-edited leukemia cells with IFNy-stimulated human macrophages (FIG. 1A). To increase generalizability, the screen was performed in two human acute myelogenous leukemia (AML) cell lines (MV411 and M0LM13), using different CRISPR enzymes and genome-targeting libraries (Cas9 and Casl2a, respectively). After 18 hours of co-culture, the pool of remaining CRISPR-edited leukemia cells were separated from the adherent macrophages, allowing for the sequencing of the single guide RNA (sgRNA) barcodes from both the leukemia and macrophage fractions (FIG. 1A). The leukemia fraction contained barcoded cells that had escaped phagocytosis while the macrophage fraction contained barcodes derived from recently phagocytosed cells. Quality control parameters demonstrated excellent screen performance, with the majority of sgRNAs being well represented in all conditions (FIGs. 7A- 7B). Guide RNA representation in each replicate was highly correlated to the original library (Pearson correlation > 0.8 in leukemia fraction and macrophage fractions) (FIGs. 7C-7F).

[0345] A comparison of the sgRNA abundances in the co-cultured leukemia and macrophage fractions to leukemia cells cultured alone was performed (FIGs. 1B-1C, FIGs. 1K-1L, FIGs. 8A-8D). Genetic deletion of APMAP in MV411 or M0LM13 cells resulted in depletion in the Atorney Docket No.: 167741-053601 / PCT

[0346] Electronic Deposit Date: October 27, 2025 leukemia fraction and enrichment in the macrophage fraction, providing confidence that the coculture system could identify known regulators of macrophage phagocytosis (FIGs. 1B-1C, FIGs. 8A-8B). Integrated analyses of leukemic and macrophage fractions in each screen revealed additional genes that scored as shared negative regulators of macrophage phagocytosis, including genes involved in O-linked glycosylation (SPN, C1GALT1, C1GALT1C1), TNF alpha signaling (PPP6C), and endosomal trafficking (VPS25), as well as genes that scored as cell linespecific regulators, including CDC7, ATP6V1A, MZT1 and VPS29 in MV411, and FADD, IKBKG, MAP3K7 and NHLRC2 in M0LM13 (FIG. 8A-D).

[0347] Example 3: Genetic loss of CD47 in human leukemia did not potently enhance phagocytosis by human macrophages

[0348] Treatment of leukemia cells with an anti-CD47 antibody (clone MIAP410) potently enhanced phagocytosis by unstimulated macrophages (FIG. 9A). In contrast, genetic deletion of CD47 in leukemia cells did not result in depletion in the leukemia fraction or enrichment in the macrophage fraction in the antibody-independent cellular phagocytosis (AICP) screens (FIGs. 1B-1C, FIGs. 8A-8D). Similarly, genetic deletion of QPCTL, a CD47-modifying enzyme required for optimal binding of CD47 to SIRPa, did not enhance phagocytosis (FIGs. 1B-1C). To ensure that these findings did not represent a false-negative result, CD47 was deleted in the MV41 1 and M0LM13 cell lines (FIGs. 9B-9C) and the functional impact of CD47 loss in competitive phagocytosis experiments by co-culturing 1 : 1 mixes of control and CD47-deficient leukemias with M-CSF differentiated human macrophages stimulated with LPS or IFNy was evaluated (FIG. ID) Consistent with the screen results, CD47 deletion did not enhance the phagocytosis of human leukemia cells relative to control leukemias by human macrophages (FIG. IE). Similarly, addition of soluble recombinant SIRPa, which antagonizes binding of CD47 on leukemia cells to SIRPa on human macrophages, did not enhance phagocytosis (FIG. 9D) To determine whether this represents a difference in the activity of this checkpoint between species, CD47-deficient human leukemias were co-cultured with macrophage colony-stimulating factor (M-CSF) differentiated mouse bone marrow-derived macrophages and a significant enhancement of phagocytosis was observed (FIG. IF). Therefore, CD47 expression in human AML cells potently inhibited phagocytosis by mouse but not human macrophages.

[0349] Given that genetic deletion of CD47 had a marginal impact on phagocytosis by human macrophages, an assessment was performed to determine whether the mechanism by which anti- CD47 antibodies enhance phagocytosis was mediated through recruitment of FcyR and subsequent antibody-dependent cellular phagocytosis (ADCP). Leukemia cells treated with anti- Atorney Docket No.: 167741-053601 / PCT

[0350] Electronic Deposit Date: October 27, 2025

[0351] CD47 antibody (MIAP410) exhibited dramatically enhanced phagocytosis when co- cultured with human macrophages (FIG. 1G, grey bars). However, when macrophages were pretreated with an Fc receptor-blocking cocktail of antibodies (anti-CD16 / CD32 / CD64), there was a significant decrease in the percent of phagocytosed cells, suggesting that antibodies targeting human CD47 exerted pro-phagocytic effects through ADCP (FIG. 1G, white bars). These findings suggested that anti-CD47 antibodies enhanced phagocytosis primarily via antibodydependent recruitment of macrophage FcyRs, rather than CD47 serving as a “don’t eat me” macrophage inhibitory checkpoint.

[0352] Example 4: MHC class I potently inhibited antibody-dependent cellular phagocytosis

[0353] A screening approach was next used to identify regulators of antibody-dependent cellular phagocytosis (ADCP) (FIG. 1H). Given the high expression of CD47 on leukemia cell lines and the findings that genetic loss of CD47 did not impact human antibody-independent cellular phagocytosis (AICP) (FIGs. 1B-1E), an anti-CD47 antibody (clone MIAP410) with strong FcyR activity was used as an opsonizing antibody to elicit ADCP. Excellent screen performance with good sgRNA representation across all conditions and replicates (Pearson correlation > 0.8 in the leukemia fraction and >0.7 in the macrophage fraction) was observed (FIGs. 10A-10F). Loss of CD47 in leukemia cells was the strongest resistance factor in this screen, consistent with immune escape due to antigen loss (FIG. II). Several genes involved in MHC class I expression (NLRC5, TAPI, TAP2, TAPBP, RFX5, and B2M) were identified as negative regulators of ADCP in leukemia cells co-cultured with macrophages (FIG. II, FIGs. HA and 11B). Competition assays confirmed that genetic deletion of NLRC5, TAPI, or TAPBP in MV411 leukemia cells resulted in increased phagocytosis in co-culture with human macrophages stimulated with anti- CD47 antibody but not with IFNy (FIG. 1 J), demonstrating that MHC -I expression on AML cells was a regulator of ADCP but not AICP. Similar enhancement of phagocytosis upon treatment of leukemia cells with an opsonizing antibody targeting CD33, another highly expressed leukemia cell surface receptor, was observed (FIG. 12A).

[0354] MHC -I proteins interact with the myeloid-specific inhibitory receptors LILRB1 and LILRB2, which contain immunoreceptor tyrosine-based inhibitory motifs that restrain immune signaling. Mouse macrophages do not express the LILRB family of genes and instead express the murine ortholog, Pirb. This species difference in receptor expression highlights the failure of cross-species co-culture screens to resolve the MHC-I::LILRB1 interaction as a key regulator of antibody-dependent cellular phagocytosis (ADCP) . In human macrophages, genetic loss of LILRB 1 or antibody-mediated blockade of LILRB 1 has been shown to enhance ADCP. As there Atorney Docket No.: 167741-053601 / PCT

[0355] Electronic Deposit Date: October 27, 2025 are several clinical trials exploring strategies to disrupt the MHC-I:LILRB axis to enhance immunotherapy response, an assessment of whether genetic loss of MHC-I in leukemia cells enhanced ADCP through loss of interaction with LILRB1 and / or another member of the LILRB family, LILRB2, was performed. CRISPR / Cas9 was used to delete LILRB 1 or LILRB2 in human macrophages, and it was found that deletion of either gene reduced the preferential phagocytosis of MHC-I-deficient leukemias by macrophages (FIGs. 12B-12C), demonstrating that MHC-I expression can restrain ADCP of AML cells through not just LILRB 1 but also LILRB2 on human macrophages.

[0356] Example 5: Mucin-type O-linked glycosylation was a negative regulator of antibodyindependent cellular phagocytosis (AlCP)and antibody-dependent cellular phagocytosis (ADCP)

[0357] To systematically rank the impact of individual genes on AICP or ADCP, data were integrated from both the leukemia and macrophage fractions of each screen across MV411 and M0LM13 cell lines. For each individual gene, an aggregate score of sgRNA depletion in the leukemia fraction and sgRNA enrichment in the macrophage fraction was calculated. It was found that PPP6C, SPN, APMAP, VPS29, and NHLRC2 were negative regulators of AICP (FIG. 2A). NLRC5, TAPBP, TAP2, PPP6C, and PTPN6 were negative regulators of ADCP (FIG. 2A). To validate the hit calling method, it was shown that genetic deletion of PTPN6 in leukemia cells strongly enhanced ADCP in co-culture competition assays (FIG. 2B, FIG. 12D).

[0358] Next, hits that regulated both AICP and ADCP were focused upon by plotting the z- normalized aggregated sgRNA scores in the ADCP and AICP screens(FIG. 2C). This approach highlighted APMAP as a regulator of both AICP and ADCP. Additionally, deletion of TMEM30A, which encodes the phosphatidylserine flippase CDC50A, enhanced overall macrophage phagocytosis, possibly, without intending to be bound by theory, due to an increase in surface phosphatidylserine exposure which activates scavenger receptors such as MERTK. Strikingly, several genes in the O-linked glycosylation pathway, including C1GALT1, C1GALT1C1, SLC39A9, and SLC35A2, emerged as major negative regulators of both AICP and ADCP (FIGs. 2C-2D). SPN, encoding CD43, a cell surface glycoprotein that undergoes extensive O-glycosylation, also scored as a negative regulator of both AICP and ADCP (FIGs. 2C-2D), implicating the O-glycosylation pathway as a major inhibitor of macrophage phagocytosis. O-linked glycosylation is a post-translational modification of cell surface and secreted proteins that attaches glycan chains terminating in the monosaccharide sialic acid (FIG. 2D). C1GALT1 encodes the T- synthase enzyme, which catalyzes an early step in the O- Atorney Docket No.: 167741-053601 / PCT

[0359] Electronic Deposit Date: October 27, 2025 glycosylation pathway, while C1GALT1C1 is a chaperone protein required for C1GALT1 stability. SLC39A9, a putative zinc transporter, regulates C1GALT1 expression, and SLC35A2 transports the donor substrate UDP-galactose, which is incorporated into O-glycan structures.

[0360] Example 6: O-linked glycosylation inhibited phagocytosis through terminal sialylation of cell surface glycoproteins

[0361] Single gene knockouts of C1GALT1 and C1GALT1C1 were generated, and changes in cell surface glycosylation were evaluated with fluorescently-labeled lectins, which bind unique carbohydrate motifs with high specificity. As expected, C1GALT1- and (’ / ( / . / / / ' / (’ / -deficient leukemias expressed increased levels of the C1GALT1 substrate Tn antigen, decreased levels of the downstream enzymatic product T antigen, and decreased levels of sialic acid (FIG. 13A). Knockout of neither C1GALT1 nor C1GALT1C1 altered cell growth kinetics in vitro (FIG. 13B). However, upon co-culture with macrophages, C1GALT1- and (’ / ( / . / / / / ' / (’ / -deficient leukemias were more sensitive to both antibody-independent cellular phagocytosis (AICP) and antibodydependent cellular phagocytosis (ADCP) (FIG. 3A, FIG. 13C). Similarly, genetic deletion of SLC39A9 or SLC35A2 also enhanced phagocytosis by IFNy-stimulated macrophages in competition assays (FIG. 3B) In contrast, CRISPR-mediated genetic deletion of MGAT1, a key component of the N-linked glycosylation pathway, had no impact on AICP (FIGs. 13D-13E). These data implicate mucin-type O-linked glycosylation, but not N-linked glycosylation, as an inhibitory pathway for human macrophage phagocytosis.

[0362] To assess the impact of O-linked glycosylation on macrophage phagocytosis in vivo, a model of ADCP for both in vitro and in vivo studies was established. ADCP was decoupled from confounding effects of interfering with the function of endogenous leukemia antigens by engineering MV411 leukemia cells to ectopically express mouse CD8a (FIG. 3C, FIG. 13F). In competition assays, ectopic expression of mouse CD8a in control leukemias (sgCtrl-CD8a OE) rendered them sensitive to anti-mouse CD8a-mediated ADCP when mixed with control leukemias that overexpressed luciferase (sgCtrl -Luciferase OE) (FIG. 13G). C1GALT1- defi cient leukemias overexpressing CD8a (sgClGALTl-CD8a), but not (7 / - / / -deficient leukemias overexpressing CD8a, were selectively phagocytosed compared to sgCtrl-CD8a OE leukemias (FIG. 13H) Thus, ectopic expression of mouse CD8a on MV411 leukemias enabled anti-CD8a-mediated cellular phagocytosis and preserved the inhibitory impact of O-linked glycosylation in vitro. These studies were extended in vivo by adoptively transferring leukemias into sublethally irradiated NSG mice which lack CD4+and CD8a+T lymphocytes but harbor mouse macrophages that are capable of phagocytosis (FIG. 3D). Systemic administration of Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 opsonizing anti-CD8a antibodies to sgCtrl-CD8a leukemia-bearing NSG mice in vivo resulted in a statistically significant prolongation in survival compared to mice that did not receive anti- CD8a antibodies (FIG. 131). Notably, it was observed that mice bearing sgClGALTl-CD8a OE leukemias exhibited prolonged survival compared to NSG mice bearing sgCtrl-CD8a OE leukemias (FIG. 3E), demonstrating that the O-linked glycosylation pathway enabled leukemia evasion of macrophages in vivo.

[0363] O-glycans often terminate in sialic acids, which are negatively charged sugar residues with immune inhibitory activity. As terminal sialic acids on cell surface glycoproteins are lost after genetic deletion of C1GALT1 or C1GALT1C1 , cell surface sialylation was enzymatically and genetically perturbed to determine if sialic acid modifications were responsible for inhibiting macrophage phagocytosis. First, both control and C1GALT1- or C7GA£77C7-deficient leukemias were pre-treated with V. cholerae sialidase, a pan-sialidase that removes all terminal sialic acid linkages (a2,3 / a2,6 / a2,8). Not intending to be bound by theory, it was hypothesized that enzymatic removal of terminal sialic acids from control leukemias would render them equally sensitive to phagocytosis as C1GALT1- or C1GALT1 Cl -deficient leukemias. Consistent with this hypothesis, increasing concentrations of sialidase abrogated the selective phagocytosis of C1GALT1- or C1GALT1C1- deficient leukemias in competition assays (FIG. 3F). Furthermore, selective treatment of control leukemia cells with a concentration of sialidase that completely removed cell surface sialic acids reversed the relative preference of human macrophages for C1GALT1- or C1GALT1 Cl -deficient leukemias that had not been treated with sialidase (FIG. 3G).

[0364] Sialic acid incorporation into glycans requires conversion of sialic acid to CMP-Sia by CMAS and the transport of CMP-Sia into the Golgi by SLC35A1. Consistent with the sialidase result, knockout of CMAS or SLC35A1 in leukemia cells enhanced phagocytosis (FIG. 13J). While not intending to be bound by theory, these results demonstrated that the inhibitory effects of O-linked glycosylation on macrophage phagocytosis were mediated by the incorporation of terminal sialic acids to cell surface glycans.

[0365] Example 7: CD43 was a major O-linked glycoprotein that inhibited phagocytosis of leukemia

[0366] To determine the identity of the O-glycosylated cell surface proteins on leukemia cells that directly inhibit phagocytosis, all known cell surface and secreted proteins were ranked by mRNA expression and effect on macrophage phagocytosis. As O-glycosylation involves the addition of sugar molecules to a serine (Ser) or threonine (Thr), the proportion of Ser / Thr Atorney Docket No.: 167741-053601 / PCT

[0367] Electronic Deposit Date: October 27, 2025 residues were also plotted for each protein, as each represents a potential O-glycosylation site. Strikingly, the mucin CD43 (SPN), whose extracellular domain is composed of >30% serine / threonine residues, scored the highest in this analysis (FIG. 4A).

[0368] Genetic deletion of SPN (CD43) in MV411 and M0LM13 leukemias enhanced phagocytosis by fFNy-stimulated macrophages in competition assays compared to control leukemias (FIG. 4B), while CD43 -deficient leukemias engineered to overexpress CD8a (sgCD43-CD8a OE) were more sensitive to opsonizing antibody-mediated phagocytosis compared to sgCtrl-Luc leukemias (FIG. 4C). Conversely, CD43 overexpression in control MV41 1 leukemia cells, which have high endogenous expression of CD43, inhibited phagocytosis by fFNy-stimulated macrophages in vitro in competition assays with control MV41 1 leukemias overexpressing the control antigen luciferase (FIG. 4D). In contrast, overexpression of C1GALT1 in control leukemias had no impact on phagocytosis, suggesting, without intending to be bound by theory, that Cl GALT 1 enzymatic activity was not rate-limiting in leukemias (FIG. 4D). Furthermore, genetic deletion of CD43 in CD8a OE leukemias resulted in a statistically significant prolongation of survival in vivo in mice treated with anti- CD8a compared to sgCtrl-CD8a OE leukemias (FIG. 4E). Similar to the observations with C1GALT1- or C / GN / / / 7C / -deficient leukemias, CD43 -deficient leukemias exhibited no growth defects in vitro (FIG. 14A). Thus, CD43 expression on leukemia cells inhibited macrophage phagocytosis both in vitro and in vivo.

[0369] Without intending to be bound by theory, it was hypothesized that loss of O- glycosylation impairs sialylation of CD43 so sialylated CD43 was measured using an anti-CD43 antibody (clone MEM59) with known specificity for sialylated but not desialylated CD43. As expected, sialylated CD43 were not detected on CD43 -deficient leukemias or control leukemia cells treated with sialidase (FIG. 14B). Near-complete loss of sialylated CD43 in C1GALT1- or C1GALT1 Cl -deficient leukemias was observed, both by flow cytometry and Western blot (FIG. 4F, left; FIG. 4G) Loss of O-linked glycosylation impaired CD43 sialylation with a second antibody specific for sialylated but not desialylated human CD43 (clone AT1413) (FIG. 4F, right). In contrast, no changes in total CD43 levels in C1GALT1- or C7GA 77C7-deficient leukemias were seen, but a mobility shift consistent with loss of O-glycans was observed (FIG. 4H) While overexpression of Cl GALT 1 in Cl GALT 1 -deficient leukemias completely reversed their susceptibility to macrophage phagocytosis, overexpression of CD43 in Cl GALT 1 -deficient leukemias had no effect (FIG. 41), consistent, without intending to be bound by theory, with the hypothesis that intact sialylation of CD43 was required for inhibition of phagocytosis and this deficiency could not be rescued simply by increasing the total expression level of CD43. Thus, Atorney Docket No.: 167741-053601 / PCT

[0370] Electronic Deposit Date: October 27, 2025 without intending to be bound by theory, O-linked glycosylation inhibited macrophage phagocytosis of leukemia through post-translational addition of inhibitory terminal sialic acid residues to CD43.

[0371] To determine whether CD43 was the primary mediator of the inhibitory effect of O- glycosylation on macrophage phagocytosis, genetic epistasis experiments were performed by deleting C1GALT1, CD43, or both genes in leukemia cells prior to co-culture with macrophages. As expected, genetic loss of CD43 in a control background (sgCtrT) strongly enhanced phagocytosis (FIG. 4J). However, in cells that lacked C1GALT1 or its chaperone C1GALT1C1 (sgClGALTl, sgClGALTlCl), genetic loss of CD43 had no effect, suggesting, without intending to be bound by theory, that the inhibitory effect of CD43 was dependent on an intact O-glycosylation pathway (FIG. 4J, FIG. 14C). Thus, sialylated CD43 was a downstream effector through which C1GALT1 and C1GALT1C1 inhibited macrophage phagocytosis.

[0372] An experiment was undertaken to demonstrate that deletion of the O-glycan pathway (see, e.g., FIG. 17A) or CD43 in acute myeloid leukemia (AML) cells increased human macrophage phagocytosis. The experiment was carried out as shown in FIG. 17B. Results from the experiment are shown in FIG. 17C, where knockout of C1GALT1, C1GALT1C1, or CD43 increased human macrophage phagocytosis of the edited cells.

[0373] Example 8: CD43 inhibition of phagocytosis was not mediated by the known sialic acidbinding lectins SIGLEC-7 or SIGLEC-9

[0374] SIGLECs are a family of inhibitory sialic acid-binding cell surface receptors that are expressed by several mature immune cells, including monocytes, granulocytes, macrophages, and natural killer (NK) cells. Without intending to be bound by theory, it was hypothesized that SIGLEC receptors on human macrophages deliver inhibitory signals from sialylated CD43 to restrain phagocytosis. Abundant expression of several SIGLEC family members in M-CSF differentiated human macrophages, including SIGLEC-7 and SIGLEC-9, were detected (FIG. 15A- 15B) As SIGLEC-7 and SIGLEC-9 on macrophages have been implicated as functionally important sensors of sialylation , an assessment of whether sialylated CD43 on leukemia cells inhibits phagocytosis through SIGLEC-7 and / or SIGLEC-9 was performed. Surprisingly, genetic loss of CD43 in leukemias did not impact binding of either SIGLEC as measured by recombinant SIGLEC-7 or SIGLEC-9 Fc staining (FIG. 15C). Furthermore, neither genetic deletion of both SIGLEC-7 and SIGLEC-9 in macrophages nor dual antibody blockade with anti-SIGLEC-7 / anti-SIGLEC-9 neutralizing antibodies impacted phagocytosis (FIG. 15D-15F). Thus, SIGLEC-7 and SIGLEC-9 were not necessary for mediating the inhibitory effects of Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025 sialylated CD43 on human macrophage phagocytosis. In addition to binding to SIGLEC receptors, sialylated O-glycans may impact biophysical interactions through steric or electrostatic hindrance. CD43 has a heavily sialylated rod-like ectodomain that extends outwards from the plasma membrane and imparts a negative charge. To assess whether the biophysical properties of CD43 could impair intercellular interactions, cell: cell interaction avidity measurements were performed using macrophages co-cultured with either control or CD43 -deficient leukemias. CD43 -deficient leukemias bound human macrophages with higher avidity than control leukemias (FIG. 15G). These results suggested that sialylated CD43 may restrain macrophage phagocytosis through modulation of macrophageleukemia binding.

[0375] Experiments were undertaken to demonstrate that SIGLEC- 1, SIGLEC-7, and SIGLEC-9 did not exclusively recognize CD43 and that SIGLEC- 1, SIGLEC-2, or SIGLEC-9 inhibition or genetic ablation did not alter preferential phagocytosis of CD43 knockout cancer cells by macrophage. A fusion protein containing the extracellular domain of SIGLEC- 1 and an Fc domain of an antibody was capable of binding to MV411 or M0LM13 cancer cells regardless of whether or not the cells were edited to knock out expression of C1GALT1 or CD43 (FIG. 18). Similarly, it was determined that SIGLEC-7 recognized O-glycans expressed on the surface of MV41 1 or MOLM-13 cancer cells, but CD43 loss did not eliminate SIGLEC-7 binding to the cells (FIG. 19). Also, it was determined that SIGLEC-9 recognized ligands other than CD43 (FIG. 20). Consistent with these findings, when MV411 leukemia cells co-cultured with macrophage were contacted with anti-SIGLECl antibodies, the anti-SIGLECl antibodies (SIGLEC 1 blockade) did not eliminate the preference of the macrophage for phagocytosis of C1GALT1 knockout or CD43 knockout MV411 cells (FIG. 21). These findings suggest that there was no inhibitory interaction between SIGLEC 1 and CD43 that regulated macrophage phagocytosis.

[0376] Example 9: Anti-CD43 antibodies as a therapeutic strategy for acute myelogenous leukemia (AML)

[0377] To explore the suitability of CD43 as a therapeutic target in AML, expression of CD43 was examined in patient-derived normal and malignant hematopoietic cells. The expression of CD43 in primary AML patient samples was characterized using single-cell RNA sequencing (scRNA-seq) to avoid the confounding effects of mixtures of malignant and normal myeloid cells. First, a scRNA-seq dataset of bone marrow aspirates from healthy donors and AML patients was re-analyzed and the expression of CD43 (SPN) was compared. SPN expression was highest in malignant hematopoietic stem cell (HSC)-like and malignant hematopoietic progenitor Atorney Docket No.: 167741-053601 / PCT

[0378] Electronic Deposit Date: October 27, 2025 cell (HPC)-like cells compared to normal HSC-like and HPC-like cells (FIGs. 5A-5C). Western blot analysis also showed that expression of sialylated CD43 was higher in AML compared to normal peripheral blood mononuclear cells or purified monocytes, and high surface expression in AML patients by flow cytometry was confirmed (FIGs. 5D-5E). In contrast, expression of O- glycosylation enzymes (C1GALT1, C1GALT1C1, SLC39A9, SLC35A2) was similar between normal and malignant hematopoietic cells (FIG. 16A). These results indicated that CD43 was overexpressed in human leukemia cells relative to normal hematopoietic cells, making it attractive as a therapeutic target in AML.

[0379] Given the high expression of sialylated CD43 in both human leukemia cells and primary AML blasts, the impact of targeting sialylated CD43 with antibodies was evaluated. It was observed that the addition of anti- CD43 antibodies with high specificity for sialylated CD43 (clones MEM59 and 10G7) to co-culture assays enhanced phagocytosis of MV411, M0LM13, and HEL AML cell lines in a dose-dependent manner, as measured by flow cytometry or fluorescence microscopy (FIG. 5F, FIGs. 16B-16C). These experiments were extended beyond AML cell lines to primary patient-derived AML blasts, and it was similarly observed that anti- CD43 antibody treatment enhanced their phagocytosis (FIG. 5G). Taken together, these results indicated that CD43 antibody blockade phenocopied genetic loss of CD43.

[0380] Not intending to be bound by theory, anti-CD43 antibodies may enhance macrophage phagocytosis through at least two distinct mechanisms: 1) direct interference with the inhibitory effects of sialylated CD43 on human macrophages; 2) activation of the FcyR after opsonization of leukemia cells with anti-CD43 antibodies. If anti-CD43 antibodies act entirely through activation of the FcyR, then abrogating the ability of anti-CD43 antibodies to bind the FcyR should reverse the enhancement of phagocytosis after CD43 blockade. It was observed that anti- CD43 antibodies with impaired binding to FcyR (D265A mouse IgGl “Fc dead”) still enhanced phagocytosis in a dose-dependent manner (FIG. 16D). Furthermore, unlike anti-CD47 antibodies, anti-CD43 antibodies enhanced macrophage phagocytosis even after blockade of all FcyR (CD16 / CD32 / CD64) on human macrophages with neutralizing antibodies (FIG. 5H). Therefore, without intending to be bound by theory, unlike anti-CD47 antibodies, anti-CD43 antibodies enhanced human macrophage phagocytosis through a combination of FcyR- independent and FcyR-dependent mechanisms.

[0381] Because myeloid cell dysfunction has been observed in patients with AML, it was next tested whether macrophages derived from patients with active AML were responsive to anti- CD43 mediated enhanced phagocytosis. AML patient-derived macrophages showed increased phagocytosis either following CD43 antibody blockade or knockout of CD43 in MV411 cells Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0382] (FIGs. 5I-5K). These results indicated that AML patient-derived macrophages retained their ability to respond to loss of sialylated CD43, resulting in increased phagocytosis.

[0383] Macrophages are an important effector population of innate immunity and are highly abundant in the tumor microenvironment in most types of cancer. In the context of certain activation cues, they can exert potent anti-tumor activity through cytokine production, antigen presentation to T cells, and importantly, direct phagocytosis and elimination of tumor cells. Genome-scale CRISPR screens in co-culture assays of human leukemias and human monocyte- derived macrophages were used to discover the pathways that restrain human macrophage antibody-independent and antibody-dependent phagocytosis. The screens recovered previously identified regulators of phagocytosis, such as APMAP, confirming the reliability of the screening approach. Additionally, the experiments of the Examples provided above revealed that genetic loss of CD47 alone was insufficient to enhance phagocytosis by human macrophages, whereas genetic loss of CD47 did not stimulate phagocytosis by murine macrophages. Additionally, without intending to be bound by theory, a Sirpa polymorphism in Non-Obese Diabetic scid gamma (NSG) mice that confers strong recognition of human CD47 may in part explain encouraging results in pre-clinical leukemia xenograft models that contrast with the lack of clinical benefit of CD47 blocking antibodies in recent phase 3 trials. The role of the MHC- I:LILRBl / 2 axis as the dominant regulator of antibody-dependent cellular phagocytosis was missed in prior studies due to species-mismatched co-culture assays. It remains possible that CD47-blocking antibodies could have therapeutic benefit through antibody-dependent cellular phagocytosis (ADCP), as opposed to only serving as a macrophage-inhibitory checkpoint as originally proposed.

[0384] Without intending to be bound by theory, the above Examples demonstrate that O-linked glycosylation and sialylation restrain both antibody- independent and antibody-dependent phagocytosis of leukemia, largely through sialylation of the surface glycoprotein CD43. Genetic deletion or antibody targeting of sialylated CD43 enhanced phagocytosis of primary acute myelogenous leukemia (AML)blasts by macrophages from healthy donors and AML patients.

[0385] O-glycan modifications that terminate in sialic acid, particularly on the highly expressed cell surface mucin CD43, potently inhibited macrophage phagocytosis. Overexpression of CD43 in leukemia cells that lacked the ability to undergo mucin-type O-linked glycosylation had no impact on macrophage phagocytosis, suggesting, without indenting to be bound by theory, that post-translational glyco-modifications (and not the underlying protein structure) may be responsible for mediating immune evasion. SIGLEC receptors are an important class of sialic Atorney Docket No.: 167741-053601 / PCT

[0386] Electronic Deposit Date: October 27, 2025 acid-binding inhibitory immune receptors, and SIGLEC-7 has been implicated a functionally important cognate receptor for sialylated CD43. The Examples provided above demonstrate that genetic deletion of SIGLEC-7, or the related receptor SIGLEC-9, in human macrophages nor antibody-mediated blockade of SIGLEC-7 / 9 abrogated the enhancement of phagocytosis of Cl GALT 1 -deficient or CD43 -deficient leukemias. Without intending to be bound by theory, loss of CD43 may enhance phagocytosis either through other inhibitory receptors with carbohydrate recognition domains (galectins, C-type lectins, or other SIGLECs) or perhaps through a receptor-independent mechanism. CD43 has >30 reported extracellular O-linked glycosylation sites, and extends 45 nm from the cell surface. Without intending to be bound by theory, this large, negatively charged structure may form a barrier that sterically or electrostatically hinders high avidity interactions between leukemia cells and immune effector cells.

[0387] The Examples of the present disclosure highlight the importance of sialylated CD43 as a regulator of macrophage phagocytosis of acute myelogenous leukemia (AML).

[0388] Example 10: Genetic deletion of CD43 or antibody-mediated blockade of CD43 enhanced natural killer (NK) cell cytoxicity in vitro

[0389] Experiments were undertaken to demonstrate that genetic deletion of CD43 or an antibody-mediated blockade of CD43 enhanced natural killer (NK) cell-mediated killing of cancer cells in vitro. The experiments was carried out as shown in FIG. 22A. In one experiment, unedited leukemia cells were co-cultured with unstimulated NK cells and either further unedited leukemia cells or leukemia cells edited to knock out expression of CD43. In another experiment, unedited leukemia cells were co-cultured with unstimulated NK cells and contacted with an anti- CD43 antibody (antibody-mediated blockade of CD43). The leukemia cells (target cells) were co-cultured with the NK cells (effector cells) at an effector-to-target cell ratio (E:T) of 1 :4 or 1 :2. It was determined that genetic deletion of CD43 in the leukemia cells and the antibody -mediated blockade of CD43 of the leukemia cells enhanced killing of the leukemia cells by NK cells (FIGs. 22B and 22C)

[0390] Example 11: Genetic deletion of CD43 enhanced T cell cytoxicity in vitro and in vivo

[0391] Experiments were undertaken to demonstrate that genetic deletion of CD43 enhanced T cell-mediated killing of cancer cells in vitro and in vivo.

[0392] An experiment was carried out as described in FIG. 23A. Unedited leukemia cells were co-cultured with activated T cells and either further unedited leukemia cells or leukemia cells Atorney Docket No.: 167741-053601 / PCT

[0393] Electronic Deposit Date: October 27, 2025 edited to knockout expression of CD43. The leukemia cells (target cells) were co-cultured with the activated T cells (effector cells) at an effector-to-target cell ratio (E:T) of 1 :4 or 1:2. The leukemia cells were either edited to over express an anti-CD3 antibody and CD80 (+anti-CD3 / 80 ORF) or not (no CD3 / 80). It was determined that CD43 inhibited human T cell cytotoxicity in vitro (FIG. 23B)

[0394] An experiment was carried out as described in FIG. 24A. Unedited MV411 leukemia cells (ctrl Leukemia cells) or MV411 leukemia cells edited to knock out expression of CD43 were administered to NSG mice. The MV411 leukemia cells were edited to express an anti-CD3 antibody and CD80. The NSG mice were subsequently either administered (+T cells) or not administered (no T cells) human T cells and survival of the mice was monitored over time (FIG. 24D). The unedited MV411 leukemia cells surface-expressed CD43 (FIGs. 24B and 24C). Mice administered T cells and CD43 knockout (KO) MV411 leukemia cells showed the highest survival rate of all conditions evaluated, thereby demonstrating that CD43 inhibited human T cell cytotoxicity in vivo.

[0395] The following materials and methods were used in the above Examples.

[0396] Isolation of human monocytes and differentiation into human macrophages

[0397] Peripheral blood mononuclear cells (PBMCs) were isolated using FICOLL™ polysaccharide gradient separation of whole blood from healthy donors. Monocytes were isolated from PBMCs using negative selection using the EASYSEP™ Human Monocyte Enrichment Kit (STEMCELL TECHNOLOGIES™). Following isolation, monocytes were resuspended in RPMI 1640 medium with 10% fetal bovine serums, penicillin, streptomycin, glutamine and 50 ng / mL of M-CSF (PEPROTECH™). Monocytes were allowed to adhere to the tissue culture plate and media was exchanged every 2-3 days. Following differentiation, the macrophages were either stimulated with interferon-y (PEPROTECH™) or used directly for coculture assays.

[0398] In vitro macrophage co-culture assays

[0399] Macrophages were plated in 24-well plates with 200,000 macrophages per well and cocultured with leukemia cell lines at an effector to target ratio of 2: 1. Control or knockout leukemia cell lines were engineered to express either blue fluorescent protein (BFP) or the red fluorescent protein RFP657 and 1 : 1 mixes of control and knockout lines with different fluorophores were added to macrophages. Following 18 hours of co-culture with or without Atorney Docket No.: 167741-053601 / PCT

[0400] Electronic Deposit Date: October 27, 2025 macrophages, leukemia cells were harvested, washed and stained for CD1 Ib-FITC as a macrophage marker prior to performing flow cytometry to distinguish the relative ratios of BFP or RFP expressing cells remaining in the culture. After staining with antibodies targeting CD1 lb, cells were resuspended in phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS) and 5 mM EDTA along with a fixed proportion of counting beads (BIOLEGEND® 424902) to normalize event acquisition across conditions. Samples were acquired on the BECKMAN COULTER® CYTOFLEX® instrument and analyzed using FLOWJO™ software.

[0401] Mouse bone marrow-derived macrophage differentiation

[0402] Total bone marrow cells were flushed from the femurs and tibias of C57BL / 6 mice with ice cold DMEM complete medium (DMEM + 10% FBS + 0.5% P / S). Cells were strained through a nylon mesh filter before red blood cell lysis with ACK buffer. Mouse bone marrow cells were then counted, resuspended in complete DMEM + 25 ng / mL mouse M-CSF, and plated in 15 cm dishes. Media was replenished on day 3 before adherent bone marrow-derived macrophages were lifted with 2.5 mM EDTA in PBS and plated for downstream phagocytosis experiments.

[0403] Cell culture

[0404] MV41 1, M0LM13, and HEL cell lines were cultured in RPMI 1640 (SIGMA™) supplemented with 10% fetal bovine serum and antibiotics. The identity of each cell line was confirmed by short tandem repeat (STR) genotyping. All cell lines were negative for Mycoplasma.

[0405] Generation of CRISPR edited lines

[0406] For CRISPR knockout generation, cell lines stably expressed S. pyogenes Cas9 or enCas 12a with a blasticidin selection cassette. Cell lines were infected with lentivirus containing sgRNAs targeting the gene of interest and a puromycin resistance cassette. Cell lines were selected with the 2-4 pg / mL of puromycin for 48 hours and cultured for approximately one week before characterization of the knockout phenotype using Western blotting, flow cytometry, or sequencing of the sgRNA targeting DNA locus. The program CRISPRESSO2 (see Pinello et al., “Analyzing CRISPR genome-editing experiments with CRISPResso.” Nat. Biotechnol. 34:695- 697 (2016) and Clement et al., “CRISPResso2 provides accurate and rapid genome editing sequence analysis.” Nat. Biotechnol. 37: 224-226 (2019), the disclosures of which are hereby Atorney Docket No.: 167741-053601 / PCT

[0407] Electronic Deposit Date: October 27, 2025 incorporated by reference in their entirety for all purposes) was used to quantify editing at the DNA locus targeted by the sgRNA of interest.

[0408] Cas9 / RNP Editing of Monocytes sgRNAs targeting genes of interest or a non-targeting control were synthesized as modified RNAs from the company SYNTHEGO™. sgRNAs were precomplexed in a 1 : 1 ratio with Alt-R-V3 Cas9 from the company IDT™ at 37 deg. C for 15 minutes. Cas9 / RNP complexes were electroporated into healthy donor derived monocytes immediately after isolation using the LONZA NUCLEOFECTOR® 4D high-efficiency transfection system and a P3 Primary Cell NUCLEOFECTOR® high-efficiency transfection system kit (V4XP-3024). Following electroporation, monocytes were cultured in RPMI 1640 media with 10% FBS and 1% penicillin / streptomycin / glutamine supplemented with 50 ng / mL of M-CSF to induce macrophage differentiation. After 5-7 days, macrophages were harvested and flow cytometry was performed to validate knockout of the genes of interest.

[0409] Genome-wide CRISPR co-culture screening

[0410] For the genome-scale in vitro CRISPR screens, a MV411 cell line stably expressing enCasl2a or the M0LM13 cell line stably expressing S. pyogenes Cas9 was used. Cas9 or enCasl2a activity was verified to be greater than 70%. MV411 expressing enCasl2a was infected with set C and set D of the enCasl2a Humagne gene knockout library and the M0LM13 Cas9 expressing cell line was infected with the Brunello gene knockout library. All pooled infections were performed to achieve infection rates of 15-30% to ensure single copy sgRNA infection at sufficient cell numbers to yield >1000x coverage per sgRNA. Transduced cells were selected with 4 pg / mL of puromycin for 48 hours and aliquots were frozen following selection for an early time point control of library representation. Transduced and selected cells were expanded for 5-7 days prior to performing co-culture with macrophages for 18 hours. Cells were maintained in culture to achieve >2000x representation and co-cultures were performed to maintain 500x-1000x representation. Following co-culture, remaining leukemia cells were separated from the macrophages and harvested. Macrophages which are tightly adherent to the tissue culture dish were harvested using TrypLE™ and physical agitation. DNA was isolated using the QIAGEN BLOOD MAXI KIT™ DNA isolation kit and sgRNAs were PCR amplified using Argon and Kermit 135 and P7 ILLUMINA® primers, prior to sequencing on an ILLUMINA® HISEQ® sequencing device. Atorney Docket No.: 167741-053601 / PCT

[0411] Electronic Deposit Date: October 27, 2025

[0412] Co-culture CRISPR screen analysis

[0413] Guide sequences were demultiplexed and quantified using the program PoolQ 3.6.1. Read counts were library normalized per 1 million reads and log2 transformed with a pseudocount of one. Gene-targeting guides were z normalized by the control sgRNA distribution. Guide fold changes for the n top performing guides (4 for M0LM13 Cas9 Brunello library; 2 for MV411 enCasl2 Humagne library) were calculated as residuals fit to a natural cubic spline with 4 degrees of freedom. As previously described, significant depleted or enriched sgRNAs were identified using the STARS algorithm (see Doench et al., “Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.” Nat. Biotechnol. 34: 184-191 (2016), the disclosure of which is hereby incorporated by reference in its entirety for all purposes) considering the n top performing mapped guides per gene. Average p-values for genes in the library were calculated with the hypergeometric distribution; a control distribution for this calculation was created by grouping together n random control guides (n = 4 for Brunello; n = 2 for Humagne) into pseudogenes.

[0414] Flow cytometry

[0415] For flow cytometry of cell lines, cells were isolated and washed with PBS with 2% fetal bovine serum (FBS) and 5 mM EDTA prior to staining with the indicated fluorescently labeled antibodies at concentrations recommended by the manufacturer. Assays were performed with 105-106 cells and analyses were acquired using a BECKMAN COULTER™ CYTOFLEX™ benchtop flow cytometer instrument and analyzed using FLOWJO™ data analysis software.

[0416] Lectin staining

[0417] Flow cytometry -based lectin staining (Vicia villosa Lectin, Anti-Peanut Agglutinin, Sambucus nigra Lectin, Maackia amurensis Lectin II) was performed on human leukemia cell lines and primary acute myelogenous leukemia (AML) blasts. Approximately 105-106 cells were isolated per each condition and washed with PBS with 0.5% bovine serum albumin (BSA) prior to staining at 4°C with VVA-FITC™ (1 : 1000, VECTOR LABORATORIES™, catalog FL- 1231-2), VVA-Biotin (1 :500, VECTOR LABORATORIES™, catalog B-1235-2), PNA-Biotin (1 :500, VECTOR LABORATORIES™, catalog BA-0074-.5), SNA-Biotin (1 :400, VECTOR LABORATORIES™, catalog B-1305-2), or MAL II-Biotin (1 :200, catalog B-1265-1). Samples that were stained with biotinylated lectins were washed twice and stained with streptavidin- FITC™ (1 :200) or streptavidin-BV421 (1 :200) at 4°C. Atorney Docket No.: 167741-053601 / PCT

[0418] Electronic Deposit Date: October 27, 2025

[0419] SIGLEC Fc staining

[0420] Flow cytometry -based staining of human leukemia cell lines and primary acute myelogenous leukemia (AML) blasts was carried out using an adapted form of methods described in Wisnovsky et al., “Genome-wide CRISPR screens reveal a specific ligand for the gly can-binding immune checkpoint receptor Siglec-7.” Proc. Natl. Acad. Sci. U. S. A. 118: e2015024118 (2021) , and Flynn et al., “Small RNAs are modified with N-glycans and displayed on the surface of living cells.” Cell 184: 3109-3124. e22 (2021), the disclosures of which are hereby incorporated by reference in their entirety for all purposes. Briefly, approximately 105- 106cells were washed with 0.5% bovine serum albumin (BSA), blocked with human Fc blocking solution (TruX, BIOLEGEND® catalog 422302) before incubation at 4°C with pre-complexes consisting of 1 pg / mL SIGLEC 7-Fc (R&D™, catalog 1138-SL) or 1 pg / mL SIGLEC 9-Fc (R&D™, catalog 1139-SL) and goat anti-human IgG ALEXA FLUOR® 488 (JACKSON IMMUNORESEARCH™, 109-545-003). Pre-complexes were formed for 1 hr at 4°C.

[0421] Fluorescent live cell imaging

[0422] Macrophage colony-stimulating factor (M-CSF) differentiated human macrophages were plated in imaging-compatible 24 well flat-bottom plates (Ibidi) and either unstimulated or stimulated with 20 ng / mL IFNy approximately 24 hours prior to co-culture with leukemia cells. GFP’ MV411 leukemia cells were labeled with PHRODO RED AM™ (INVITROGEN™) per the manufacturer’s instructions, opsonized with isotype (mouse IgGl), anti-CD43 (MEM59), or anti-CD47 (MIAP410), and added to unstimulated or IFNy-stimulated human macrophages. Live cell imaging was performed directly in the 24 well flat-bottom plates on the MICA™ microscope (LEICA MICROSYSTEMS™). Images were processed using a FIJI™ image processing package.

[0423] Bulk RNA-sequencing of human macrophages

[0424] Bulk RNA-sequencing analysis was performed. Briefly, ILLUMINA™ adapter sequences were trimmed using Trimmomatic (v0.36) software and pre- / post-trimming quality control was done with FastQC (vO.11.7) software. Reads were quantified by pseudoalignment to hg38 using Kallisto (v0.46.0), and RNA-seq gene counts were quantified using the tximport package (vl.24.0) in R. From raw counts, a pseudocount of one was added to every data point to prevent zero values in a later log2 transformation step. The data was transcripts per million (TPM) normalized to account for differences in transcript length and sequencing depth. Afterwards, the data was log2 transformed to ensure normality. The three biological replicates Atorney Docket No.: 167741-053601 / PCT

[0425] Electronic Deposit Date: October 27, 2025 for each condition were then mean collapsed resulting in a gene expression value. To generate heatmaps of gene expression data, the gene expression values for the SIGLEC genes were plotted across the three interferon conditions using the R package ComplexHeatmap (v2.18.0). Hierarchical clustering was determined using the package’s default complete linkage method with the Euclidean distance.

[0426] Single cell RNA-sequencing analysis

[0427] The van Galen et. al., 2019 scRNA-Seq dataset (see Galen, “Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity,” Cell, 176: 1265-1281. e24 (2019), the disclosure of which is hereby incorporated by reference in its entirety for all purposes) was extracted from the Curated Cancer Cell Atlas, including cell type annotations, with the data converted to h5ad format via Scanpy (see Wolf et al., “SCANPY: large-scale single-cell gene expression data analysis.” Genome Biol. 19: 15 (2018), the disclosure of which is hereby incorporated by reference in its entirety for all purposes ). The data was then filtered to remove cells composed of > 10% mitochondrial genes and doublets with Scrublet (see Wolock et al., “Scrublet: Computational identification of cell Doublets in Single- cell transcriptomic data.” Cell Syst. 8: 281-291. e9 (2019), the disclosure of which is hereby incorporated by reference in its entirety for all purposes ). The data was normalized via the shifted logarithm approach, implemented by the usage of the Scanpy preprocessing ‘normalize:totaT and Tog Ip’ functions. The data was then loaded into a SingleCellExperiment (see Amezquita et al., “Orchestrating single-cell analysis with Bioconductor.” Nat. Methods 17: 137-145 (2020), the disclosure of which is hereby incorporated by reference in its entirety for all purposes) object using zellkonverter (see Zappia, Zellkonverter, Bioconductor, 2020; doi.org / 10.18129 / B9.BIOC.ZELLKONVERTER). Cells were then Z-scored for their normalized expression of genes of interest and grouped by their cell type and cell subtype, with cells originating from post-treatment patient samples being removed. The mean Z-score and the percentage of cells expressing each gene of interest for each cell type / subtype group was then calculated and plotted.

[0428] In vivo models of leukemia

[0429] MV41 1 leukemias (sgCtrl, sgClGALTl, sgCD43) were infected with mouse CD8a lentivirus (CD8a-pLX313) and confirmed to express high levels of mouse CD8a by flow cytometry prior to use in experiments. 0.5x106 MV411 leukemias were injected intravenously into sublethally irradiated (225 Gy) NOD Prkdcscld I12rgnull(NSG) mice. Beginning on day 5 Atorney Docket No.: 167741-053601 / PCT

[0430] Electronic Deposit Date: October 27, 2025 after injection, mice were injected with anti-mouse CD8 (clone 53-6.7) intraperitoneally until mice reached their endpoint.

[0431] Glycopeptide Ranking

[0432] To identify and prioritize genes within the O-linked glycosylation pathway, a “Macrophage Enrichment Score” was first calculated by summing the absolute max-normalized gene log-fold changes from the macrophage arms of the “IFN-high vs Isotype” comparisons in the non- antibody-dependent cellular phagocytosis (ADCP) ZFNy-stimulated MV411 enCasl2 Humagne and M0LM13 Cas9 Brunello screens. Cell line RNA-sequencing for M0LM13 (ACH-000362) and MV411 (ACH-000045) was collected from the Cancer Cell Line Encyclopedia (CCLE). Data was subsetted to genes coding for proteins classified as “membrane” or “membrane and secreted isoforms” (Human Protein Atlas) and as O-linked glycosylated peptides. Numbers of glycosylation sites per protein were derived from GlyCosmos (see Yamada et al., “The GlyCosmos Portal: a unified and comprehensive web resource for the glycosciences.” Nat. Methods 17: 649-650 (2020), the disclosure of which is hereby incorporated by reference in its entirety for all purposes); only peptides with a recorded number of sites were maintained in the analysis.

[0433] Culturing of acute myelogenous leukemia (AML) patient samples

[0434] Peripheral blood mononuclear cells were isolated from peripheral blood from patients with acute myeloid leukemia and peripheral blood blast count >20% who provided informed consent prior to inclusion in the study. Isolated cells were cultured in short-term expansion media consisting of STEMSPAN™ II (STEMCELL TECHNOLOGIES™ Cat No. 9655), CC100 (STEMCELL TECHNOLOGIES™ Cat No. 2690), 100 ng / mL human TPO (PEPROTECH™ Cat No. 10773-602), L-glutamine (LIFE TECHNOLOGIES™ Cat No. 25030081), and Penicillin-Streptomycin (LIFE TECHNOLOGIES™ Cat No. 15140163). Isolated primary AML cells were cultured either in the presence or absence of macrophages.

[0435] Cell binding avidity assays

[0436] 107fFNy-stimulated human macrophages were attached to poly-L-lysine coated chips to form a monolayer >80% confluency. Control or CD43 -deficient MV411 leukemia cells were labeled with CELLTRACE™ Far Red dye (THERMO FISHER™, catalog C34564) and 200 pL of cells were added to the human macrophage monolayers at a concentration of 106 / mL. After a 5 minute incubation period, increasing amounts of force was applied with the Z-MOVI® Cell Atorney Docket No.: 167741-053601 / PCT

[0437] Electronic Deposit Date: October 27, 2025

[0438] Avidity Analyzer (LUMICKS™) and cell detachment was quantified. Analysis was performed using Ocean software.

[0439] Western blotting

[0440] Cell pellets were lysed in radioimmunoprecipitation assay (RIP A) buffer supplemented with 5 mM EDTA and a protease inhibitor cocktail (THERMO SCIENTIFIC™). Cell pellets were incubated and rotated on a rocker at 4 deg. C for 30 minutes. Total protein amounts were quantified by Bicinchoninic Acid (BCA) assay. Samples were denatured in sample buffer containing dithiothreitol (DTT) and boiled at 95 deg. C for 5-10 minutes. Approximately 30-40 pg of protein was loaded into each well of 4-12% gradient Bis-Tris gels and resolved by SDS- PAGE. Gels were transferred onto polyvinylidene difluoride (PVDF) membranes via wet transfer for 2 hours at 4 deg. C. Membranes were blotted overnight with primary antibodies directed against sialylated CD43, total CD43, and / or GAPDH. Membranes were washed, stained with fluorescently tagged secondary antibodies (LICOR™), and visualized on a LICOR™ western blot imaging machine.

[0441] Sequences

[0442] Tables 1 and 2 list sequences for sgRNA molecules used in the above Examples.

[0443] Table 1: Cas9 sgRNA sequences Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0444] Table 2: enCaslla sgRNA sequences

[0445] Antibodies The following antibodies were used for flow cytometry: anti-human CD1 lb (clone

[0446] MI / 70, BIOLEGEND®), anti-human CD43 (MEM59, THERMO FISHER®), anti-human CD43 (10G7, BIOLEGEND®), anti-mouse CD8a (53-6.7, BIOLEGEND®), anti-human CD34 (clone 581, BIOLEGEND®). The following antibodies were used for Western blot analysis: antihuman MGAT1 (clone EPR1247, ABCAM™), anti-human sialylated CD43 (clone MEM59, THERMO FISHER™), anti-human CD43 (clone PAS-80067, THERMO FISHER®), anti- PTPN6 (clone E1U6R, CELL SIGNALING TECHNOLOGY™). Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 2025

[0447] Other Embodiments

[0448] From the foregoing description, it will be apparent that variations and modifications may be made to the embodiments and aspects of the disclosure described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0449] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. Atorney Docket No.: 167741-053601 / PCTElectronic Deposit Date: October 27, 2025CLAIMSWhat is claimed is:

1. A method for increasing phagocytosis and / or killing of a cancer cell by an immune cell, the method comprising contacting the cancer cell with an antibody that selectively binds to a CD43 protein expressed on the surface of the cancer cell.

2. The method of claim 1, wherein the cancer cell is a myeloma cell or a lymphoma cell.

3. The method of claim 1, wherein the cancer cell is a leukemia cell.

4. The method of claim 1, wherein the phagocytosis is macrophage-mediated phagocytosis.

5. The method of claim 1, wherein the antibody selectively binds a glycoform of CD43 expressed on the surface of the cancer cell.

6. The method of claim 1, wherein the antibody is a VHH antibody.

7. The method of claim 1, wherein the immune cell is a natural killer (NK) cell or a T cell.

8. The method of claim 1, wherein the immune cell is a macrophage.

9. A method for increasing cancer cell phagocytosis and / or killing by immune cells in a subject in need thereof, the method comprising: administering to the subject an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell, wherein the cancer cell is characterized as expressing CD43, thereby increasing phagocytosis of the cancer cell.

10. The method of claim 9, wherein the cancer cell is a myeloma cell or a lymphoma cell.

11. The method of claim 9, wherein the cancer cell is a leukemia cell.

12. The method of claim 9, wherein the phagocytosis is macrophage-mediated phagocytosis.Atorney Docket No.: 167741-053601 / PCTElectronic Deposit Date: October 27, 202513. The method of claim 9, wherein the antibody selectively binds a glycoform of CD43 expressed on the surface of the cancer cell.

14. The method of claim 9, wherein the antibody is a VHH antibody.

15. The method of claim 9, wherein the immune cell is a natural killer (NK) cell or a T cell.

16. The method of claim 9, wherein the immune cell is a macrophage.

17. A method for treating a CD43-positive cancer in a selected subject, the method comprising: administering to the subject an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell, wherein the subject is selected as having a cancer expressing CD43, thereby treating cancer in the subject.

18. A kit comprising an antibody that selectively binds to a CD43 protein expressed on the surface of a cancer cell and instructions for using said antibody in the method of any one of claims 1-17.

19. A method for identification of genes modulating phagocytosis of a cell by a macrophage, the method comprising: a) editing a cell to knock-out expression of a gene and yield an edited cell, b) co-culturing the cell with macrophages to yield a co-culture, and c) using nucleotide sequencing to determine whether the edited cell is enriched or depleted in a macrophage fraction of the co-culture, wherein enrichment or depletion of the edited cell in the macrophage fraction relative to a reference cell identifies the gene as modulating phagocytosis.

20. The method of claim 19, wherein the nucleotide sequencing is single-cell sequencing.

21. The method of claim 19, wherein expression of the gene is knocked out using CRISPR.

22. The method of claim 21, wherein the method comprises contacting the cell with a library of single guide RNAs (sgRNAs), where each sgRNA targets a unique gene for knock-out.Atorney Docket No.: 167741-053601 / PCT Electronic Deposit Date: October 27, 202523. The method of claim 21, wherein the cells express a polynucleotide programmable endonuclease.

24. The method of claim 19, wherein the cells are cancer cells.

25. The method of claim 24, wherein the cancer cells are leukemia cells.

26. The method of claim 19, wherein a) further comprises selecting for an edited cell before co-culturing the edited cell with macrophages.

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