MODULATION OF MYELOID FCgamma RECEPTOR DEPLETION

Abstract

Methods are provided for modulation of FcΥR expression on myeloid cells during administration of anti-CD47 antibodies to achieve improved therapies.

Description

MODULATION OF MYELOID FCy RECEPTOR DEPLETIONCROSS REFERENCE TO OTHER APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 528,304 filed July 21 , 2023 the contents of which are hereby incorporated by reference in its entirety.BACKGROUND

[0002] Erythroblasts are enucleated in the bone marrow and enter the circulation as reticulocytes, which there mature into red blood cells (RBCs). RBCs circulate once every 45 seconds for -120 days in humans, or once every 15 seconds for -45 days in mice, before they are phagocytosed by macrophages primarily in the spleen and liver. In their constant interactions with near-vascular macrophages, RBCs require protection from nonspecific phagocytic destruction. This protection comes in the form of the ‘don’t eat me’ signal, CD47. CD47 is a conserved surface protein expressed by all cells in the human body. It is also the first described ‘don’t eat me’ signal in that it binds to the macrophage receptor SIRPa to inhibit phagocytosis. This ‘don’t eat me’ function was first described on RBCs, though it was later found to be important for circulating hematopoietic stem cell and leukemic stem cell survival. It was also shown that antibodies that block the CD47-SIRPa interaction can be leveraged to promote phagocytic destruction of leukemic stem cells. Since then, CD47 has been found to be overexpressed in many cancers, and several CD47 antibodies have entered clinical trials.

[0003] CD47 antibody is a doubly potent therapy, as it promotes phagocytosis both by blocking the anti-phagocytic CD47-SIRPa interaction, and by stimulating pro-phagocytic macrophage-expressed Fc-gamma receptors (FcgRs). FcgRs are the principle immune receptors for antibodies and mediate downstream effector functions of antibody binding.

[0004] As RBCs require surface CD47 to survive in the circulation and are sensitive to antibody-mediated phagocytosis, a hurdle for CD47 antibody therapies has been overcoming the toxicity associated with nonspecific destruction of RBCs. It was found that the administration of a low priming dose of CD47 antibody protects against anemia and enables much greater subsequent dosing. However this protocol can be associated with phagocytic impairment. Methods of modulating this effect are of interest.SUMMARY

[0005] Compositions and methods are provided relating to antibodies that bind to CD47 and block the interaction between CD47 and SIRPa. Administration of an anti-CD47 antibody in vivo can result in binding of the antibody to red blood cells (RBC). The antibody-antigen complex formed by this binding is “pruned” from the RBC surface, resulting in a dramatic lossof erythroid surface CD47. RBC CD47 loss also corresponds to a global reduction in myeloid and NK cell FcyR expression. In particular, significant FcyRIIB / lll loss was observed for myeloid and NK cell populations of the spleen, liver, bone marrow, and peritoneum. There is a corresponding substantial phagocytic impairment associated with the loss of FcyR. Many CD47 antibody therapies aim to promote macrophage phagocytosis through both the blockade of the CD47-SIRPa interaction and the engagement of macrophage-expressed FcyRs, and the observed reduction of myeloid FcyR expression and impairment of macrophage phagocytosis may limit therapeutic efficacy. Disabling productive high affinity Fc receptor engagement can restore activity to the antibody and provide an improved therapeutic profile.

[0006] In an embodiment, compositions and methods are provided for the in vivo therapeutic use of antibodies that bind to CD47 and block the interaction between CD47 and SIRPoc, where productive high affinity Fc receptor engagement by the anti-CD47 antibody is reduced or disabled. In some such embodiments, the Fc region of the anti-CD47 antibody is modified to reduce or disable binding to Fey receptors expressed by myeloid cells, e.g. macrophages, monocytes, dendritic cells, etc. present in an individual being treated with the anti-CD47 antibody. In some embodiments the antibody comprises a human Fc region sequence, which may be selected from the lgG1 , lgG2a, lgG2b, lgG3, lgG4 Fc sequence, which Fc region is an Fc region modified to have reduced binding to high affinity Fc receptors. In some embodiments the Fc has been modified by one or more amino acid changes to reduce Fc receptor binding, including without limitation an lgG1 Fc sequence comprising L234A / L235A amino acid substitutions; or modification of asparagine 297 of the Fc domain to reduce glycosylation. In other embodiments the antibody lacks an Fc region, e.g. being provided as an F(ab)2antibody.

[0007] In some embodiments, an anti-CD47 antibody is administered in combination with an effective dose of an FcyR blocking agent. In some such embodiment the anti-CD47 antibody is administered in a regimen comprising one or more initial sub-therapeutic (priming) doses, where the antibody priming dose is administered in combination with an effective dose of an FcyR blocking agent. In some embodiments the blocking agent is administered at a dose and timing such that it is present in an effective dose when the “pruning complex” of CD47 antibody and antigen is present in the circulation. In some embodiments the FcyR blocking agent is an antibody. In some embodiments the antibody specifically binds to one or both of human FcyRII and human FcyRIII. In other embodiments the FcyR blocking agent is a small molecule.

[0008] Such compositions and methods can be used for the treatment of human disease, where the anti-CD47 antibody increases phagocytosis of target cells, for example in combination with a second antibody that binds to an antigen on the targeted cell surface,including without limitation a cancer cell, an infected cell, a fibrotic cell, a cardiovascular cell, a hematopoietic stem cell, etc. In some embodiments the target cell is a cancer cell.

[0009] In some embodiments a method of treatment is provided, comprising contacting an individual with an effective dose of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors, or an anti-CD47 antibody administered in combination with an FcyR blocking agent, wherein the effective dose provides for binding the antibody to a phagocytic cell, thereby increasing phagocytosis of target cells expressing CD47. Treatment may be systemic or localized, e.g. delivery by intratumoral injection, etc.

[0010] In some embodiments, the use of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors or an anti-CD47 antibody administered in combination with an FcyR blocking agent, provides for improved clinical efficacy, relative to methods where the antibody has an unmodified Fc region sequence (e.g. with normal FcyR binding) or in the absence of treatment with the FcyR blocking agent. In such methods the anti-CD47 antibody is administered to an individual comprising red blood cells expressing the cognate CD47 antigen. In some embodiments, phagocytosis of targeted cells is increased at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or more.

[0011] In an embodiment, myeloid cells, e.g. macrophages, are monitored for expression of FcyR following administration of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors, or an anti-CD47 antibody administered in combination with an FcyR blocking agent. In some embodiments the receptor is FcyRIIB / lll. In some embodiments expression of FcyR following such treatment, relative to methods where the antibody has a wild-type Fc region sequence or in the absence of treatment with the FcyR blocking agent, is increased at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or more.

[0012] In other embodiments, methods are provided for treatment of antibody associated diseases, comprising administering an effective dose of an antibody that binds to an antigen present on red blood cells, in a dose effective to cause FcyR loss in reticulo-endothelial myeloid cell populations. In some embodiments the antigen is CD47. In some embodiments the antibody comprises a human Fc region sequence, which may be selected from the lgG1 , lgG2a, lgG2b, lgG3, lgG4 Fc sequence. In some embodiments the antibody induces loss of FcyRIIB / lll for a period of at least about 2 weeks, where the level of cell surface FcyRIIB / lll on splenic or circulating myeloid cells is reduced by at least about 25%, at least about 50%, at least about 75% or more.

[0013] In some embodiments the antibody-associated disease is associated with autoimmune antibodies, including without limitation Grave’s ophthalmopathy, multifocal motor neuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Kawasaki disease, and immune thrombocytopenia (ITP). In some embodiments the patient is monitored for loss of FcyRII B / l 11 during the course of treatment.

[0014] In some embodiments the dose of antibody administered for treatment is a dose of from about 0.5 mg / kg to about 10 mg / kg. In some embodiments the dose of antibody administered for treatment is a dose of from about 1 mg / kg to about 5 mg / kg, e.g. about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg. Administration may be repeated, e.g. weekly, bi-weekly, monthly, etc.

[0015] In some embodiments a method is provided for treating an individual for a disease associated with auto-antibodies selected from Grave’s ophthalmopathy, multifocal motor neuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Kawasaki disease, and ITP, the method comprising administering a dose of an anti-CD47 antibody comprising a human Fc sequence of from 1 mg / kg to 5 mg / kg, effective to induce at least a 50% loss of cell surface FcyRI I B / l 11 on splenic or circulating myeloid cells.

[0016] In other embodiments, a method is provided for protecting red blood cells (RBC) from immune destruction following transplantation, the method comprising treating the RBC with a dose of an anti-CD47 antibody comprising a human Fc sequence effective to induce loss of cell surface CD47 on the RBC; and transfusing the treated RBC to an individual in need thereof. The level of cell surface CD47 on RBC is reduced by at least about 25%, at least about 50%, at least about 75% or more. In some embodiments, an anti-CD47 antibody is administered to the individual prior to transfusing the treated RBC. In some embodiments, RBCs coated with anti-CD47 antibodies are administered to the individual prior to transfusing the treated RBC.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0018] FIGS. 1A-1 E. CD47 antibody elicits FcyR-mediated RBC CD47 loss in mice. A, Flow cytometry data for RBC CD47 expression for each mouse after 14 days of CD47 antibody treatment, isotype control antibody treatment, or no treatment. B, Normalized mouse RBCCD47 expression after 1 , 14, and 28 days of treatment, (D1 : n=5 mice, ****P<0000.1 , D14: n=5 mice, ****P<0.000001 , D28: n=5 mice, ****P<0.000001 ; unpaired two-tailed t-tests for CD47 antibody treated vs. istoype control antibody treated groups), C, Normalized mouse RBC-bound antibody (lgG1 ) after 1 , 14, and 28 days of treatment. D, Normalized mouse RBC CD47 expression after no treatment (-), CD47 antibody treatment (+), or CD47 antibody and FcgRIlB / lll antibody (+ / +) treatment, (n=5 mice; for - v. + / +, ****P< 0.0001 , for + / + v. +, ****P<0.0001 ; unpaired two-tailed t-tests). E, Normalized mouse RBC-bound antibody (lgG1 ) after CD47 antibody treatment (+) or CD47 antibody and FcgRIlB / lll antibody (+ / +) treatment, (n=5 mice, ****P<0.0001 ; unpaired two-tailed t-test).

[0019] FIGS. 2A-2E: CD47 antibody elicits delayed CD47 loss by immature erythroid cells. A, Normalized flow cytometry data for mouse erythroblast, reticulocyte and RBC CD47 expression and (B) antibody bound (lgG1 ) at days 1 , 14 and 28 of CD47 antibody therapy, n=5 mice. Antibody bound is normalized to average naive CD47 expression. C, Flow cytometry data for erythroblast CD47 expression for each mouse after 14 days of CD47 antibody treatment, isotype control antibody treatment, or no treatment. D, Normalized mouse erythroblast CD47 expression after no treatment (-), CD47 antibody treatment (+), or CD47 antibody and FcgRIlB / lll antibody treatment (+ / +), (n=5 mice; [*P<0.02 for: - v. + / + ], [***p=0.Q01 , for + / + v. +]; unpaired two-tailed t-tests). E, Normalized mouse erythroblast remaining antibody bound after CD47 antibody treatment (+) or CD47 antibody and FcgRIlB / lll antibody treatment (+ / +), (n=5 mice; ****P<0.0001 ; unpaired two-tailed t-test).

[0020] FIGS. 3A-3B. CD47 antibody induces global myeloid FcyR loss. A, Normalized expression of each assessed FcgR in columns representing, from left-to-right, splenic macrophages, NK cells, neutrophils, red pulp macrophages, and bone marrow myeloid cells after administration of a loading dose of CD47 antibody, n=5 mice. B, Normalized FcgRIlB / lll expression of murine myeloid and NK cell populations studied after 1 , 14, and 28 days of CD47 antibody treatment, n=5 mice. Dotted lines represent average respective expression in isotype antibody treated animals.

[0021] FIGS. 4A-4D. CD47 antibody inhibits phagocytosis of circulating RBCs in vivo. A, Schematic of experimental setup for in vivo phagocytosis assay. RBCs were collected from CD47 antibody treated mice, CFSE labeled, and infused into CD47 antibody treated and naive mice. Blood and spleens were collected and cells analyzed by flow cytometry for CFSE+ signal. B, Representative flow cytometry plots showing phagocytosis of CFSE+ RBCs by splenic red pulp macrophages (RPMs) in a naive mouse (left) and CD47 antibody treated mouse (right). C, In vivo RBC survival is quantified as the normalized count of CFSE+ RBCs per blood volume. Data is shown for CFSE+ RBC count per blood volume for naive (-) and CD47 antibody treated (+) mice, (n=5 mice, ****P<0.0001 , unpaired two-tailed t-test. D, Phagocytosis by RPMs is quantified as percent of RPMs that are CFSE+. Data is shown forRPMs in naive (-) and CD47 antibody treated (+) mice, (n=5 mice, ****P<0.0001 , unpaired two-tailed t-test).

[0022] FIGS. 5A-5B. RBC-binding antibodies reduce myeloid FcyR and impair phagocytosis. RBC-binding antibodies reduce myeloid FcgR and impair phagocytosis. A, Mice are treated with an antibody against an RBC-expressed antigen and splenic myeloid populations are assessed for FcgR expression by flow cytometry. Expression is normalized to average naive expression, n=5 mice. B, In vivo phagocytosis of fluorescently-labeled antibody-opsonized red blood cells (RBCs). Mice are treated with an antibody against an RBC-expressed antigen and after 24 hours are infused with fluorescently labeled antibody-opsonized RBCs. Red pulp macrophage phagocytosis is quantified as the fraction of fluorescence-positive cells as assessed by flow cytometry, n=5 mice.

[0023] FIGS. 6A-6D. Effects of CD47 antibody therapy on mouse red blood cells. A, Mouse red blood cell (RBC) counts after 0, 1 , and 14 days of CD47 antibody treatment or isotype control antibody treatment, n=5 mice. B, Mouse RBC CD47 expression and C, remaining bound antibody after 24 hour in vitro incubation with (+) or without (-) CD47 antibody, n=5 mice. D, RBC CD47 expression after CD47 antibody therapy (-) or CD47 antibody F(ab’)2 therapy (+), (n=5 mice, **P<0.005, unpaired two-tailed t-test).

[0024] FIG. 7. Gating strategy for erythroid progenitor flow cytometry. Flow cytometry gating strategy for assessment of mouse erythroid progenitor cells. After removal of doublets, erythroblasts and reticulocytes were assessed as DAPI-Ter1 19+CD71 hi, and DAPI- Ter1 19+CD71 mid, respectively.

[0025] FIGS. 8A-8D. Gating strategy for mouse myeloid and NK cell FcyR flow cytometry. A, Gating strategy for mouse splenic myeloid and NK cell assessment. After removal of debris and doublets, neutrophils were assessed as DAPI-CD45+CD11 b+Gr1 +, macrophages as DAPI-CD45+CD1 1 b+F4 / 80+, RPMs as DAPI-CD45+CD11 bi nt F4 / 80+ , and NK cells as DAPI- CD45+CD1 1 b+NK1 .1 +. B, Bone marrow immature myeloid cells and neutrophils were assessed as DAPI-CD45+CD11 b+Gr1 +, C, liver macrophages as DAPI- CD45+CD1 1 b+F4 / 80+, D, and peritoneal macrophage populations as DAPI- CD45+CD1 1 bint F4 / 80i nt and DAPI-CD45+CD11 bhi F4 / 80hi .

[0026] FIGS. 9A-9C. Experimental design and representative plots of myeloid and FcyR flow cytometry. A, Schematic of experimental design for CD47 antibody therapy with tissue collection at days 1 , 14 and 28. Mice were treated 3x weekly, with 100ug doses in the first week, followed by 300ug doses in subsequent weeks. B, Schematic of the four murine Fc gamma receptors analyzed by flow cytometry. C, Representative flow cytometry histograms comparing FcgRIlB / lll expression in splenic myeloid and NK cell populations at day 14 of CD47 antibody or isotype control antibody treatment, data scaled modally.

[0027] FIG. 10. Normalized baseline FcgRIlB / lll expression. Flow cytometry FcgRIlB / lll expression data for all myeloid and NK cell populations assessed, for mice treated with lgG1 isotype control antibody, n=5 mice. Data for each population are normalized to the average for that population, and data from mice treated with CD47 antibody are also normalized to this average.

[0028] FIGS. 11 A-1 1 C. CD47 antibody therapy induces inconsistent changes in myeloid and NK cell FcyR I, I IB and IV expression. CD47 antibody therapy induces inconsistent changes in myeloid and NK cell FcgR I, IIB, and IV expression. A, B, C, Normalized myeloid and NK cell expression of FcgRI (A), FcgRHB (B), and FcgRIV (C) after 1 , 14, and 28 days of CD47 antibody therapy, n=5 mice. Dotted lines represent average expression in isotype control treated mice. Peritoneal macrophage populations did not demonstrate notable expression of FcgRIV, and are not shown in (C).

[0029] FIGS. 12A-12C. Myeloid FcyR expression and RBC CD47 expression recover after cessation of CD47 antibody therapy. Myeloid FcgR expression and RBC CD47 expression recover after cessation of CD47 antibody therapy. A, Normalized FcgRIlb / lll expression in myeloid and NK cell populations after cessation of CD47 antibody therapy. B, Representative flow cytometry histograms for RBC CD47 from a CD47 antibody treated mouse (top) and a naive mouse (bottom) 30 days after cessation of treatment. C, Quantified fraction of circulating RBCs that are CD47-deficient at 0, 30, and 60 days post cessation of treatment, n=5 mice.

[0030] FIG. 13. C57BL / 6 mouse splenic myeloid and NK cell FcyR IIB / III expression after single dose of antibody against RBC antigen. C57BL / 6 mouse splenic myeloid and NK cell FcgR IIB / III expression after single dose of antibody against RBC antigen. Normalized murine C57BL / 6 FcgRIlb / lll expression in myeloid and NK cell populations after single dose of antibody against an RBC-expressed antigen, n=5 mice.DETAILED DESCRIPTION

[0031] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0034] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0036] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0037] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0038] CD47 is a broadly expressed transmembrane glycoprotein with a single Ig-like domain and five membrane spanning regions, which functions as a cellular ligand for SIRPa with binding mediated through the NH2-terminal V-like domain of SIRPa. SIRPa is expressed primarily on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells, and their precursors, including hematopoietic stem cells. Structural determinants on SIRPa that mediate CD47 binding are discussed by Lee et al. (2007) J. Immunol. 179:7741 -7750; Hatherley et al. (2008) Mol Cell. 31 (2):266-77; Hatherley et al. (2007) J.B.C. 282:14567-75; and the role of SIRPa cis dimerization in CD47 binding is discussed by Lee et al. (2010) J.B.C. 285:37953-63.

[0039] The CD47 gene is 48,771 bases in size and is composed of 1 1 exons encoding a 5234 base mRNA and 5 additional alternatively spliced transcripts, located at 3q13.12 in the human genome. Splicing of alternative 3’-UTRs in the transcripts control localization of newly translated CD47 proteins. The 5’ sequence from -272 to the ATGs contains binding sites for transcription factors including TFAP2A (AP-2), MAZ, CREB1 , SP1 , and E2F. Its expression is regulated by a a-Pal / NRF-1 region. CD47 expression is increased in many cancers with progression of disease including ovarian carcinoma, T-cell leukemia and lymphoma, and multiple myeloma. Increased CD47 expression is linked to poor prognosis in many cancers.

[0040] Alternative exon splicing produces CD47 isoforms with short or long C-terminal cytoplasmic tails. The CD47 long isoform precursor contains 323 amino acids, has a mass of35214 Da, and contains an 18 residue N-terminal signal peptide. The mature long isoform protein comprises residues 19-323, and the short isoform comprises residues 19-305. The N-terminal residue of the mature protein Gln-19 is enzymatically modified to pyrrolidone carboxylic acid (pyroGlu), which is required for binding to SIRPA. Mature CD47 is an integral membrane protein that contains an extracellular immunoglobulin domain and a transmembrane domain with 5 membrane spanning segments related to the presenilins. The long isoform cytoplasmic tail contains a ubiquitinylation site at Lys-317. The protein contains disulfide bonds linking Cys at positions 33 to 263, which links the IgV domain to the transmembrane domain, and 41 to 1 14 within the IgV domain. Heterogeneous N-linked glycosylation is found at asparagine residues 23, 34, 50, 73, 1 1 1 , and 206.

[0041] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule, including a functional Fc sequence. Antibodies may comprise a human gamma Fc region sequence, e.g. lgG1 , lgG2a, lgG2b, lgG3, lgG4.

[0042] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and / or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0043] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0044] "Antibody fragment", and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a"single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1 ) single-chain Fv (scFv) molecules; nanobodies or domain antibodies comprising single Ig domains from human or non-human species or other specific single-domain binding modules including non-antibody binding proteins such as, but not limited to, adnectins and anticalins; and multispecific or multivalent structures formed from antibody fragments.

[0045] The invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The immunoglobulin single variable domain includes fully human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences. An immunoglobulin variable domain and structure of an immunoglobulin single variable domain can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region 1" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively.

[0046] Such immunoglobulin single variable domains may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring sequences. Such immunoglobulin single variable domains may include "humanized" or otherwise "sequence optimized" VHHs, "camelized" immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, i.e., camelized VHs), as well as human VHs, human VLs, camelid VH Hs that have been altered by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein.

[0047] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and / or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0048] Variable regions of interest include at least one CDR sequence from the variable regions of an anti-CD47 antibody, usually at least 2 CDR sequences, and more usually 3 CDR sequences on the light and on the heavy chain. One of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see“Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001 ;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181 :6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[0049] “Fv” is the minimum antibody fragment, which contains a complete antigen-recognition and antigen-binding site. The CD3 binding antibodies of the invention comprise a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association; however additional antibodies, e.g. for use in a multi-specific configuration, may comprise a VH in the absence of a VL sequence. Even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although the affinity may be lower than that of two domain binding site.

[0050] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')2antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0051] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. See, for example, Jones et al, (1986) Nature 321 :522-525; Chothia et al (1989) Nature 342:877; Riechmann et al (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol. 151 , 2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986- 4995; and Presta et al (2001 ) Thromb. Haemost. 85:379-389. For further details, see U.S. Pat. Nos. 5,225,539; 6,548,640; 6,982,321 ; 5,585,089; 5,693,761 ; 6,407,213; Jones et al (1986) Nature, 321 :522-525; and Riechmann et al (1988) Nature 332:323-329.

[0052] Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs, cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (“SMIPs™"), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload, e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc., or other pendant group [e.g., poly-ethylene glycol, etc.

[0053] Exemplary antibody agents include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies (f.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals (“SMIPs™"), single chain antibodies, cameloid antibodies, and antibody fragments. As used herein, the term “antibody agent” also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g. bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. In some embodiments, the term encompasses stapled peptides. In some embodiments, the term encompasses one or more antibody-like binding peptidomimetics. In some embodiments, the term encompasses one or more antibody-like binding scaffold proteins. In come embodiments, the term encompasses monobodies or adnectins.

[0054] "Antibody fragment", and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1 ) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chainvariable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and / or can contain any hinge region sequence found in an intact antibody, and / or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).

[0055] The antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0056] An "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1 ) to greater than 75% by weight of antibody as determined by the Lowry method, and most preferably more than 80%, 90% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0057] The term "epitope tagged" when used herein refers to an anti- CD47 antibody (or fragment) fused to an "epitope tag". The epitope tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the anti- CD47 antibody. The epitope tag preferably is sufficiently unique so that the antibody specific for the epitope does not substantially crossreact with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9- 30 residues). Examples include the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3(6):547-553 (1990)). An additional example is a “histidine tag” or “histidine-rich affinity peptide”, which is a metal ion affinity peptide that is rich in histidines (e.g., 6xHis tag, HAT tag, 6xHN tag, and the like). A histidine tag can also specifically bind to an anti-His antibody.

[0058] Anti-CD47 antibodies. In some embodiments, an anti-CD47 antibody specifically binds CD47 and reduces the interaction between CD47 on one cell and SIRPa on another cell.Unless specifically modified to reduce FcgR binding, such antibodies can down-regulate expression of Fcyrllb / lll on myeloid cells when administered in vivo to a subject comprising RBC expressing the cognate CD47 antigen. In some embodiments, a suitable anti-CD47 antibody does not activate CD47 upon binding. Suitable anti-CD47 antibodies include fully human, humanized or chimeric versions of such antibodies. Humanized antibodies (e.g., hu5F9-G4, Magrolimab) are especially useful for in vivo applications in humans due to their low antigenicity. Similarly caninized, felinized, etc. antibodies are especially useful for applications in dogs, cats, and other species respectively. Antibodies of interest include humanized antibodies, or caninized, felinized, equinized, bovinized, porcinized, etc., antibodies, and variants thereof.

[0059] In some embodiments an anti-CD47 antibody comprises a human IgG Fc region, e.g. an lgG1 , lgG2a, lgG2b, lgG3, lgG4 constant region. In an embodiment the IgG Fc region is an lgG4 constant region. The lgG4 hinge may be stabilized by the amino acid substitution S241 P or S228P (see Angal et al. (1993) Mol. Immunol. 30(1 ):105-108, herein specifically incorporated by reference). In an embodiment the IgG Fc region is an lgG1 constant region modified to reduce FcyR binding. In some embodiments, the anti-CD47 agent does not activate CD47 upon binding. When CD47 is activated, a process akin to apoptosis (i.e., programmed cell death) may occur (Manna and Frazier, Cancer Research, 64, 1026-1036,Feb. 1 2004). Thus, in some embodiments, the anti-CD47 agent does not directly induce cell death of a CD47-expressing cell.

[0060] Anti-CD47 antibodies useful in the methods of the disclosure and currently in clinical trials include, without limitation those listed below.Hu5F9-G4 / Magrolimab (Forty Seven Inc / Gilead) CD47 mAbCC-90002 (Celgene) CD47 mAbIBI188 (Innovent Biologies) CD47 mAbSRF231 (Surface Oncology) CD47 mAbAO-176 (Arch Oncology) CD47 mAbIMC-002 (ImmuneOncia) CD47 mAbIMC-002 (ImmuneOncia Therapeutics LLC) anti-CD47 mAbLemzoparlimab (l-Mab Bio-Tech (Tianjin) Co., Ltd) anti-CD47 mAbLetaplimab (Innovent Biologies Inc) anti-CD47 mAbLigufalimab (Akeso Biopharma Inc) anti-CD47 mAbMIL-95 (KeyMed Biosciences Co Ltd) anti-CD47 mAbSHR-1603 (Jiangsu Hengrui Medicine Co Ltd) anti-CD47 mAbZL-1201 (Zai Lab Limited) anti-CD47 mAb

[0061] Such antibodies may be modified as disclosed herein to reduce or disable binding toFey receptors expressed by myeloid cells. In some embodiments the antibody is magrolimab, also referred to as 5F9 antibody, see for example U.S. Patent no 9,017,675, and Liu et al. (2015) PLoS One. 10(9) :e0137345, each specifically incorporated by reference.

[0062] In some embodiments, the anti-CD47 antibody comprises an HCDR1 region comprising of sequence (SEQ ID NO: 1 ), an HCDR2 region comprising of sequence (SEQ ID NO: 2), an HCDR3 region comprising of sequence (SEQ ID NO: 3), an LCDR1 region comprising of sequence (SEQ ID NO: 4), an LCDR2 region comprising of sequence (SEQ ID NO: 5), and an LCDR3 region comprising of sequence (SEQ ID NO: 6). In some embodiments such an antibody comprises a modified human IgG constant region sequence, including a human lgG1 sequence.SEQ ID NO:1 Asn Tyr Asn Met HisSEQ ID NO:2 Thr lie Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gin Lys Phe LysSEQ ID N0:3 Gly Gly Tyr Arg Ala Met Asp TyrSEQ ID N0:4 Arg Ser Ser Gin Ser He Vai Tyr Ser Asn Gly Asn Thr Tyr Leu GlySEQ ID N0:5 Lys Vai Ser Asn Arg Phe SerSEQ ID N0:6 Phe Gin Gly Ser His Vai Pro Tyr Thr

[0063] Fc receptors. The human IgG receptor family consists of a number of high affinity Fc receptors, including hFcyRI, hFcyRIIA, hFcyRIIC, hFcyRIIIA, hFcyRIIB, hFcyRIIIB; and a low affinity receptor, hFcRn, involved in recycling and transport of IgG. Activation of the high affinity receptors may require the FcR subunit to be expressed and functional at the cell surface. Other high affinity Fc receptors include, for example, FcocRI (CD89), Fcoc / y.R, FCERI, etc.

[0064] Expression of the Fc receptors varies among immune effector cells. hFcyRI (CD64) is restricted to monocytes / macrophages and dendritic cells (DCs) and, inducibly, expressed on neutrophils and mast cells; hFcyRIIA (CD32A) is expressed on all myeloid cells but not on lymphocytes; hFcyRIIB (CD32B) is highly expressed only on circulating B cells and basophils and expressed on tissue macrophages and DCs, but not on mast cells; hFcyRIIC (CD32C) is expressed on NK cells, monocytes, and neutrophils; hFcyRIIIA (CD16A) is expressed on NK cells and monocytes / macrophages; hFcyRIIIB (CD16B) is expressed on neutrophils and subsets of basophils.

[0065] The low affinity hFcRn, which importantly contributes to the biological half-life of antibodies in the blood, is expressed on antigen-presenting cells, monocytes / macrophages, neutrophils, vascular endothelial cells, intestinal epithelial cells, and syncytiotrophoblasts.

[0066] The Fey receptors differ in their affinity for IgG and likewise the different IgG subclasses have unique affinities for each of the Fey receptors. These interactions are further tuned by glycans (oligosaccharide), e.g. at position CH2-84.4 of IgG. For example, by creating steric hindrance, fucose containing CH2-84.4 glycans reduce IgG affinity for FcyRII I A.

[0067] Fc domain or region. The Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Engineering the Fc region of a therapeutic monoclonal antibody or Fc fusion protein allows the generation of molecules that are better suited to the pharmacology activity required of them. The half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.

[0068] A “wild-type Fc region” possesses the effector functions of a native-sequence Fc region, in particular for the purposes of the present invention interacting with one or more of the high affinity receptors e.g. the FcyRI; FcyRIIA; FcyRIIBI ; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors; and can be assessed using various assays as disclosed, for example, in definitions herein. A “dead” Fc is one that has been mutagenized to retain activity with respect to, for example, prolonging serum half-life, but which has reduced or absent binding to a high affinity Fc receptor, including without limitation a human FcyR.

[0069] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human lgG1 Fc region (non-A and A allotypes); native-sequence human lgG2 Fc region; native-sequence human lgG3 Fc region; and native-sequence human lgG4 Fc region, as well as naturally occurring variants thereof.

[0070] A “variant Fc region” or “engineered Fc region” comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith. Typically an lgG4 sequence will comprise an S228P substitution.

[0071] In one aspect, the scope of the invention encompasses the use of anti-CD47 antibodies wherein such antibodies have been selected for or engineered for reduced FC- FcyR interaction. For example, in some embodiments, reduced FC-FcyR interaction encompasses reduced binding to an FcyR or reduced activation of FcyR- mediated processes by the Fc region(s) of the antibody, such reduction, for example, being reduced in comparison to interaction of like anti-CD47 antibodies that have not been so engineered. Such reduced FC-FcyR interaction encompasses any reduction in antibody Fc region interaction with an FcyR, including a reduction in interaction with any of FcyRI, FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), FcyRIIIB (CD16b). In some embodiments, all or substantially all FC-FcyR interaction is inhibited, and such antibodies may be referred to herein as “Fc dead” or “dead Fc” antibodies.

[0072] Antbiodies engineered for reduced FC-FcyR interaction may comprise antibodies having mutations or amino acid substitutions that inhibit or ablate interaction of the Fc with an FcyR .Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1 q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991 )). Substitution into human lgG1 of lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. etal., 2001 . J Biol Chem. 276(9):6591 -604).

[0073] Binding of IgG to the FcyRs or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and C1 q binding, and have unique sequences in lgG2 and lgG4. Substitutions into human lgG1 or lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 have been shown to greatly reduce ADCC and CDC. Numerous mutations have been made in the CH2 domain of human lgG1.

[0074] The triple amino acid substitution L234A, L235A, and G237A largely eliminates FcyR and complement effector functions (see, for example, US20100266505).

[0075] In some embodiments the Fc region has been modified by the choice of expression host, enzymatic treatment of amino acid substitutions to have reduced glycosylation and binding to FcyR, relative to the native protein.

[0076] In an embodiment, amino acid substitutions that reduce binding to FcyR are made, which include, without limitation, modification of the glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction. The residue is conserved across human Fc region sequences and may be made in any of lgG1 , lgG2a, lgG2b, lgG3,lgG4. In some embodiments an lgG1 Fc sequence is modified as N297 to reduce glycosylation. Known amino acid substitutions at N297 include, for example, N297A / Q / D / H / G / C, which changes result in the loss of a glycosylation site on the protein. Enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of a glycosylation inhibitor and the expression of Fc domains in bacteria have a similar loss of glycosylation and consequent binding to FcyRs. See, for example, Wang et al. IgG Fc engineering to modulate antibody effector functions. Protein Cell. 2018 Jan;9(1 ):63- 73, herein specifically incorporated by reference. Additional mutations include mutations which disrupt FC-FcyR interaction including substitutions or deletions at any of: Met252, Ile253, Ser254, Asn434, His435, and Tyr436, in igG1 , lgG2, and lgG4; His435 and Tyr436 in lgG3: Gln268, Glu269; Pro329 in lgG1 ; and Asp270, Pro271 , Pro329 and Ser330 in lgG4.

[0077] The lgG1 LALA variant, L234A / L235A, also has significantly reduced FcyR binding; as does E233P / L234V / L235A / G236 + A327G / A330S / P331 S. See, for example, Armour et al. (1999) Eur J Immunol. 29(8):2613-24. The set of mutations: K322A, L234A and L235A are sufficient to almost completely abolish FcyR and C1 q binding. A set of three mutations, L234F / L235E / P331 S (dubbed TM), have a very similar effect.

[0078] Other Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto.

[0079] The Fc may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in an aglycosylated or deglycosylated form. In one embodiment, the methods of the invention encompass the use of an anti-CD47 antibody wherein the Fc region comprises a silylated Fc, for example silylated at Asn297. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line. Such cell lines can include microorganisms, e.g. Pichia Pastoris, and mammalians cell line, e.g. CHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes, or can be rendered unable to express glycosylation enzymes (See e.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J. Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269 (27): 17872 (1994); Ujita- Lee et al., J. Biol. Chem., 264 (23): 13848 (1989); Imai-Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07 / 055916). As one example of a cell engineered to have altered sialylation activity, the alpha-2, 6-sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into sf9 cells. Antibodies expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining Fc molecules havinga modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography (See e.g., WO 07 / 1 17505). The presence of particular glycosylation moieties has been shown to alter the function of Immunoglobulins. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the C1 q part of the first complement component C1 and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity (See e.g., Kaneko, et al, Science 313:760 (2006)), whereas removal of fucose from the IgG leads to enhanced ADCC activity (See e.g., Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

[0080] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this invention can comprise an antibody with or without K447.

[0081] FcyR Blocking agents. In one aspect, the scope of the invention encompasses the administration of an FcyR blocking agent. Such agent comprises an agent which inhibits Fc- FcyR interaction, for example reducing or inhibiting the binding of an Fc domain to an FcyR or reducing the activation of processes mediated by Fc- FcyR interaction. Fey receptors (FcyRs) are proteins found on the surface of various immune cells, including macrophages, neutrophils, natural killer (NK) cells, and dendritic cells. These receptors bind the Fc region of immunoglobulin G (IgG) antibodies, facilitating the antigenic pruning and the associated loss of myeloid phagocytic activity described herein. Agent that block Fc-FcyR interaction include, for example, monoclonal antibodies, for example antibodies that bind to an FcyR or which otherwise competitively inhibit Fc-FcyR interaction.

[0082] In one embodiment, the FcyR blocking agent comprises an anti- FcyR antibody. In one embodiment, the anti- FcyR antibody is an anti-FcyRII (CD32a) antibody, for example, Bl- 1206. In one embodiment, the anti- FcyR antibody is an anti-FcyRII I (CD16s) antibody, for example, CB16, 3G8, B73.1 , MEM-154, VIB9600 (VielaBio) and GMA161 (Sanofi).

[0083] In one embodiment, the FcyR blocking agent comprises a soluble FcyR or Fc-binding domain thereof. Soluble FcyRs mimic the extracellular portion of FcyRs, acting as decoys to bind circulating IgG and prevent it from engaging with cell surface FcyRs. Soluble FcyRsinclude multimers of FcyRs or Fc-binding domains thereof. Exemplary agents in this class include, for example, Valziflocept (Shire / Takeda).

[0084] In one embodiment, the FcyR blocking agent comprises a recombinant Fc or receptorbinding domain thereof, which inhibits Fc-FcyR interactions by binding to FcyR or competitively inhibiting Fc-FcyR interactions. In one implementation, the FcyR blocking agent comprises a multimer of recombinant Fc’s or receptor-binding domains thereof. Exemplary agents include, for example, Pf06755347 (Pfizer), CSL730 (Momenta Pharma), Pan Fc Receptor Interacting Molecule (PRIM)(Shire), Hexagard (as describe in Sci Rep 2015; 5: 9526); Fc TP-L309C (CSL777) (as described in J Immunol, 200 (8) (2018), pp. 2542-2553), and hexavalent lgG1 / lgG4 Fc hybrid molecules, for example as described in Sci Rep, 7 (1) (2017), Article 17049.

[0085] Fc fusion proteins combine the Fc region of an antibody with another biologically active molecule, which can block FcyRI l / l 11 by competitive inhibition.

[0086] Small molecule inhibitors of FcyR are also known. For example Syk inhibitors include Fostamatinib, an oral spleen tyrosine kinase inhibitor that has been shown to inhibit signaling downstream of FcyR activation. Entospletinib is another Syk inhibitor. Idelalisib and Duvelisib are phosphoinositide 3-kinase (PI3K) inhibitors that affects signaling pathways downstream of FcyRs. It is used in the treatment of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. BTK inhibitors such as Ibrutinib and Acalabrutinib also modulate of FcyR signaling.Dosina

[0087] In some embodiments, for therapeutic applications, anti-CD47 antibodies, which may be administered in combination with a second antibody after priming, are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved.

[0088] In some embodiments, methods for treating a subject with a therapeutic dose of anti- CD47 agent include a step of administering a primer agent to subject, followed by a step of administering a therapeutically effective dose of an anti-CD47 agent to the subject. In some embodiments, the step of administering a therapeutically effective dose is performed after at least about 3 days (e.g., at least about 4 days, at least about 5 days, at least about 6 days, atleast about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days) after beginning the administration of a primer agent. This period of time is, for example, sufficient to provide for enhanced reticulocyte production by the individual. See, for example, U.S. Patent no. 9,623,079, herein specifically incorporated by reference.

[0089] In some embodiments, a priming dose is defined as a dose that causes an anemia that is not worsened by subsequent doses. A priming dose of an anti-CD47 agent can depend on the specific agent used, but is generally from about 0.5 to about 5 mg / kg. The term “priming dose” or as used herein refers to a dose of an anti-CD47 agent that primes a subject for administration of a therapeutically effective dose of anti-CD47 agent such that the therapeutically effective dose does not result in a severe loss of RBCs (reduced hematocrit or reduced hemoglobin). The specific appropriate priming dose of an anti-CD47 agent can vary depending on the nature of the agent used and on numerous subject-specific factors (e.g., age, weight, etc.).

[0090] Examples of suitable priming (sub-therapeutic) doses of an anti-CD47 agent include, but are not necessarily limited to a range from about 0.05 mg / kg to about 10 mg / kg (e.g., from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 7.5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 4 mg / kg, from about 0.1 mg / kg to about 3 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 0.5 mg / kg to about 7.5 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 4 mg / kg, from about 0.5 mg / kg to about 3 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 7.5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 4 mg / kg, from about 1 mg / kg to about 3 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 7.5 mg / kg, or about 10 mg / kg).

[0091] In some embodiments a priming dose is fractionated into two or more subdoses, delivered over a period of time from about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 10 days, about 2 weeks.

[0092] In other embodiments, an initial dose of a CD47 binding agent, e.g. a priming dose, is administered by continuous fusion, e.g. as an osmotic pump, delivery patch, etc., where the dose is administered over a period of at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days.

[0093] In some embodiments a priming dose may be delivered through a sub-cutaneous route, by injection, patch, osmotic pump, and the like as known in the art.

[0094] Following administration of the priming agent, and allowing a period of time effective for protection of RBC, a therapeutic dose of an anti-CD47 agent is administered. The therapeutic dose can be administered in number of different ways. In some embodiments, two or more therapeutically effective doses are administered after a primer agent is administered, e.g. in a weekly dosing schedule. In some embodiments a therapeutically effective dose of ananti-CD47 agent is administered as two or more doses of escalating concentration, in others the doses are equivalent.

[0095] In some embodiments of the invention, the therapeutic (maintenance) dose is sufficient to achieve a circulating level of greater than 100 pg / ml for a sustained period of time. In some such embodiments the anti-CD47 agent is magrolimab, or a modified version thereof with reduced FcyR activity. In some embodiments the sustained period of time is up to about 1 week. In some embodiments the sustained period of time is up to about 10 days. In some embodiments the sustained period of time is up to about 2 weeks. In some embodiments the maintenance dose is from about 10 mg / kg to about 25 mg / ml, from about 12.5 mg / kg to about 22.5 mg / kg, from about 15 mg / kg to about 20 mg / kg, from about 17.5 mg / kg to about 20 mg / kg, from about 10 mg / kg to about 20 mg / kg. The maintenance dose may be administered at a periodicity that provides for sustained serum levels of greater than about 100 .g / ml, where administration may be weekly, every 8 days, every 9 days, every 10 days, every 1 1 days, every 12 days, every 13 days, every two weeks, every 3 weeks, and may provide for followup therapy of less frequent administration, e.g. monthly, semi-monthly, bi-monthly, etc

[0096] The administration of a therapeutically effective dose of an anti-CD47 agent can be achieved in a number of different ways. In some cases, two or more therapeutically effective doses are administered after a primer agent is administered. Suitable administration of a therapeutically effective dose can entail administration of a single dose, or can entail administration of doses daily, semi-weekly, weekly, once every two weeks, once a month, annually, etc. In some cases, a therapeutically effective dose is administered as two or more doses of escalating concentration (i.e., increasing doses), where (i) all of the doses are therapeutic doses, or where (ii) a sub-therapeutic dose (or two or more sub-therapeutic doses) is initially given and therapeutic doses are achieved by said escalation. As one non-limiting example to illustrate escalating concentration (i.e., increasing doses), a therapeutically effective dose can be administered weekly, beginning with a sub-therapeutic dose (e.g., a dose of 5 mg / kg), and each subsequent dose can be increased by a particular increment (e.g., by 5 mg / kg), or by variable increments, until a therapeutic dose (e.g., 30 mg / kg) is reached, at which point administration may cease or may continue (e.g., continued therapeutic doses, e.g., doses of 30 mg / kg). As another non-limiting example to illustrate escalating concentration (i.e., increasing doses), a therapeutically effective dose can be administered weekly, beginning with a therapeutic dose, and each subsequent dose can be increased by a particular increment, or by variable increments, until a therapeutic dose (e.g., 30 mg / kg, 100 mg / ml, etc.) is reached, at which point administration may cease or may continue (e.g., continued therapeutic doses, e.g., doses of 30 mg / kg, 100 mg / ml, etc.). In some embodiments,administration of a therapeutically effective dose can be a continuous infusion and the dose can altered (e.g., escalated) over time.

[0097] Dosage and frequency may vary depending on the half-life of the anti-CD47 agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, e.g. in the use of antibody fragments, in the use of antibody conjugates, in the use of SIRPa reagents, in the use of soluble CD47 peptides etc. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., s.c., and the like.

[0098] A “maintenance dose” is a dose intended to be a therapeutically effective dose. For example, in experiments to determine the therapeutically effective dose, multiple different maintenance doses may be administered to different subjects. As such, some of the maintenance doses may be therapeutically effective doses and others may be sub-therapeutic doses.

[0099] A “loading dose” may be used to achieve a therapeutic level of antibody before switching to a maintenance dose. A loading dose can be the same be the same or higher or lower than the maintenance dose, but will generally provide for a higher overall delivery of the agent over a given period of time. For example, a loading dose can be the same or lower than a maintenance dose, but delivered more frequently, e.g. daily, every other day, every third day, twice weekly, weekly, and the like. Alternatively a loading dose can be a higher dose than a maintenance dose, and delivered at the same periodicity, or more frequently, e.g. daily, every other day, every third day, twice weekly, weekly, and the like.

[0100] A "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. For purposes of this invention, a therapeutically effective dose of an anti-CD47 agent is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease.

[0101] According to the present invention, compositions can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, aerosol, or intramuscular means. The most typical route of administration is intravenous although other routes can be equally effective.

[0102] For parenteral administration, compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols suchas propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises polypeptide at 1 mg / mL, formulated in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01 % polysorbate 20, adjusted to pH 7.4 with HCI or NaOH.

[0103] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0104] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0105] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0106] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0107] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1 ).

[0108] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, the mammal is a human, e.g. a human diagnosed with an antibody-associated disease. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0109] "Comparable cell" shall mean a cell whose type is identical to that of another cell to which it is compared. Examples of comparable cells are cells from the same cell line.[001 10] "Inhibiting" the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.[001 11] "Treating" a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent.[001 12] "Suitable conditions" shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When this term is used in connection with nucleic acid hybridization, the term shall mean conditions that permit a nucleic acid of at least 15 nucleotides in length to hybridize to a nucleic acid having a sequence complementary thereto. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions" as used herein means physiological conditions.[001 13] The definition of an appropriate patient sample encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as endometrial cells, etc. A sample if interest in bronchial lavage sample. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, spleen or lympho node biopsysamples, and the like. A “biological sample” includes a sample obtained from a patient's sample cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s sample cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising sample cells from a patient. A biological sample comprising a sample cell from a patient can also include normal, non-diseased cells.[001 14] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of antibody-associated disease, including autoantibody associated diseases.[001 15] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic (i.e., first therapeutic agent) and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect. First therapeutic agents contemplated for use with the methods of the present invention include any other agent for use in the treatment of disease, including tumor specific antibodies, FcyR blocking agents, and the like.[001 16] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of the therapeutic agent and antibody agent at such time that both the known therapeutic agent and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention. Therapeutic agents contemplated for concomitant administration according to the methods of the present invention include any other agent for use in the treatment of disease.[001 17] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.[001 18] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictatedby (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).[001 19] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0120] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0121] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0122] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

[0123] In some embodiments treatment efficacy is determined by monitoring the presence of FcyR on myeloid cells, e.g. circulating monocytes, splenic myeloid cells, etc.Methods of Use

[0124] The scope of the invention encompasses a method and associated compositions utilized in performance of the method, wherein a condition (for example, cancer) in a subject is treated by: administration of an agent to the subject that blocks the interaction between CD47 and SIRPa; and wherein productive high affinity interactions between an Fc moiety ofthe agent and Fey receptors of myeloid cells are inhibited, reduced, or disabled. The inhibited, reduced, or disabled Fc- FcyR interaction is an interaction between the administered anti- CD47 / SIRPa agent and any FcyRs present in the subject being treated with the agent and expressed by myeloid cells, for example, by any of macrophages, monocytes, dendritic cells or other myeloid cell types. Productive high affinity interaction means, in one embodiment, that the interaction is biologically significant, resulting in activation of FcyR-mediated processes, for example to myeloid FcgR-mediated trogocytosis or other processes that result in the impairment of phagocytic activity by myeloid cells, for example, macrophages.

[0125] In one implementation, the method of the invention is achieved by administration of an agent that blocks the interaction between CD47 and SIRPoc, wherein the agent does not comprises an Fc region with reduced or inhibited capacity to engage Fey receptors expressed by one or more myeloid cell types. In some embodients, the agent comprises an antibody fragment lacking an Fc region. In some embodiments, the agent is an antibody comprising an Fc region wherein the Fc region of the antibody is modified to reduce or disable binding to Fey receptors expressed by myeloid cells.

[0126] In some embodiments the antibody comprises a human Fc region sequence, which may be selected from the lgG1 , lgG2a, lgG2b, lgG3, lgG4 Fc sequence, which Fc region is an Fc region with reduced binding to high affinity Fc receptors. In some embodiments the Fc has been modified by one or more amino acid changes to reduce Fc receptor binding, including without limitation the lgG1 Fc sequence comprises the L234A / L235A amino acid substitutions; or modification of the glycosylation on asparagine 297 of the Fc domain. In some embodiments, the antibody is Hu5F9-G4 / Magrolimab or a fragment thereof.

[0127] In a second implementation of the general method, a condition (for example, cancer) in a subject is treated by: administration of a first agent to the subject that blocks the interaction between CD47 and SIRPa; in combination with administration of a second agent comprising an FcyR blocking agent.

[0128] In some embodiments, an anti-CD47 antibody is administered in a regimen comprising one or more initial sub-therapeutic (priming) doses, where the antibody priming dose is administered in combination with an effective dose of an FcyR blocking agent. In some embodiments the blocking agent is administered at a dose and timing such that it is present in an effective dose to inhibit myeloid FcyR-mediated trogocytosis or when the “pruning complex” of CD47 antibody and antigen is present in the circulation. In some embodiments, the FcyR blocking agent is administered a period of time prior to administration of the anti- CD47 antibody, for example, in embodiments a period of about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, one week, two weeks, three weeks or more. In some embodiments, the FcyR blocking agent is administered substantially simultaneously or concurrently withadministration of the anti-CD47 antibody. In some embodiments, the FcyR blocking agent is administered a period of time after administration of the anti-CD47 antibody, for example, in embodiments a period of about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or one week, two weeks, three weeks, or more. In some embodiments the FcyR blocking agent is an antibody. In some embodiments the antibody specifically binds to human FcyRII. In some embodiment the antibody specifically binds to human FcyRIII. In other embodiments the FcyR blocking agent is a small molecule.

[0129] In some embodiments a method of treatment is provided, comprising contacting an individual with an effective dose of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors, or an anti-CD47 antibody administered in combination with an FcyR blocking agent, wherein the effective dose provides for binding the antibody to a phagocytic cell thereby increasing phagocytosis of target cells expressing CD47. Treatment may be systemic or localized, e.g. delivery by intratumoral injection, etc.

[0130] In some embodiments, the use of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors, or an anti-CD47 antibody administered in combination with an FcyR blocking agent, provides for improved clinical efficacy, relative to methods where the antibody has a wild-type Fc region sequence or in the absence of treatment with the FcyR blocking agent. In such methods the anti-CD47 antibody is administered to an individual comprising red blood cells expressing the cognate CD47 antigen. In some embodiments, phagocytosis of targeted cells is increased at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or more.

[0131] In an embodiment, myeloid cells, e.g. macrophages, are monitored for expression of FcyR following administration of a modified anti-CD47 antibody with reduced binding to high affinity Fc receptors, or an anti-CD47 antibody administered in combination with an FcyR blocking agent. In some embodiments the receptor is FcyRIIB / lll. In some embodiments expression of FcyR following such treatment, relative to methods where the antibody has a wild-type Fc region sequence or in the absence of treatment with the FcyR blocking agent, is increased at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or more.

[0132] In some embodiments an individual is treated for cancer. The types of cancer that can be treated using the subject methods of the present invention include but are not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoidtumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia.

[0133] Leukemias and lymphomas are of particular interest. These are cancers that start in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. For example, leukemias can originate in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (e.g., acute versus chronic) and for the type of white blood cell that is effected (e.g., myeloid versus lymphoid). Myeloid leukemias are also called myelogenous or myeloblastic leukemias. Lymphoid leukemias are also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells may collect in the lymph nodes, which can become swollen. Examples of leukemias include, but are not limited to: Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia (CML), and Chronic lymphocytic leukemia (CLL).

[0134] Lymphomas are cancers that begin in cells of the immune system. For example, lymphomas can originate in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphomas. One kind is Hodgkin lymphoma (HL), which is marked by the presence of a type of cell called the Reed-Sternberg cell. There are currently 6 recognized types of HL. Examples of Hodgkin lymphomas include: nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL, and nodular lymphocyte predominant HL.

[0135] The other category of lymphoma is non-Hodgkin lymphomas (NHL), which includes a large, diverse group of cancers of immune system cells. Non-Hodgkin lymphomas can be further divided into cancers that have an indolent (slow-growing) course and those that have an aggressive (fast-growing) course. There are currently 61 recognized types of NHL. Examples of non-Hodgkin lymphomas include, but are not limited to: AIDS-relatedLymphomas, anaplastic large-cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt’s lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-Cell lymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatric lymphoma, peripheral T-Cell lymphomas, primary central nervous system lymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, and Waldenstrom's macroglobulinemia.

[0136] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0137] Chemotherapy may include Abitrexate (Methotrexate Injection), Abraxane (Paclitaxel Injection), Adcetris (Brentuximab Vedotin Injection), Adriamycin (Doxorubicin), Adrucil Injection (5-FU (fluorouracil)), Afinitor (Everolimus) , Afinitor Disperz (Everolimus) , Alimta (PEMET EXED), Alkeran Injection (Melphalan Injection), Alkeran Tablets (Melphalan), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab Injection), Avastin (Bevacizumab), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Busulfex Injection (Busulfan Injection), Campath (Alemtuzumab), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), Clolar (Clofarabine Injection), Cometriq (Cabozantinib), Cosmegen (Dactinomycin), Cytosarll (Cytarabine), Cytoxan (Cytoxan), Cytoxan Injection (Cyclophosphamide Injection), Dacogen (Decitabine), DaunoXome (Daunorubicin Lipid Complex Injection), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex Injection), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex Injection), Droxia (Hydroxyurea), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Erbitux (Cetuximab), Erivedge (Vismodegib), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Injection), Eulexin (Flutamide), Fareston (Toremifene), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix Injection), Fludara (Fludarabine),Folex (Methotrexate Injection), Folotyn (Pralatrexate Injection), FUDR (FUDR (floxuridine)), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine wafer), Halaven (Eribulin Injection), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Ifex (Ifosfamide), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin Injection), Ixempra (Ixabepilone Injection), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel Injection), Kadcyla (Ado-trastuzumab Emtansine), Kyprolis (Carfilzomib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lupron (Leuprolide), Lupron Depot (Leuprolide), Lupron DepotPED (Leuprolide), Lysodren (Mitotane), Marqibo Kit (Vincristine Lipid Complex Injection), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mesnex (Mesna), Mesnex (Mesna Injection), Metastron (Strontium-89 Chloride), Mexate (Methotrexate Injection), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Neosar Injection (Cyclophosphamide Injection), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (nilutamide)), Nipent (Pentostatin), Nolvadex (Tamoxifen), Novantrone (Mitoxantrone), Oncaspar (Pegaspargase), Oncovin (Vincristine), Ontak (Denileukin Diftitox), Onxol (Paclitaxel Injection), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab Injection), Platinol (Cisplatin), Platinol (Cisplatin Injection), PlatinolAQ (Cisplatin), PlatinolAQ (Cisplatin Injection), Pomalyst (Pomalidomide), Prednisone Intensol (Prednisone), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Sandostatin (Octreotide), Sandostatin LAR Depot (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Sterapred (Prednisone), Sterapred DS (Prednisone), Stivarga (Regorafenib), Supprelin LA (Histrelin Implant), Sutent (Sunitinib), Sylatron (Peginterferon Alfa-2b Injection (Sylatron)), Synribo (Omacetaxine Injection), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel Injection), Taxotere (Docetaxel), Temodar (Temozolomide), Temodar (Temozolomide Injection), Tepadina (Thiotepa), Thalomid (Thalidomide), TheraCys BCG (BCG), Thioplex (Thiotepa), TICE BCG (BCG), Toposar (Etoposide Injection), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin Injection), Trexall (Methotrexate), Trisenox (Arsenic trioxide), Tykerb (lapatinib), Valstar (Valrubicin Intravesical), Vantas (Histrelin Implant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Vepesid (Etoposide), Vepesid (Etoposide Injection), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin Injection), Xalkori (Crizotinib), Xeloda (Capecitabine), Xtandi (Enzalutamide), Yervoy (Ipilimumab Injection),Zaltrap (Ziv-aflibercept Injection), Zanosar (Streptozocin), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid), Zortress (Everolimus), Zytiga (Abiraterone), Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab / MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.

[0138] Antibiotics, e.g. antibiotics with the classes of aminoglycosides; carbapenems; and the like; penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc. penicillins in combination with p-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc:; tetracyclines; cephalosporins; quinolones; lincomycins; macrolides; sulfonamides; glycopeptides including the anti-infective antibiotics vancomycin, teicoplanin, telavancin, ramoplanin and decaplanin. Derivatives of vancomycin include, for example, oritavancin and dalbavancin (both lipoglycopeptides). Telavancin is a semi-synthetic lipoglycopeptide derivative of vancomycin (approved by FDA in 2009). Other vancomycin analogs are disclosed, for example, in WO 2015022335 A1 and Chen et al. (2003) PNAS 100(10): 5658-5663, each herein specifically incorporated by reference. Non-limiting examples of antibiotics include vancomycin, linezolid, azithromycin, daptomycin, colistin, eperezolid, fusidic acid, rifampicin, tetracyclin, fidaxomicin, clindamycin, lincomycin, rifalazil, and clarithromycin.

[0139] Radiotherapy means the use of radiation, usually X-rays, to treat illness. X-rays were discovered in 1895 and since then radiation has been used in medicine for diagnosis and investigation (X-rays) and treatment (radiotherapy). Radiotherapy may be from outside the body as external radiotherapy, using X-rays, cobalt irradiation, electrons, and more rarely other particles such as protons. It may also be from within the body as internal radiotherapy, which uses radioactive metals or liquids (isotopes) to treat cancer.

[0140] For the treatment of cancer, the anti-CD47 antibody may be combined with one or more antibodies specific for a tumor antigen. Of these, tumor-associated antigens (TAAs) are relatively restricted to tumor cells, whereas tumor-specific antigens (TSAs) are unique to tumor cells. TSAs and TAAs typically are portions of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.

[0141] Tissue specific differentiation antigens are molecules present on tumor cells and their normal cell counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs fall into several different categories. Hematopoietic differentiation antigens are glycoproteins that are usually associated with cluster of differentiation (CD) groupings and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates that are found on the surface of both normal and tumor cells. Antigens that are involved in growth and differentiation signaling are often growthfactors and growth factor receptors. Growth factors that are targets for antibodies in cancer patients include CEA, epidermal growth factor receptor (EGFR; also known as ERBB1 )’ ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1 R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1 ; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B) and receptor activator of nuclear factor-DB ligand (RANKL; also known as TNFSF11 ). Antigens involved in angiogenesis are usually proteins or growth factors that support the formation of new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin aV|33 and integrin a5p1 . Tumor stroma and the extracellular matrix are indispensable support structures for a tumor. Stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

[0142] Specific antibodies include, without limitation: anti-CD20 antibodies, e.g. Ofatumumab (Arzerra), Obinutuzumab (Gazyva), Rituximab (Rituxan); anti-HER2: Trastuzumab (Herceptin); anti-EGFR: Cetuximab (Erbitux); anti-VEGF: Bevacizumab (Avastin); anti-CD38: Daratumumab (Darzalex); anti-SLAMF7: Elotuzumab (Empliciti); anti-CD19, e.g. Blinatumomab (Blincyto) a bispecific antibody targeting CD19 and CD3; anti-CD30, e.g. Brentuximab vedotin (Adcetris); anti-CD33, e.g. Gemtuzumab ozogamicin (Mylotarg); anti- CD22, e.g. Inotuzumab ozogamicin (Besponsa); anti-CD79b, e.g. Polatuzumab vedotin (Polivy), and the like as known in the art.

[0143] For the treatment of cancer, the anti- CD47 antibody may be combined with one or more antibodies that inhibit immune checkpoint proteins. Of particular interest are immune checkpoint proteins displayed on the surface of a tumor cell. The immune-checkpoint receptors that have been most actively studied in the context of clinical cancer immunotherapy, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1 ; also known as CD279) - are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors implies that antitumor immunity can be enhanced at multiple levels and that combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.

[0144] The two ligands for PD1 are PD1 ligand 1 (PDL1 ; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells and through binding to its receptor PD1 on T cells it inhibits T cell activation / function. See, for example, Avelumab as a therapeutic antibody.

[0145] Agents that agonize an immune costimulatory molecule are also useful in the methods of the invention. Such agents include agonists or CD40 and 0X40. CD40 is a costimulatory protein found on antigen presenting cells (APCs) and is required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of theTNF receptor family. The primary activating signaling molecules for CD40 are IFNyand CD40 ligand (CD40L). Stimulation through CD40 activates macrophages.

[0146] Anti CCR4 (CD194) antibodies of interest include humanized monoclonal antibodies directed against C-C chemokine receptor 4 (CCR4) with potential anti-inflammatory and antineoplastic activities.

[0001] As used herein, endpoints for treatment will be given a meaning as known in the art and as used by the Food and Drug Administration.

[0002] Overall survival is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Survival is considered the most reliable cancer endpoint, and when studies can be conducted to adequately assess survival, it is usually the preferred endpoint. This endpoint is precise and easy to measure, documented by the date of death. Bias is not a factor in endpoint measurement. Survival improvement should be analyzed as a risk-benefit analysis to assess clinical benefit. Overall survival can be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval. A benefit of the methods of the invention can include increased overall survival of patients.

[0003] Endpoints that are based on tumor assessments include DFS, ORR, TTP, PFS, and time-to-treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumor measurements). Disease-Free Survival (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. The most frequent use of this endpoint is in the adjuvant setting after definitive surgery or radiotherapy. DFS also can be an important endpoint when a large percentage of patients achieve complete responses with chemotherapy.

[0004] Objective Response Rate . ORR is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study.

[0005] Time to Progression and Progression-Free Survival. TTP and PFS have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. The precise definition of tumor progression is important and should be carefully detailed in the protocol.

[0006] Other conditions that may be treated include, without limitation, atherosclerosis (see U.S. 10,329,354); infection (see US 9,771 ,428); depletion of hematopoietic stem cells (see US 10,406,179) and fibrosis (see US 11 ,286,301 ), each herein specifically incorporated by reference.Treatment of Autoimmune Disease

[0147] In other embodiments, methods are provided for treatment of antibody associated diseases, comprising administering an effective dose of an antibody that binds to an antigen present on red blood cells, in a dose effective to cause FcyR loss in reticulo-endothelial myeloid cell populations. In some embodiments the antigen is CD47. In some embodiments the antibody comprises a human Fc region sequence, which may be selected from the lgG1 , lgG2a, lgG2b, lgG3, lgG4 Fc sequence, which binds to an FcyR. In some embodiments the antibody induces loss of FcyRIIB / lllon myeloid cells for a period of at least about 2 weeks, where the level of cell surface FcyRII B / ll I on splenic or circulating myeloid cells is reduced by at least about 25%, at least about 50%, at least about 75% or more.

[0148] In some embodiments the antibody-associated disease is associated with autoimmune antibodies, including without limitation Grave’s ophthalmopathy, multifocal motor neuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Kawasaki disease, and immune thrombocytopenia (ITP). In some embodiments the patient is monitored for loss of FcyRII B / l 11 during the course of treatment.

[0149] In some embodiments the dose of antibody administered for treatment is a dose of from about 0.5 mg / kg to about 10 mg / kg. In some embodiments the dose of antibody administered for treatment is a dose of from about 1 mg / kg to about 5 mg / kg, e.g. about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg. Administration may be repeated, e.g. weekly, bi-weekly, monthly, etc.

[0150] In other embodiments, a method is provided for protecting red blood cells (RBC) from immune destruction following transplantation, the method comprising treating the RBC with a dose of an anti-CD47 antibody comprising a human Fc sequence effective to induce loss of cell surface CD47 on the RBC; and transfusing the treated RBC to an individual in need thereof. The level of cell surface CD47 on RBC is reduced by at least about 25%, at least about 50%, at least about 75% or more. In some embodiments, an anti-CD47 antibody is administered to the individual prior to transfusing the treated RBC. In some embodiments, RBCs coated with anti-CD47 antibodies are administered to the individual prior to transfusing the treated RBC.

[0151] Conditions for which the antibodies described herein find use in treatment include a number of conditions where inhibition of the action of autoantibodies is of interest. Suchdiseases may include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), autoimmune hepatitis; multifocal motor neuropathy; chronic inflammatory demyelinating polyneuropathy; Kawasaki disease; and immune thrombocytopenia (ITP).

[0152] Diseases associated undesirable antibody activity are of interest, which conditions include, without limitation, systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis, lgG4 related disease; autoimmune hepatitis; multifocal motor neuropathy; chronic inflammatory demyelinating polyneuropathy; Kawasaki disease; and immune thrombocytopenia (ITP) etc.

[0153] There are multiple mechanisms by which autoantibodies contribute to the pathogenesis of autoimmune disease. First, immune complexes can form or deposit in tissues where they can activate complement and induce CDC to cause tissue damage. RA is associated with the production of rheumatoid factor, autoantibodies that bind the Fc region of IgG, and anti-citrullinated protein antibodies (ACPAs). In RA, both rheumatoid factor containing and ACPA-containing immune complexes activate complement pathways in joints, leading to the production of C5a and the generation of the membrane attack complex (MAC), which both contribute to joint damage; IgM rheumatoid factor can also increase complement activation mediated by ACPA-containing immune complexes. Autoantibodies can promote tissue damage via ADCC by co-engagement of antigens on the target tissue and Fc receptors (FcRs) on macrophages, neutrophils, natural killer (NK) cells and other effector cell types. Autoantibody containing immune complexes can also activate immune cells through dual engagement of FcRs and Toll-like receptors (TLRs) (on macrophages and dendritic cells) or dual engagement of the B cell receptor (BCR) and TLRs. Immune complexes facilitate antigen loading onto dendritic cells via immune complexes, enabling these cells to efficiently activate T cells.

[0154] Rheumatoid Arthritis (RA) is a chronic syndrome characterized usually by symmetric inflammation of the peripheral joints, potentially resulting in progressive destruction of articular and periarticular structures, with or without generalized manifestations (Firestein (2003) Nature 423(6937) :356-61 ; Mclnnes and Schett. (201 1 ) N Engl J Med. 365(23):2205-19). The cause is unknown. A genetic predisposition has been identified, and, in some populations, localized to a pentapeptide in the HLA-DR betal locus of class II histocompatibility genes. Environmental factors may also play a role. For example, cigarette smoking places individuals possessing HLA-DR4 containing the "shared epitope" polymorphism at approximately 10-20 fold increased risk of developing RA. Cigarette smoking is thought to induce anti-citrullinated protein antibody (ACPA) responses, which are measured using the commercial cyclic- citrullinated peptide (CCP) assay (Klareskog et al. (2006) Arthritis Rheum. 54(1 ) :38-46). In addition, periodontitis and infection with P. gingivalis might also play a role in the initiation of autoimmune responses that result in development of RA (Rutger and Persson. 2012, J OralMicrobiol. 4). Immunologic changes may be initiated by multiple factors. About 0.6% of all populations are affected, women two to three times more often than men. Onset may be at any age, most often between 25 and 50 yr.

[0155] Prominent immunologic abnormalities that may be important in pathogenesis include antibodies and immune complexes found in joint fluid cells and in vasculitis. Plasma cells produce antibodies that contribute to these complexes. Lymphocytes that infiltrate the synovial tissue are primarily T helper cells, which can produce pro-inflammatory cytokines. Macrophages and their cytokines (e.g., tumor necrosis factor, granulocyte-macrophage colony-stimulating factor) are also abundant in diseased synovium. Increased adhesion molecules contribute to inflammatory cell emigration and retention in the synovial tissue. Increased macrophage-derived lining cells are prominent along with some lymphocytes and vascular changes in early disease.

[0156] In chronically affected joints, the normally delicate synovium develops many villous folds and thickens because of increased numbers and size of synovial lining cells and colonization by lymphocytes and plasma cells. The lining cells produce various materials, including collagenase and stromelysin, which can contribute to cartilage destruction; interleukin-1 , which stimulates lymphocyte proliferation; and prostaglandins. The infiltrating cells, initially perivenular but later forming lymphoid follicles with germinal centers, synthesize interleukin-2, other cytokines, RF, and other immunoglobulins. Fibrin deposition, fibrosis, and necrosis also are present. Hyperplastic synovial tissue (pannus) may erode cartilage, subchondral bone, articular capsule, and ligaments. PMNs are not prominent in the synovium but often predominate in the synovial fluid.

[0157] Onset is usually insidious, with progressive joint involvement, but may be abrupt, with simultaneous inflammation in multiple joints. Tenderness in nearly all inflamed joints is the most sensitive physical finding. Synovial thickening, the most specific physical finding, eventually occurs in most involved joints. Symmetric involvement of small hand joints (especially proximal interphalangeal and metacarpophalangeal), foot joints (metatarsophalangeal), wrists, elbows, and ankles is typical, but initial manifestations may occur in any joint. RA is characterized by the development of focal bone erosions through degradation and remodeling of bone at the joint margins and in subchondral bone of patients with RA. A hallmark of a subset of RA is the development of autoantibodies, including rheumatoid factors (RF) and anti-citrullinated protein antibodies (ACPA). RF, antibodies to human Fey, are present in about 70% of patients with RA. However, RF, often in low titers, occurs in patients with other diseases, including other connective tissue diseases such as systemic lupus erythematous, granulomatous diseases, chronic infections such as viral hepatitis, subacute bacterial endocarditis, and tuberculosis, and cancers. Low RF titers can also occur in a small percentage of the general population, and more commonly in the elderly.Another disease indicator is the presence of ACPA, which are measured using the clinical anti-CCP (cyclic citrullinated peptide) antibody test. Anti-CCP antibodies are approximately 60% sensitive and 95% specific for the diagnosis of RA, and like RF, predict a worse prognosis.

[0158] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by malar rashes, oral ulcers, photosensitivity, serositis, seizures, low white blood cell counts, low platelet counts, seizures, a positive anti-nuclear antibody (ANA) test, and other positive autoantibodies. SLE is an autoimmune disease characterized by polyclonal B cell activation, which results in a variety of anti-protein and non-protein autoantibodies that result in immune complexes and inflammation which contributes to tissue damage (see, e.g., Kotzin et al., 1996, Cell 85:303-06 for a review of the disease). SLE has a variable course characterized by exacerbations and remissions and is difficult to study. For example, some patients may demonstrate predominantly skin rash and joint pain, show spontaneous remissions, and require little medication. The other end of the spectrum includes patients who demonstrate severe and progressive kidney involvement (glomerulonephritis and cerebritis) that requires therapy with high doses of steroids and cytotoxic drugs such as cyclophosphamide. Hydroxychloroquine slows SLE progression, and is a mainstay therapeutic for the management of SLE.

[0159] Autoimmune hepatitis is a disease in which the body's immune system attacks liver cells. This immune response causes inflammation of the liver, also called hepatitis. Researchers think a genetic factor may make some people more susceptible to autoimmune diseases. About 70 percent of those with autoimmune hepatitis are female. The disease is usually quite serious and, if not treated, gets worse overtime. Autoimmune hepatitis is typically chronic, meaning it can last for years, and can lead to cirrhosis-scarring and hardening-of the liver. Eventually, liver failure can result.

[0160] In some embodiments the methods of the invention comprise the step of identifying individuals "at-risk" for development of, or in the "early-stages" of, an inflammatory disease. "At risk" for development of an inflammatory disease includes: (1) individuals whom are at increased risk for development of an inflammatory disease, and (2) individuals exhibiting a "pre-clinical" disease state, but do not meet the diagnostic criteria for the inflammatory disease (and thus are not formally considered to have the inflammatory disease).

[0161] Individuals "at increased risk" for development (also termed "at-risk" for development) of an inflammatory disease are individuals with a higher likelihood of developing an inflammatory disease or disease associated with inflammation compared to the general population. Such individuals can be identified based on their exhibiting or possessing one or more of the following: a family history of inflammatory disease; the presence of certain geneticvariants (genes) or combinations of genetic variants which predispose the individual to such an inflammatory disease; the presence of physical findings, laboratory test results, imaging findings, marker test results (also termed "biomarker" test results) associated with development of the inflammatory disease, or marker test results associated with development of a metabolic disease; the presence of clinical signs related to the inflammatory disease; the presence of certain symptoms related to the inflammatory disease (although the individual is frequently asymptomatic); the presence of markers (also termed "biomarkers") of inflammation; and other findings that indicate an individual has an increased likelihood over the course of their lifetime to develop an inflammatory disease or disease associated with inflammation. Most individuals at increased risk for development of an inflammatory disease or disease associated with inflammation are asymptomatic, and are not experiencing any symptoms related to the disease that they are at an increased risk for developing.

[0162] Included, without limitation, in the group of individuals at increased risk of developing an inflammatory disease or a disease associated with inflammation, are individuals exhibiting "a pre-clinical disease state". The pre-disease state may be diagnosed based on developing symptoms, physical findings, laboratory test results, imaging results, and other findings that result in the individual meeting the diagnostic criteria for the inflammatory disease, and thus being formally diagnosed. Individuals with "pre-clinical disease" exhibit findings that suggest that the individual is in the process of developing the inflammatory disease, but do not exhibit findings, including the symptoms, clinical findings, laboratory findings, and / or imaging findings, etc. that are necessary to meet the diagnostic criteria for a formal diagnosis of the inflammatory disease. In some embodiments, individuals exhibiting a pre-clinical disease state possess a genetic variant or a combination of genetic variants that place them at increased risk for development of disease as compared to individuals who do not possess that genetic variant or that combination of genetic variants. In some embodiments, these individuals have laboratory results, or physical findings, or symptoms, or imaging findings that place them at increased risk for development of an inflammatory disease. In some embodiments, individuals with preclinical disease states are asymptomatic. In some embodiments, individuals with pre- clinical disease states exhibit increased or decreased levels of the expression of certain genes, certain proteins, inflammatory markers, metabolic markers, and other markers.

[0163] In some embodiments a disease treated by the methods of the disclosure is an autoimmune disease in which IVIG is useful therapeutically. Intravenous immunoglobulin (IVIG) is a concentrate of the pooled immunoglobulins derived from 1000 to 100000 healthy donors depending upon the manufacturer. The composition of IVIG products closely corresponds to that of immunoglobulins in the normal human plasma, especially IgG (along with its subclasses), IgA, traces of other Igs, cytokines, and soluble receptors.

[0164] In autoimmune and inflammatory conditions, two to four-fold increases in doses of IVIG, when compared to replacement doses, can bring a variety of protective changes. For example, immune thrombocytopenic purpura (ITP) is an autoimmune condition characterized by isolated thrombocytopenia causing life-threatening bleeding. IVIG therapy has been shown to raise the platelet count within four days of administration, reducing the need for frequent and repeated platelet transfusions. Glucocorticoids, along with IVIG, are now considered first- line therapy in this condition and have greatly improved the lives of these patients. Other conditions include Grave’s ophthalmopathy, multifocal motor neuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and Kawasaki disease.

[0165] Autoimmunity is essentially an overt immune response against the body’s own tissues, and IgG autoantibodies are considered the main players in most conditions. Self-antigen is recognized by Fab fragment of IgG autoantibody, and Fc fragment relays this signal to Fc- gamma-receptors (FcgRs) on various immune cells. IgG autoantibodies cause inflammation by interacting with FcgRs, neonatal FcR (FcRn), and complement proteins. Autoantibodies disrupt a myriad of functions, including cellular lysis (as in ITP), triggering micro thrombosis, ADCC, complement-dependent cytotoxicity, uncontrolled neutrophil activation, stimulation of hormonal receptor (Graves disease), receptor blockade of neural transmission (MG), induction of inflammation (rheumatoid arthritis) and altered cell signaling.

[0166] Monomeric IgG in high dose IVIG causes blockade of activating FcgRs by saturating them on immune cells attenuating immune complex-mediated inflammation and autoimmunity They also cause an immediate increase in platelet counts in ITP patients.

[0167] Neonatal FcRs (FcRn) on endothelial cells bind to auto-IgG and prolong its half-life by preventing its catabolism. IVIG can saturate FcRn, thereby promoting the rapid elimination of the pathogenic endogenous IgGs.

[0168] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0169] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is notintended to limit the scope of the present invention which will be limited only by the appended claims.

[0170] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.EXPERIMENTAL

[0171] 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 present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.EXAMPLE 1CD47 antibody therapy decreases RBC clearance through inhibition of phagocytic capacity of macrophages

[0172] Red blood cells (erythrocytes, RBCs) circulate for -120 days in humans and -45 days in mice before they are phagocytosed primarily by macrophages in the splenic red pulp. They are protected from non-specific and premature destruction by red pulp macrophages by their expression of CD47, a ‘don’t eat me’ signal that binds to macrophage expressed SIRPa to inhibit phagocytosis. However, as monoclonal antibodies have been developed to block the CD47-SIRPa interaction for cancer immunotherapy, it has been necessary to overcome the toxicity associated with the off-target destruction of erythrocytes. It was found that a small loading dose of CD47 antibody confers to erythrocytes protection against much greater subsequent dosing. Here we identify a mechanism of this protection in mice, a mechanism by which CD47 antibody therapy may hinder its own anti-cancer function, but which can explain the more generally observed efficacy of antibodies against RBC-expressed antigens in protecting against some autoimmune disorders. We begin by demonstrating that CD47 antibody induces Fc gamma receptor (FcgR) mediated loss of erythroid CD47, with global concomitant myeloid FcgR loss in mice. We then show that CD47 antibody therapy impairs red pulp macrophage phagocytosis of erythrocytes in vivo. Finally, we show that observed myeloid FcgR loss and phagocytic impairment are the more general result of treatment with an antibody against an RBC-expressed antigen. Our findings indicate that CD47 antibodycancer immunotherapy, which is intended to engage macrophage FcgRs and to promote cancer cell phagocytosis, may hinder its own anti-cancer efficacy by its off-target activity against RBCs.

[0173] Erythroblasts are enucleated in the bone marrow and enter the circulation as reticulocytes, which there mature into red blood cells (RBCs). RBCs circulate once every 45 seconds for -120 days in humans, or once every 15 seconds for -45 days in mice, before they are phagocytosed by macrophages primarily in the spleen and liver. In their constant interactions with near-vascular macrophages, RBCs require protection from nonspecific phagocytic destruction. This protection comes in the form of the ‘don’t eat me’ signal, CD47. CD47 is a conserved surface protein expressed by all cells in the human body. It is also the first described ‘don’t eat me’ signal in that it binds to the macrophage receptor SIRPa to inhibit phagocytosis. This ‘don’t eat me’ function was first described on RBCs, though it was later found to be important for circulating hematopoietic stem cell and leukemic stem cell survival. It was also shown that antibodies that block the CD47-SIRPa interaction can be leveraged to promote phagocytic destruction of leukemic stem cells. Since then, CD47 has been found to be overexpressed in many cancers, and several CD47 antibodies have entered clinical trials.

[0174] CD47 antibody is a doubly potent therapy, as it promotes phagocytosis both by blocking the anti-phagocytic CD47 — SIRPa interaction and by stimulating pro-phagocytic macrophage-expressed Fc-gamma receptors (FcgRs). FcgRs are the principle immune receptors for antibodies and mediate downstream effector functions of antibody binding.

[0175] As RBCs require surface CD47 to survive in the circulation and are sensitive to antibody-mediated phagocytosis, a hurdle for CD47 antibody therapies has been overcoming the toxicity associated with nonspecific destruction of RBCs. It was found that the administration of a low priming dose of CD47 antibody (1 mg / kg) protects against anemia and enables much greater (>=45mg / kg) subsequent dosing.

[0176] To begin to elucidate the mechanism behind this tolerance, we collected circulating RBCs and bone marrow erythroid progenitors from mice treated with a CD47 antibody. We found that RBCs in treated animals show a log-fold reduction in surface CD47 expression. Delayed and less pronounced loss of CD47 by immature erythroid cells in the bone marrow suggests that loss of CD47 happens to mature circulating RBCs.

[0177] To understand the mechanisms of this loss, we began by identifying a similar observation made in the literature for the CD20 antibody Rituximab, which was shown to induce CD20 loss in target cells by at least two distinct mechanisms: (1 ) cell intrinsic internalization and (2) myeloid FcgR-mediated trogocytosis, a form of antigenic pruning. Although a highly expressed RBC antigen like CD47 represents a much greater antigenic load than a B cell antigen like CD20, we hypothesized that RBC CD47 loss occurs by analogousmechanisms. Indeed, we find in vitro that cell-intrinsic loss of CD47 explains some but not all of the observed RBC CD47 loss. We demonstrate, first with an FcgR-blocking antibody and then with a CD47 antibody F(ab')2 fragment unable to engage FcgRs, the influence of FcgR- mediated loss of RBC CD47.

[0178] As FcgR-mediated antigenic pruning implies myeloid-mediated removal of the antigen- antibody-FcgR complex, we collected myeloid cell rich tissues from CD47 antibody treated mice to assess global myeloid and NK cell populations for apparent FcgR loss. Indeed, we found that RBC CD47 loss corresponds to a global reduction in myeloid and NK cell FcgR expression, with much of this reduction occurring after the priming dose. Further, we showed near complete inhibition of phagocytosis of red blood cells by splenic red pulp macrophages in treated mice. Finally, we showed that this collective myeloid impairment protects circulating RBCs from antibody-mediated destruction by macrophages and is the more general result of treatment with an antibody against an RBC-expressed antigen.

[0179] While antibodies have been shown to modulate the surface expression of RBC antigens in the past, the observed log-fold loss of erythroid surface CD47 represents a fundamental change in the innate immune biology of the CD47-SIRPa axis, which is essential to RBC survival in the circulation under naive conditions. The myeloid FcgR loss and phagocytic impairment that coincide with this antigenic loss, and which appear to be the more general result of treatment with an antibody against an RBC-expressed antigen, have to our knowledge not previously been seen to result from antibody therapy. Indeed, this general dampening of myeloid function may explain the observed therapeutic benefits conferred by some RBC-targeting antibodies against immune thrombocytopenia.

[0180] Our data have immediate implications for the ongoing clinical programs targeting CD47 for cancer immunotherapy. As CD47 antibody therapy aims to promote macrophage phagocytosis through both the blockade of the CD47-SIRPa interaction and the engagement of macrophage-expressed FcgRs, the observed reduction of myeloid FcgR expression and impairment of macrophage phagocytosis may limit therapeutic efficacy in cancer.Methods

[0181] Mice. All mice were obtained from The Jackson Laboratory, and all mouse experiments adhered to the ethical care guidelines set forth by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). 6-10-week-old female C57BL / 6J mice and 8-12-week- old female BALB / c mice were used for all experiments.

[0182] Mouse Antibody Treatments. Mice for erythroid CD47 and myeloid and NK cell FcgR expression analysis were treated with anti-CD47 [clone MIAP410] or lgG1 [clone MOPC-21 ] three times weekly by intra-peritoneal injection. 100ug doses of antibody were administered in the first week, and 300ug doses in subsequent weeks. Mice for F(ab’)2 experimentsreceived a single 100ug dose of anti-CD47 [clone MIAP410] or a single 67ug dose of anti- CD47 [clone MIAP410] F(ab’)2. ‘FcgR-blocked’ mice were treated 3x weekly with 200ug anti- FcgRIlB / lll [clone 2.4G2] for one week, then 3x weekly with 200ug anti-FcgRI IB / 111 [clone 2.4G2] and 100ug anti-CD47 [clone MIAP410] for one week, and compared to mice treated 3x with 100ug anti-CD47 [clone MIAP410] in the second week. For comparisons including multiple unique RBC-binding antibodies, each antibody was administered as a single 50ug dose, 24 hours prior to subsequent experimentation and analysis. CD47 antibody-treated mice used for phagocytosis assays and time course experiments were treated for two weeks, as described above.

[0183] Mouse Tissue Collection. 300ul blood was collected by retro-orbital bleed into PBS with 5mM EDTA. Mice were then euthanized according to APLAC guidelines. Tissues were collected and single cell suspensions generated as follows. Peritoneal cells were collected by peritoneal lavage: 5ml of PBS were injected into the mouse peritoneal cavity, the abdomen was massaged for 30 seconds, and cell suspension was then collected with a syringe. Spleens were resected and pressed through 100um strainers. Livers were resected and mechanically dissociated with a straight razor and then enzymatically dissociated in 25 ml M199 medium with 2.2% Type II Collagenase and 0.2% DNAsel on a shaker at 60 RPM for 45 minutes, with clumps broken up every 15 min with a serological pipette. For bone marrow cell isolation, leg and hip bones were ground by mortar and pestle, and cell suspension was passed through a 100um strainer. All cell suspensions were washed two times with MACS buffer + 5mM EDTA prior to counting and freezing or staining.

[0184] Flow Cytometry. Cells were diluted to 20e6 cells / ml and 50-75ul cell suspension (2- 3e6 cells) were stained for flow cytometric analysis. All samples were blocked prior to staining with unlabeled anti-CD16 / 32 (clone 2.4G2; FcR block) except for those samples stained for FcgR expression. Erythroid cells were always stained fresh. For erythroid surface CD47 quantification, cells were first incubated in vitro with the CD47 antibody used for in vivo treatments, unlabeled anti-CD47 [clone MIAP410]. APC-anti-mouse lgG1 was then used to quantify true erythroid surface CD47 signal. To determine the amount of antibody remaining bound to erythroid cells in vivo, cells were directly stained with APC-anti-mouse IgG 1 . Tissues for myeloid cell FcgR staining were frozen in 10% DMSO + 90% FBS and then thawed and washed once with DMEM + 10% FBS and twice with MACS buffer + 5mM EDTA prior to staining. For myeloid surface FcgRI, FcgRIlB, FcgRHB / lll, and FcgRIV quantification, true signal was determined by subtracting sample-specific isotype antibody signal. FcgR isotype control samples were blocked with anti-CD16 / 32 prior to staining to prevent nonspecific Remediated binding of isotype antibodies.

[0185] Data Normalization and Analysis. Flow cytometric analysis was performed in FlowJo 10. For each experiment, flow cytometry signal was normalized to average isotype, naive or other control signal. All statistical tests were performed in GraphPad Prism 9.

[0186] In Vivo Phagocytosis. For in vivo phagocytosis experiments, RBCs were collected by retro-orbital bleed and washed 2x with PBS with 5mM EDTA. RBCs were then stained at 30e6 cells / ml with 20pM carboxyfluorescein succinimidyl ester (CFSE) for 1 .5 hours at 37C, quenched with DMEM + 10% FBS, and washed 2 times with PBS + 5mM EDTA. Equal numbers of RBCs (150e6) in equal volumes (1 OOul) were then infused into mice retro-orbital ly. After one hour, mice were euthanized and blood and spleen were collected and single cell suspensions generated as described above. Equal volumes of blood were analyzed by flow cytometry to determine the true counts of CFSE-labeled RBCs per blood volume across animals. Splenic phagocytosis was quantified as the percentage of red pulp macrophages (F4 / 80+, CD1 1 b-int) that were CFSE+.Results

[0187] Erythrocytes (i.e., red blood cells, RBCs) circulate for -120 days in humans and -45 days in mice. The senescent red blood cells are then filtered and phagocytosed by near- vascular macrophages. Young red blood cells are protected from premature phagocytosis by CD47, a ‘don’t eat me’ signal that interacts with macrophage expressed SIRPa to inhibit phagocytosis. Monoclonal antibodies that block the CD47-SIRPa interaction have been developed for cancer immunotherapy. While CD47 antibody therapy does promote macrophage phagocytosis of red blood cells, it was found that a small loading dose of CD47 antibody (~1 mg / kg) protects red blood cells from much greater subsequent dosing (>=45mg / kg). We sought to elucidate the mechanism of this protection.

[0188] CD47 antibody therapy induces FcgR-mediated loss of erythroid CD47 in mice. That RBCs can be tolerized to antibody-mediated blockade of their surface CD47 appears to contradict the finding that surface CD47 is necessary for RBC survival in the circulation. Indeed, while CD47-deficient RBCs are rapidly phagocytosed after infusion into naive mice, complete blood counts for mice treated with a loading dose of CD47 blocking antibody showed <10% reduced red blood cell counts, a difference that persisted through two weeks of treatment and with escalated dosing (FIG. 6A). RBCs from CD47 antibody treated mice were analyzed by flow cytometry for their surface CD47 expression and for the proportion of their surface CD47 that was antibody bound after treatment. Surprisingly, these cells showed a >5- fold reduction in surface CD47 that was sustained through four weeks of treatment, with most of this reduction occurring after the loading dose (Fig 1 A-B).

[0189] As CD47 antibody therapy appeared to induce RBC CD47 loss, to ascertain the mechanism(s) of this loss, we first looked to previously described mechanisms of antibody-mediated antigenic loss observed after Rituximab (anti-CD20) therapy. Rituximab was shown to induce target cell CD20 loss by two distinct mechanisms: (1) cell intrinsic internalization and (2) myeloid FcgR-mediated pruning. To determine the potential influence of a cell intrinsic mechanism on RBC CD47 loss, we incubated mouse RBCs with CD47 antibody for 24 hours. While RBC CD47 expression was reduced in vitro, it was not reduced to the extent seen in vivo (FIG. 6B). Further, the remaining RBC surface CD47 was fully antibody bound in vitro (-60% surface CD47), while RBCs collected from treated mice showed little remaining antibody bound (<10% surface CD47) (FIG. 6C, Fig 1 C). As aged RBCs are primarily cleared by macrophages in the spleen, we asked whether myeloid FcgR-mediated pruning might explain the near-complete antigenic loss. To test for FcgR-mediated RBC CD47 loss, we treated mice with FcgRHB / lll blocking antibody prior to and throughout the course of CD47 antibody treatment. These ‘FcgR-blocked’ mice showed reduced loss of RBC CD47 and increased remaining RBC-bound antibody (Fig 1 D-E). Together, the partial prevention of RBC CD47 loss and increase in remaining RBC-bound antibody suggest that FcgR-blocking antibody impairs antibody-mediated RBC CD47 loss. A CD47 antibody F(ab’)2 fragment unable to engage FcgRs was also shown to elicit reduced RBC CD47 loss, further supporting the influence of an FcgR-mediated mechanism (FIG. 6D).

[0190] Macrophage interactions mediate RBC development, survival, and ultimately clearance. As the survival of circulating CD47-deficient RBCs represented a fundamental change in the treatment of circulating RBCs by macrophages, we next asked whether RBC CD47 loss occurred during erythroid development or at the mature cell stage, especially as developing erythroid cells interact closely with macrophages in the bone marrow. To test this, we analyzed bone marrow immature erythroid cells from CD47 antibody treated mice by flow cytometry for their surface CD47 expression (FIG. 7 [gating strategy]). After the administration of a loading dose of CD47 antibody, erythroblasts and reticulocytes showed no reduction in CD47 and no antibody bound CD47. At two weeks of treatment, these immature populations began to show CD47 loss and showed significant antibody binding. At four weeks of treatment, erythroblasts showed ~2-fold loss of CD47, less than the proportional loss seen in mature red blood cells after the administration of a single loading dose (Fig 2A-C). The delayed loss of erythroblast CD47 suggests that the immediate change induced in RBC CD47 does not represent a change in erythroid development. The delayed binding of antibody to erythroblasts in the bone marrow suggests that delayed antibody-mediated loss of erythroblast CD47 may be due to the antibody’s difficulty in penetrating the bone marrow. Further, ‘FcgR-blocked’ mice treated with FcgRIlB / lll-blocking antibody also showed reduced loss of erythroblast CD47 and increased remaining erythroblast-bound antibody, supporting that CD47 loss by immature erythroid cells in the bone marrow is also partially FcgR-mediated (Fig 2D-E).

[0191] Erythroid CD47 loss coincides with global myeloid FcgR loss. FcgR-mediated antigenic pruning has not previously been shown to result in global myeloid FcgR loss. However, we hypothesized that a highly expressed red blood cell antigen might constitute a sufficient antigenic load to elicit concomitant myeloid FcgR loss upon uptake of the antigen- antibody-FcgR complex. Thus, we collected myeloid-cell rich tissues, including spleen, liver, bone marrow, and peritoneum from CD47 antibody treated mice, and analyzed the myeloid and NK cell populations in these tissues for their FcgR expression by flow cytometry, using antibodies against FcgRI, FcgRHB, FcgRIIB / l II and FcgRIV (FIG. 8A-D [gating strategy], FIG. 9A-B [experimental design]). After a loading dose of CD47 antibody, when the bulk of RBC CD47 loss was observed, myeloid populations assessed showed consistent loss of FcgRIlB / lll, but not of FcgRI, FcgRHB, or FcgRIV (Fig 3A). The increased or unchanged expression of FcgRHB across myeloid populations suggests that the observed FcgRIlB / lll loss is accounted for by FcgRIII loss. FcgRIII is considered the principal activatory FcgR, and its acute loss may impair myeloid immune function, especially antibody-mediated phagoctyosis. Significant FcgRIlB / lll loss was observed for myeloid and NK cell populations of the spleen, liver, bone marrow, and peritoneum (Fig 3B, FIG. 9C). FcgRIlB / lll expression was normalized to average expression in isotype antibody treated mice (FIG. 10). Most myeloid populations assessed showed unchanged or increased expression of FcgRI, FcgRHB, and FcgRIV after the administration of a loading dose, with decreased expression after two weeks of treatment (FIG. 11 A-C). The mechanism behind the delayed loss of FcgRI, FcgRHB, and FcgRIV is unclear, but may represent a general downregulation in response to initial FcgRIII loss.

[0192] CD47 antibody therapy inhibits in vivo phagocytosis of red blood cells. Red pulp macrophages, the principal effector cells of red blood cell phagocytosis, demonstrated significant FcgRIlB / lll loss with CD47 antibody therapy. However, while myeloid FcgR loss provides an explanation for how circulating antibody-coated cells could be protected from antibody-mediated phagocytosis, it does not explain the protection of CD47-deficient cells. Indeed, while myeloid FcgR expression surged 30 days after cessation of treatment (FIG. 12A), CD47-deficient red blood cells persisted, with -1 / 3 of circulating red blood cells still CD47-deficient at that time (FIG. 12B-C). We hypothesized that the survival of CD47-deficient red blood cells could be explained by phagocytic impairment of red pulp macrophages (RPMs). To test this, an in vivo phagocytosis assay was performed as follows. Briefly, red blood cells were collected from CD47 antibody treated mice, fluorescently labeled with CFSE ex vivo, and then infused into naive and CD47 antibody treated mice (Fig 4A). Blood and spleens were collected from these mice and analyzed by flow cytometry, and phagocytosis in the spleen was quantified as the proportion of red pulp macrophages with CFSE+ signal (Fig 4B). Analysis of the blood for surviving CFSE+ cells revealed that labeled red blood cells were almost entirely cleared from the circulations of naive mice, while they persisted in treated mice(Fig 4C). CFSE+ RPMs were observed in naive mice, and not in CD47 antibody treated mice (Fig 4D). These data support that CD47 antibody therapy impairs RPM phagocytosis of RBCs in CD47 antibody treated mice, protecting circulating CD47-deficient RBCs in those mice.

[0193] Antibodies against RBC antigens induce myeloid FcgR loss and inhibit BBC phagocytosis. Our findings show that CD47 antibody therapy protects red blood cells from macrophage-mediated destruction, with implicit potential benefit for patients with autoimmune disorders affecting blood cells. Interestingly, several antibodies against red blood cell- expressed antigens were previously shown to be protective against antibody-mediated destruction of platelets in BALB / c mice (immune thrombocytopenia, ITP), and Rh-antibody is a recognized treatment for ITP. As myeloid FcgR loss and phagocytic impairment can explain these observed benefits, we asked whether observed FcgR loss might represent the general result of treatment with an antibody against a red blood cell expressed antigen. To test this, BALB / c mice were treated with one of several antibodies against red blood cell expressed antigens, and indeed, similar and consistent FcgRI I B / l 11 loss was observed for splenic myeloid populations in mice treated with antibodies against CD44 and Ter1 19. Curiously, of the antibodies tested, only an alternative CD47 antibody did not elicit myeloid FcgRI I B / l 11 loss (Fig 5A). As this was the only antibody tested of the Rat lgG2a subtype, the different response may be a result of differential antibody-FcgR engagement. We also performed this experiment in C57BL / 6 mice and observed less consistent myeloid FcgR loss in response to these antibodies (FIG. 13), perhaps due to genetic differences in isotype-specific antibody recognition by FcgRs in these mice.

[0194] We then asked whether inhibited RPM phagocytosis of RBCs was also the more general result of treatment with an antibody against an RBC-expressed antigen. To test this, we treated mice with one of several antibodies against red blood cell expressed antigens, then infused the mice with CFSE-labeled Ter119-antibody-opsonized red blood cells, and measured phagocytosis as before. Each of the antibodies tested resulted in a 3-5-fold reduction in phagocytosis by red pulp macrophages (Fig 5B).

[0195] CD47 antibody therapy promotes the phagocytosis of cancer cells both by blocking the anti-phagocytic CD47-SIRPa interaction and by stimulating pro-phagocytic macrophage- expressed Fc-gamma receptors (FcgRs). As red blood cells require surface CD47 to survive in the circulation and are sensitive to antibody-mediated phagocytosis, a hurdle for CD47 antibody therapies has been overcoming the toxicity associated with nonspecific destruction of red blood cells. It was found that the administration of a low priming dose of CD47 antibody (1 mg / kg) protects against anemia and enables much greater subsequent dosing (>=45mg / kg).

[0196] To elucidate the mechanisms behind this tolerance, we collected circulating red blood cells and bone marrow erythroid progenitors from mice treated with a CD47 antibody andfound that CD47 antibody therapy induced log-fold erythroid CD47 loss in mice. We found that this loss was partially FcgR-mediated and resulted in global myeloid FcgR loss and in impaired phagocytosis of circulating RBCs.

[0197] Antibody-mediated modulation of surface antigens has been observed in the past for RBCs and was found to be a mechanism of lymphoma resistance to Rituximab, a therapeutic CD20 antibody. RBC survival despite CD47 loss following systemic administration of CD47 antibody implies a fundamental change in the activity of the CD47-SIRPa axis that otherwise protects circulating RBCs from premature phagocytosis. The myeloid FcgR loss and phagocytic impairment that we see alongside this antigenic loss, and which appear to be the more general result of treatment with an antibody against an RBC-expressed antigen, provide a mechanism for the survival of RBCs following CD47 antibody therapy.

[0198] Our data are particularly pertinent to the ongoing clinical programs targeting CD47 for cancer immunotherapy. As CD47 antibody therapy is intended to promote macrophage phagocytosis of cancer cells by both blocking the CD47-SIRPa axis and by engaging macrophage-expressed FcgRs, reduced myeloid FcgR expression and impaired phagocytosis may limit therapeutic efficacy. Indeed, our past findings supporting the efficacy of CD47 antibody cancer immunotherapy were based in xenograft studies in immunodeficient NSG mice, whose CD47 is not bound by most anti-human CD47 antibodies and thus did not manifest impaired myeloid function resulting from off-target binding of CD47 antibody to RBCs.

[0199] Nonetheless, there may be ways to ameliorate the consequences of these off-target effects. In so much as the observed myeloid impairment is driven by immune complexing between FcgR-expressing macrophages and antibody-bound RBCs, removal or inactivation of the Fc domain can counteract this impairment.References

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[0230] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

WHAT IS CLAIMED IS:1 . A method for in vivo use of antibodies that bind to CD47 and block the interaction between CD47 and SIRPoc for increasing phagocytosis of a target cell, the method comprising: administering to an individual an effective dose of an anti-CD47 antibody where productive high affinity Fc receptor engagement by the anti-CD47 antibody is reduced or disabled.

2. The method of claim 1 , wherein the the Fc region of the anti-CD47 antibody is modified to reduce or disable binding to Fey receptors expressed by myeloid cells.

3. The method of claim 1 or claim 2, wherein the Fc region sequence has been modified by one or more amino acid changes to reduce Fc receptor binding.

4. The method of claim 3, wherein the Fc region sequence is human lgG1 comprising L234A / L235A amino acid substitutions.

5. The method of claim 3, wherein the Fc region sequence is human lgG1 comprising an N297A / Q / D / H / G / C amino acid substitution.

6. The method of claim 1 , wherein the anti-CD47 antibody is administered in combination with an effective dose of an FcyR blocking agent.

7. The method of claim 6, wherein the anti-CD47 antibody is administered in a dosing regimen comprising administration of an initial priming dose, wherein the FcyR blocking agent is administered in combination with the priming dose.

8. The method of claim 6 or claim 7, wherein the FcyR blocking agent is an antibody.

9. The method of claim 8, wherein the antibody specifically binds to human FcyRII.

10. The method of claim 8 or claim 9, wherein the antibody specifically binds to human FcyRIII.1 1 . The method of claim 6 or claim 7, wherein the FcyR blocking agent is a small molecule.

12. The method of any of claims 1 -1 1 , wherein the anti-CD47 antibody is administered in in combination with a second antibody that binds to an antigen on the target cell surface.

13. The method of any of claims 1-12, wherein the target cell is a cancer cell, an infected cell, a fibrotic cell, a cardiovascular cell, or a hematopoietic stem cell.

14. The method of claim 13, wherein the target cell is a cancer cell.

15. The method of any of claims 1 -14, wherein phagocytosis of target cells is increased at least 50% relative to treatment with an antibody having an unmodified Fc region sequence, or in the absence of treatment with the FcyR blocking agent.

16. The method of any of claims 1 -15, wherein expression of FcyR on myeloid cells following is increased at least 50% relative to treatment with an antibody having an unmodified Fc region sequence, or in the absence of treatment with the FcyR blocking agent.

17. A method for treatment of an antibody-associated disease, the method comprising: administering to an individual an effective dose of an antibody that binds to an antigen present on red blood cells, in a dose effective to cause FcyR loss in myeloid cell populations in the individual.

18. The method of claim 17, wherein the antigen is CD47.

19. The method of claim 17 or claim 18, wherein the antibody comprises a human gamma Fc region sequence.

20. The method of claim 19, wherein the Fc region sequence is selected from lgG1 , lgG2a, lgG2b, lgG3, lgG4 Fc sequences.21 . The method of any claims 17-20, wherein administering the antibody induces loss of FcyRII B / l II on the myeloid cells for a period of at least about 2 weeks.

22. The method of claim 21 , wherein the level of cell surface FcyRI I B / ll I on splenic or circulating myeloid cells is reduced by at least about 25%, at least about 50%, at least about 75% relative to an untreated control.

23. The method of any claims 17-22, wherein the antibody-associated disease is associated with autoimmune antibodies.

24. The method of of any claims 17-23, wherein the disease is selected from Grave's ophthalmopathy, multifocal motor neuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Kawasaki disease, and immune thrombocytopenia (ITP).

25. The method of any of any claims 17-24, wherein the individual is monitored for loss of FcyRII B / l II during the course of treatment.

26. The method of any claims 17-25 wherein the dose of antibody administered for treatment is a dose of from about 0.5 mg / kg to about 10 mg / kg.

27. The method of any claims 17-26, wherein the dose of antibody administered is a dose of from about 1 mg / kg to about 5 mg / kg.

28. The method of any claims 17-27, wherein administration is repeated monthly, biweekly or weekly.

29. The method of any claims 17-28, wherein the antibody is selected from Hu5F9- G4 / Magrolimab; CC-90002; IBI188; SRF231 ; AO-176; IMC-002; IMC-002; Lemzoparlimab; Letaplimab; Ligufalimab; MIL-95; SHR-1603; and ZL-1201 .

30. A method for protecting red blood cells (RBC) from immune destruction following transplantation, the method comprising: treating the RBC with a dose of an anti-CD47 antibody comprising a human Fc sequence effective to induce loss of cell surface CD47 on the RBC; and transfusing the treated RBC to an individual in need thereof.

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