EA043164B1 - METHODS AND COMPOSITIONS FOR REDUCING IMMUNOGENICITY - Google Patents

Description

1. Field of invention

Provided herein are methods and uses involving the combination of an anti-CD20 antibody, such as rituximab, with a proteinaceous therapeutic agent, such as an antibody (eg, an antibody that specifically binds to human CD47).

2. Background of the invention

Although proteinaceous therapeutic agents are used to treat a number of diseases, they can elicit an immune response, including the production of antibodies to the drug, when administered to subjects. This may result in a decrease in the effectiveness of therapeutic and/or toxic effects. Accordingly, there is a need for methods to reduce this immune response.

CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multicoil transmembrane receptor belonging to the immunoglobulin superfamily. SIRPa (alpha signaling regulatory protein) expressed on macrophages interacts with CD47 and this interaction negatively controls innate immune cell effector function such as host cell phagocytosis. The expression and/or activity of CD47 has been implicated in a number of diseases and disorders. Accordingly, there is a need for therapies targeting CD47.

3. Brief description of the invention

Provided herein are methods and compositions for reducing immunogenicity in a subject, comprising administering an anti-CD20 antibody, such as rituximab, to the subject in combination with a protein therapeutic agent, wherein the immunogenicity is reduced relative to that in the subject when the therapeutic agent is administered alone. In certain aspects, the protein therapeutic is an antibody-based therapeutic. In certain aspects, the proteinaceous therapeutic agent is a fusion protein, eg an Fc-containing fusion protein, eg a soluble receptor fusion protein. In certain aspects, the protein therapeutic agent is a cytokine. In certain aspects, the protein therapeutic agent is an interleukin. In certain aspects, the protein therapeutic agent is not an enzyme. In certain aspects, the subject is a person.

In certain aspects of the methods and compositions provided herein, the protein therapeutic agent is an antibody, where the antibody-based therapeutic agent is an antibody that binds to CD47, or an antigen-binding fragment thereof.

In specific aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH CDR1 comprising SEQ ID NO: 50, a VH CDR2 comprising SEQ ID NO: 72, a VH CDR3 comprising SEQ ID NO: 52, a VL CDR1 comprising SEQ ID NO: 53, a VL CDR2 containing SEQ ID NO: 71, and a VL CDR3 containing SEQ ID NO: 55.

In specific aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH CDR1 comprising SEQ ID NO: 50, a VH CDR2 comprising SEQ ID NO: 51, a VH CDR3 comprising SEQ ID NO: 52, a VL CDR1 comprising SEQ ID NO: 53, a VL CDR2 containing SEQ ID NO: 54, and a VL CDR3 containing SEQ ID NO: 55.

In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH containing a sequence selected from the group consisting of SEQ ID NOs: 5-30. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VL containing a sequence selected from the group consisting of SEQ ID NOs: 31-47. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH containing a sequence selected from the group consisting of SEQ ID NOs: 5-30 and a VL containing a sequence selected from the group consisting of SEQ ID NOs: 31-47.

In certain aspects, the antibody that binds to CD47, or an antigen-binding fragment thereof, is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 isotype. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is an IgG isotype selected from IgG4P and IgG4PE.

In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is a component of a pharmaceutical composition comprising an antibody that binds to CD47, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is a chimeric, humanized, or fully human antibody.

In certain aspects, the methods provided herein further comprise administering a second therapeutic agent, for example, a small molecule therapeutic agent such as a chemotherapeutic therapeutic agent. In certain aspects, said chemotherapy is radiation therapy.

In certain aspects, an antibody that binds to CD47, or an antigen binding thereof

- 1 043164 fragment is administered to the subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, or 500 mg/m ² . In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg, and an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450 or 500 mg/m ² . In certain aspects, an anti-CD20 antibody, such as rituximab, is administered prior to the protein therapeutic agent. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered 1, 2, 3, 4, 5, or 6 weeks prior to the protein therapeutic agent. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered 1, 2, 3, 4, 5, or 6 days prior to the protein therapeutic agent. In certain aspects, rituximab is administered before an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered 1, 2, 3, 4, 5, or 6 weeks prior to an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered 1, 2, 3, 4, 5, or 6 days prior to an antibody that binds to CD47, or an antigen-binding fragment thereof.

In certain aspects, the methods provided herein do not include administering a proteasome inhibitor to a subject. In certain aspects, the methods provided herein do not involve administering bortezomib to a subject. In certain aspects, the methods provided herein do not involve administering methotrexate to a subject.

Provided herein are methods of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to CD47, or an antigen-binding fragment thereof, wherein the method further comprises administering to the subject an anti-CD20 antibody, such as rituximab. In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, the subject is a person. In certain aspects, the methods provided herein further include the use of radiation therapy or chemotherapy. In certain aspects, the methods provided herein further include administering another anticancer agent. In certain aspects, the cancer is a hematological cancer. In certain aspects, the cancer is a solid cancer. In some aspects, the cancer is multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), breast cancer (e.g., triple negative breast cancer), bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular carcinoma (HCC), sarcoma, or head and neck cancer. In certain aspects, the cancer is multiple myeloma. In certain aspects, the cancer is non-Hodgkin's lymphoma. In certain aspects, non-Hodgkin's lymphoma is CD20 positive. In certain aspects, non-Hodgkin's lymphoma is recurrent or refractory. In specific aspects, the subject has previously received a therapy regimen that includes an anti-CD20 antibody, such as rituximab. Provided herein are methods of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain aspects, the subject is a person. In certain aspects, the methods provided herein further include the use of radiation therapy or chemotherapy. In certain aspects, the methods provided herein further include administering another anticancer agent. In certain aspects, the cancer is a hematological cancer. In certain aspects, the cancer is a solid cancer. In some aspects, the cancer is multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), breast cancer (e.g., triple negative breast cancer), bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular carcinoma (HCC), sarcoma, or head and neck cancer. In certain aspects, the cancer is multiple myeloma. In certain aspects, the cancer is non-Hodgkin's lymphoma. In certain aspects, non-Hodgkin's lymphoma is CD20 positive. In certain aspects, non-Hodgkin's lymphoma is recurrent or refractory. In specific aspects, the subject has previously received a therapy regimen that includes an anti-CD20 antibody, such as rituximab. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg. In certain aspects, an anti-CD20 antibody, such as rituximab, is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, or 500 mg/m ² . In certain aspects, an anti-CD20 antibody, such as rituximab, is administered prior to an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain aspects, the method does not involve administering a proteasome inhibitor to a subject. In certain aspects, the method does not involve administering bortezomib to the subject. In certain aspects, the method does not involve administering methotrexate to the subject.

In certain aspects of the cancer treatment methods provided herein, the protein therapeutic is an antibody, wherein the antibody therapeutic is an antibody that binds to CD47, or an antigen-binding fragment thereof.

In specific aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH CDR1 containing SeQ ID NO: 50, a VH CDR2 containing SEQ ID NO: 72, a CDR3

VH containing SEQ ID NO: 52, CDR1 VL containing SEQ ID NO: 53, CDR2 VL containing SEQ

ID NO: 71, and a VL CDR3 containing SEQ ID NO: 55.

In specific aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH CDR1 comprising SEQ ID NO: 50, a VH CDR2 comprising SEQ ID NO: 51, a VH CDR3 comprising SEQ ID NO: 52, a VL CDR1 comprising SEQ ID NO: 53, a VL CDR2 containing SEQ ID NO: 54, and a VL CDR3 containing SEQ ID NO: 55.

In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH containing a sequence selected from the group consisting of SEQ ID NOs: 5-30. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VL containing a sequence selected from the group consisting of SEQ ID NOs: 31-47. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a VH containing a sequence selected from the group consisting of SEQ ID NOs: 5-30 and a VL containing a sequence selected from the group consisting of SEQ ID NOs: 31-47.

In certain aspects, the antibody that binds to CD47, or an antigen-binding fragment thereof, is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 isotype. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is an IgG isotype selected from IgG4P and IgG4PE. In certain aspects, an antibody that binds to CD47, or an antigen-binding fragment thereof, is chimeric, humanized, or fully human.

4. Detailed description

4.1 Methods

4.1.1 Ways to reduce immunogenicity

Provided herein are methods of reducing immunogenicity in a subject, comprising administering an anti-CD20 antibody, such as rituximab, to the subject in combination with a protein therapeutic agent, wherein the immunogenicity is reduced relative to that in the subject when the protein therapeutic agent is administered alone. In certain embodiments, the protein therapeutic is an antibody-based therapeutic. In certain embodiments, the proteinaceous therapeutic agent is a cytokine. In some embodiments, the cytokine is bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN -β), interleukin 2 (IL-2), interleukin 11 (IL-11) or interferon gamma (IFN-γ). In certain embodiments, the implementation of the protein therapeutic agent is an interleukin. In certain embodiments, the protein therapeutic agent is not an enzyme. In certain embodiments of the methods provided herein, the protein therapeutic is an antibody-based therapeutic, wherein the antibody-based therapeutic is an antibody that binds to CD47, or an antigen-binding fragment thereof.

In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is a component of a pharmaceutical composition comprising an antibody that binds to CD47, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the methods provided herein include the use of chemotherapy. In specific embodiments, said chemotherapy is radiation therapy. In some embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to and/or simultaneously with the protein therapeutic agent. In some embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to the protein therapeutic agent. In some embodiments, an anti-CD20 antibody, such as rituximab, is administered concurrently with a proteinaceous therapeutic agent.

In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to and/or simultaneously with an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered concurrently with an antibody that binds to CD47, or an antigen-binding fragment thereof.

In certain embodiments, the methods provided herein do not include administering a proteasome inhibitor to a subject. In certain embodiments, the methods provided herein do not include administering bortezomib to a subject. In certain

- 3 043164 embodiments, the methods provided herein do not include administering methotrexate to the subject.

Immunogenicity can be measured by any method known to the person skilled in the art. In certain embodiments, immunogenicity is measured by determining the amount and/or concentration of anti-drug antibodies present in serum. In certain embodiments, immunogenicity is measured by determining the titer of anti-drug antibodies present in serum. In certain embodiments, immunogenicity is measured by determining the amount of neutralized protein therapeutic agent per volume of serum. In some embodiments, the presence of immunogenicity is detected by the occurrence of anaphylaxis, cytokine release syndrome, infusion reactions, delayed hypersensitivity, and/or cross-reactivity to endogenous proteins. In certain embodiments, immunogenicity is measured using a screening assay. In specific embodiments, the screening assay is a direct binding enzyme immunoassay (ELISA), a bridged ELISA, a radioimmunoprecipitation assay (RIPA), a surface plasmon resonance (SPR) assay, a Bethesda assay, or a bridged electrochemiluminescence assay. In certain embodiments, immunogenicity is measured using a neutralization assay. In specific embodiments, the neutralization assay is a cell-based biological assay or a non-cellular ligand competitive binding assay. In certain embodiments, anti-drug antibodies bind to a protein therapeutic agent. In certain embodiments, the anti-drug antibodies neutralize the protein therapeutic agent. In certain embodiments, the anti-drug antibodies bind to and neutralize the protein therapeutic agent.

In certain aspects, immunogenicity is measured one week after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured two weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured three weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured four weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured five weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured six weeks after the first dose of the proteinaceous therapeutic agent.

In certain aspects, immunogenicity is measured weekly after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured one week after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured two weeks after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured three weeks after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured four weeks after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured five weeks after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured six weeks after the first dose of the proteinaceous therapeutic agent and then weekly thereafter. In certain aspects, immunogenicity is measured every second week after the first dose of the proteinaceous therapeutic agent.

In certain aspects, the number of B cells is measured one week after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured two weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured three weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured four weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured five weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured six weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured one week after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured two weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured three weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured four weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured five weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured six weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured weekly after the first dose of the proteinaceous therapeutic agent. In certain aspects, the number of B cells is measured one week after the first dose of the proteinaceous therapeutic agent, and then weekly. In certain aspects, B cell counts are measured two weeks after the first dose of the protein therapeutic agent, and then weekly thereafter. In certain aspects, the number of B cells is measured three weeks after the first dose of the protein therapeutic agent, and then weekly. In certain aspects, the number of B cells is measured four weeks after the first dose of the protein therapeutic agent, and then weekly. In certain aspects, the number of B cells is measured five weeks after the first dose of the protein therapeutic agent, and then weekly. In certain aspects, the number of B cells is measured six weeks after the first dose of the proteinaceous therapeutic agent, and then weekly. In certain aspects, the number of B cells is measured every other week after the first dose of the proteinaceous therapeutic agent.

In certain aspects, immunogenicity is measured one week after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured two weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured three weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured four weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured five weeks after the first dose of the proteinaceous therapeutic agent. In certain aspects, immunogenicity is measured six weeks after the first dose of the proteinaceous therapeutic agent.

In certain aspects, immunogenicity is measured weekly after the first dose of an anti-CD20 antibody, such as rituximab. In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, immunogenicity is measured one week after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured two weeks after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured three weeks after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured four weeks after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured five weeks after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured six weeks after the first dose of rituximab and then weekly thereafter. In certain aspects, immunogenicity is measured every second week after the first dose of rituximab.

In some aspects, the number of B cells is measured one week after the first dose of an anti-CD20 antibody, such as rituximab. In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, the number of B cells is measured two weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured three weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured four weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured five weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured six weeks after the first dose of rituximab.

In some aspects, the number of B cells is measured one week after the first dose of an anti-CD20 antibody, such as rituximab. In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, the number of B cells is measured two weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured three weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured four weeks after the first dose of rituximab. In certain respects, the number

B cells are measured five weeks after the first dose of rituximab. In certain aspects, the number of B cells is measured six weeks after the first dose of rituximab.

In some aspects, the number of B cells is measured weekly after the first dose of an anti-CD20 antibody, such as rituximab. In certain aspects, the anti-CD20 antibody is rituximab. In certain aspects, the number of B cells is measured one week after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured two weeks after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured three weeks after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured four weeks after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured five weeks after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured six weeks after the first dose of rituximab, and then weekly. In certain aspects, the number of B cells is measured every second week after the first dose of rituximab.

4.1.2 Cancer treatments

Provided herein are methods of treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to CD47, or an antigen-binding fragment thereof, wherein the method further comprises administering to the subject an anti-CD20 antibody, such as rituximab. In certain embodiments, the anti-CD20 antibody is rituximab. Also provided herein are methods of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of

- 5 043164 an antibody that binds to CD47, or an antigen-binding fragment thereof.

In certain embodiments, the methods of treating cancer provided herein further include the use of radiation therapy or chemotherapy. In certain embodiments, the methods provided herein further comprise administering another anticancer agent. In certain embodiments, the cancer is a hematologic cancer. In certain embodiments, the cancer is a solid cancer. In some embodiments, the cancer is multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), breast cancer (e.g., triple negative breast cancer), bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular carcinoma (HCC), sarcoma or head and neck cancer. In certain embodiments, the cancer is non-Hodgkin's lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is CD20 positive. In certain embodiments, non-Hodgkin's lymphoma is recurrent or refractory. In certain embodiments, the subject has previously received treatment with an anti-CD20 antibody, such as rituximab.

In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to and/or simultaneously with an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain embodiments, the anti-CD20 antibody is rituximab. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered prior to an antibody that binds to CD47, or an antigen-binding fragment thereof. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered concurrently with an antibody that binds to CD47, or an antigen-binding fragment thereof.

In certain embodiments, the method does not involve administering a proteasome inhibitor to a subject. In certain embodiments, the method does not involve administering bortezomib to the subject. In certain embodiments, the method does not involve administering methotrexate to the subject. In certain embodiments, the method further comprises administering methotrexate to the subject.

In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is a component of a pharmaceutical composition comprising an antibody that binds to CD47, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In some embodiments, provided herein are methods of protecting against a condition or disorder, such as cancer, using an anti-CD47 antibody described herein alone or in combination with an anti-CD20 antibody, such as rituximab.

In specific embodiments, provided herein are methods for controlling, treating, preventing, or protecting against cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment described herein that specifically binds to CD47 (e.g., human CD47 ) alone or in combination with an anti-CD20 antibody such as rituximab. In certain embodiments, provided herein is a method of alleviating, inhibiting, or reducing the progression or severity of one or more symptoms associated with cancer.

As used herein, the term administer or administer refers to the act of injecting or otherwise physically delivering a substance (e.g., an anti-CD20 antibody, e.g., rituximab or an anti-CD47 antibody provided herein, or an antigen-binding fragment thereof) to a subject or patient (e.g., a human). ), such as by mucosal, topical, intradermal, parenteral, intravenous, subcutaneous, intramuscular delivery methods and/or any other physical delivery method described herein or known in the art. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered intravenously. In certain embodiments, the administration of an anti-CD47 antibody provided herein is administered intravenously. In certain embodiments, the administration of rituximab and the anti-CD47 antibody provided herein is administered intravenously. As used herein, the term effective amount refers to the amount of a composition (for example, an antibody or pharmaceutical composition provided herein, or an anti-CD20 antibody, such as rituximab, or a pharmaceutical composition provided herein) that is sufficient to achieve a particular result, for example, reducing and/or alleviating the severity and/or duration of a given condition, disorder, or disease (eg, cancer, metastasis, or angiogenesis) and/or a symptom associated therewith. Such term also includes an amount necessary to reduce, slow or facilitate the spread or progression of a given disease, reduce, slow or alleviate the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g. therapy other than anti-CD47 antibodies provided herein). In some embodiments, an effective amount, as used herein, refers to the amount of an anti-CD20 antibody,

- 6 043164 for example rituximab associated with a decrease in the immunogenicity of a protein therapeutic agent, as described in this document.

Doses of an anti-CD20 antibody, such as rituximab, for example, for administration in combination with a proteinaceous therapeutic agent, such as an anti-CD47 antibody, may include from about 0.1 mg/kg body weight to about 100 mg/kg body weight. Doses of rituximab, for example, for administration in combination with a proteinaceous therapeutic agent, such as an anti-CD47 antibody, may include from about 25 mg/m ² to about 1500 mg/m ² . In some embodiments, rituximab is administered to the subject at a dose of 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg or more. In some embodiments, rituximab is administered to the subject at a dose of 0.1, 0.3, 0.5, 1, 2, 4, 5, 8, 10, 15, 20, 25, 30, 50, 75, or 100 mg/day. kg. In some embodiments, rituximab is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg. In some embodiments, rituximab is administered to the subject at a dose of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 , 750, 1000, 1250, 1500 mg/m ² or more. In some embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² . In some embodiments, rituximab is administered to a subject at a dose of 375 mg/m ² . The frequency of administration of an anti-CD20 antibody, such as rituximab, may vary, for example, from twice a day to once a month. In some embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² once per week. In some embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every two weeks. In some embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every four weeks. In some embodiments, rituximab is administered to a subject at a dose of 375 mg/m ² once a week. In some embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every two weeks. In some embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every four weeks. Doses of the anti-CD47 antibody or antigen-binding fragment thereof, for example, for administration in combination with an anti-CD20 antibody, such as rituximab, may include from about 0.1 mg/kg body weight to about 100 mg/kg body weight. In some embodiments, the anti-CD47 antibody is administered to the subject at a dose of 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg. kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg or more. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.1, 0.3, 0.5, 1, 2, 4, 5, 8, 10, 15, 20, 25 , 30, 50, 75 or 100 mg/kg. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg.

Dosing frequency of the anti-CD47 antibody or antigen-binding fragment thereof may vary, for example, from twice a day to once a week. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg once a week. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg twice a week. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 8 mg/kg twice a week. In certain embodiments, an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 20 mg/kg once a week.

In some embodiments, an anti-CD20 antibody, such as rituximab, is administered to a subject at a dose of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 , 450, 475, 500, 750, 1000, 1250, 1500 mg/m ² , and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.1, 0.3, 0.5, 1 , 2, 4, 5, 8, 10, 15, 20, 25, 30, 50, 75 or 100 mg/kg. In certain embodiments, rituximab is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of , component 0.3, 1, 2, 4, 8, 15, or 20 mg/kg. In certain embodiments, rituximab is administered to a subject at a dose of 375 mg/m ² and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg. In certain embodiments, rituximab is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² once per week and an antibody that binds to CD47, or an antigen-binding fragment thereof. administered to the subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg once a week. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every two weeks, and an antibody that binds to CD47 , or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1, 2, 4, 8, 15, or 20 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof is administered to a subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered subject at a dose of 20 mg/kg once a week. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every four weeks, and an antibody that binds to CD47 , or an antigen-binding fragment thereof, is administered to a subject at a dose of 0.3, 1.2, 4, 8, 15, or 20 mg/kg twice a week. In certain embodiments, rituximab is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to a subject at a dose of 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered subject at a dose of 20 mg/kg once a week. In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 375 mg/m ² once a week, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 0.3, 1, 2, 4, 8, 15 or 20 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² once a week, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg, twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² once a week, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg, twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² once a week, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 20 mg/kg once a week.

In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 375 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 0.3.1 , 2, 4, 8, 15, or 20 mg/kg, twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every two weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 20 mg/kg once a week.

In certain embodiments, an anti-CD20 antibody, such as rituximab, is administered to the subject at a dose of 375 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 0.3.1 , 2, 4, 8, 15, or 20 mg/kg, twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 8 mg/kg twice a week. In certain embodiments, rituximab is administered to the subject at a dose of 375 mg/m ² every four weeks, and an antibody that binds to CD47, or an antigen-binding fragment thereof, is administered to the subject at a dose of 20 mg/kg once a week.

Used in this document, the term in combination in the context of the introduction of other therapies refers to the use of more than one type of therapy. The use of the term in combination does not limit the order in which therapies are administered. Therapies can be administered, for example, serially, sequentially, simultaneously or in parallel.

In certain embodiments of the methods provided herein, an anti-CD20 antibody, such as rituximab, is administered prior to a protein therapeutic agent, such as an anti-CD20 antibody.

- 8 043164 body to CD47. In certain embodiments of the methods provided herein, an anti-CD20 antibody, such as rituximab, is administered concurrently with a protein therapeutic agent, such as an anti-CD47 antibody. In certain embodiments of the methods provided herein, an anti-CD20 antibody, such as rituximab, is administered after a proteinaceous therapeutic agent, such as an anti-CD47 antibody. In certain embodiments of the methods provided herein, an anti-CD20 antibody, such as rituximab, is administered prior to and simultaneously with a protein therapeutic agent, such as an anti-CD47 antibody. As used herein, the terms subject and patient are used interchangeably. As used herein, a subject is a non-primate mammal (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), e.g. Human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a condition or disorder provided herein (eg, cancer, metastasis, or angiogenesis). In another embodiment, the subject is a mammal, such as a human, at risk of developing a condition or disorder provided herein (eg, cancer, metastasis, or angiogenesis). In another embodiment, the subject is a human. As used herein, the term hematological cancer refers to cancer of the blood and includes, among others, leukemia, lymphoma, and myeloma. Leukemia refers to blood cancer in which too many white blood cells are produced, which are ineffective in fighting infection, thus crowding out other cells that make up the blood, such as platelets and red blood cells. It is clear that cases of leukemia are classified as either acute or chronic. Some forms of leukemia include, without limitation, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome. Lymphoma may refer to, among others, Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). Myeloma may refer to multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain myeloma or Bene-Jones myeloma. In certain embodiments, the hematologic cancer is multiple myeloma. In certain embodiments, the hematologic cancer is non-Hodgkin's lymphoma. Non-limiting examples of a condition that can be treated or controlled with an anti-CD47 antibody described herein, alone or in combination with an anti-CD20 antibody, such as rituximab, include hematologic cancer and/or solid tumors, as well as diseases or disorders associated with aberrant CD47 expression, activity, and/or signaling, including but not limited to, for example, hematologic cancer and/or solid tumors. Hematologic cancers include, for example, leukemia, lymphoma, and myeloma. Some forms of leukemia include, without limitation, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome. Certain forms of lymphoma include, without limitation, Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small and large cell). Certain forms of myeloma include, without limitation, multiple myeloma (MM), giant cell myeloma, heavy chain and light chain myeloma, or Bence-Jones myeloma. Solid tumors include, for example, breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanomas, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors. Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain often localized to the inflamed area, loss of appetite, weight loss, swelling, headache, fatigue, rash, anemia, muscle weakness , muscle fatigue and abdominal symptoms such as abdominal pain, diarrhea or constipation.

In specific aspects of the present invention, anti-CD47 antibodies useful alone or in combination with an anti-CD20 antibody, e.g., rituximab, are provided in the treatment, delay in progression, inhibition, prevention of recurrence, or alleviation of a symptom of cancer (e.g., MM, NHL, AML, breast cancer ( e.g. triple negative breast cancer), bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular carcinoma (HCC), sarcoma, and head and neck cancer). For example, anti-CD47 antibodies described herein are useful alone or in combination with an anti-CD20 antibody, eg, rituximab, in the treatment of hematological malignancies and/or tumors, eg, hematological malignancies and/or tumors. For example, the anti-CD47 antibodies described herein are useful in the treatment of CD47+ tumors. As a non-limiting example, the anti-CD47 antibodies described herein are useful in the treatment of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma ( MM), breast cancer (eg, triple negative jelly breast cancer

- 9 043164 s), ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, etc. Solid tumors include, for example, breast tumors, ovarian tumors, lung tumors (eg, NSCLC), pancreatic tumors, prostate tumors, melanomas, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, tumors of the esophagus, liver tumors (eg, hepatocellular carcinoma), sarcoma, and kidney tumors.

In a specific embodiment, provided herein is a method of treating cancer (e.g., a hematological disorder/cancer or solid cancer) in a subject, comprising administering (e.g., simultaneous or sequential administration) to a subject in need thereof (i) an anti-CD47 antibody described in herein, or an antigen-binding fragment thereof specifically binding to CD47, such as human CD47, alone or in combination with an anti-CD20 antibody, such as rituximab, and (ii) another anti-cancer agent. In some embodiments, the anticancer agent is a chemotherapeutic agent (eg, a microtubule disassembly blocker, an antimetabolite, a topisomerase inhibitor, and a DNA crosslinker or damaging agent). In some embodiments, the anticancer agent is a tyrosine kinase inhibitor (eg, GLEEVEC® (imatinib mesylate) or SUTENT® (SU11248 or sunitinib)). Other non-limiting examples of tyrosine kinase inhibitors include 706 and AMNI07 (nilotinib). RAD00I, PKC412, gefitinib (IRESSA™), erlotinib (TARCEVA®), sorafenib (NEXAVAR®), pazopanib (VOTRIENT™), axitinib, bosutinib, cediranib (RECENTIN®), SPRYCEL® (dasatinib), lapatinib (TYKERB®), lestaurtinib, neratinib, nilotinib (TASIGNA®), semaxanib, toceranib (PALLADIA™), vandetanib (ZACTIMA™), and vatalanib. In a specific aspect, provided herein is a method for treating cancer (e.g., a hematological disorder/cancer or solid cancer) in a subject, comprising administering (e.g., simultaneous or sequential administration) to a subject in need thereof (i) an anti-CD47 antibody described herein. document, or an antigen-binding fragment thereof that specifically binds to CD47, such as human CD47, alone or in combination with an anti-CD20 antibody, such as rituximab, and (ii) radiation therapy.

In a specific aspect, provided herein is a method of stimulating (e.g., inducing or increasing) phagocytosis, e.g., macrophage-mediated phagocytic killing of tumor cells, comprising contacting an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47 with tumor cells. cells, alone or in combination with an anti-CD20 antibody such as rituximab. Also provided herein is a method of stimulating (e.g., inducing or increasing) phagocytosis, e.g., macrophage-mediated phagocytic killing of tumor cells in a subject in need thereof (e.g., a subject with tumor cells, such as tumor cells expressing CD47), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47, alone or in combination with an anti-CD20 antibody such as rituximab. In a specific aspect, provided herein is a method for reducing tumor volume, comprising contacting an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47 with a tumor, alone or in combination with an anti-CD20 antibody, such as rituximab. Also provided herein is a method for reducing tumor volume in a subject in need thereof (e.g., a subject with a tumor, such as a tumor expressing CD47), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47 , alone or in combination with an anti-CD20 antibody such as rituximab.

In a specific aspect, provided herein is a method for inhibiting the growth or proliferation of cancer cells, comprising contacting an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47 with cancer cells, alone or in combination with an anti-CD20 antibody, such as rituximab. Also provided herein is a method for inhibiting the growth or proliferation of cancer cells in a subject in need thereof (e.g., a subject with cancer cells, such as CD47-expressing cancer cells), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein that specifically binds to human CD47, alone or in combination with an anti-CD20 antibody such as rituximab.

4.2 Compositions

4.2.1 Protein therapeutics

The protein therapeutics used in the methods described herein, for example in combination with an anti-CD20 antibody, such as rituximab, can be any protein therapeutics known to one of skill in the art. In certain embodiments, the proteinaceous therapeutic agent is a cytokine. In some embodiments, the cytokine is bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte macro- 10 043164 fatal colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β), interleukin 2 (IL-2), interleukin 11 (IL-11), or interferon gamma (IFN-γ). In certain embodiments, the implementation of the protein therapeutic agent is an interleukin. In certain embodiments, the protein therapeutic agent is not an enzyme.

In certain embodiments, the proteinaceous therapeutic agent is a fusion protein, such as an Fc-containing fusion protein, such as a soluble receptor fusion protein.

4.2.2 Antibodies

Protein therapeutics used in the methods described herein, for example in combination with an anti-CD20 antibody such as rituximab, can be antibody based therapeutics. In some embodiments, the anti-CD20 antibody is rituximab. Rituximab (RITUXAN®, Biogen/Genentech) is a chimeric mouse/human IgG ₁ -kappa monoclonal antibody directed against the CD20 antigen. Rituximab has an approximate molecular weight of 145 kDa and a binding affinity for the CD20 antigen of approximately 8.0 nM. Rituximab is produced by a suspension culture of mammalian cells (Chinese hamster ovary) in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detected in the final product. RITUXAN® is a sterile, clear, colorless, preservative-free liquid concentrate for intravenous administration. RITUXAN® is supplied in a concentration of 10 mg/ml in disposable vials of 100 mg/10 ml or 500 mg/50 ml. The product is composed of polysorbate 80 (0.7 mg/ml), sodium chloride (9 mg/ml), sodium citrate dihydrate (7.35 mg/ml) and water for injection. The pH value is 6.5. In some embodiments, the anti-CD20 antibody is ocrelizumab. In some embodiments, the anti-CD20 antibody is ofatumumab. In some embodiments, the anti-CD20 antibody is obinutuzumab. In some embodiments, the anti-CD20 antibody is tositumomab. In some embodiments, the anti-CD20 antibody is ibritumomab. In some embodiments, the anti-CD20 antibody is ocaratuzumab. In some embodiments, the anti-CD20 antibody is veltuzumab. As used herein, and unless otherwise indicated, the terms approximately or approximately mean within plus or minus 10% of a given value or range. Where an integer is required, the terms mean within plus or minus 10% of a given value or range, rounded up or down to the nearest whole number. As used herein, the terms antibody and immunoglobulin and Ig are terms of the prior art and can be used interchangeably herein and refer to a molecule with an antigen-binding site that specifically binds an antigen. The antibodies provided herein may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, mouse antibodies (e.g., mouse or rat antibodies), chimeric antibodies, synthetic antibodies and tetrameric antibodies containing two heavy chain molecules and two light chain molecules. In specific embodiments, antibodies can include, without limitation, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody heavy chain-antibody light chain pair, intrabodies, an antibody heteroconjugate, single domain antibodies, and monovalent antibodies. In a particular embodiment, antibodies may include antigen-binding fragments or epitope-binding fragments such as, but not limited to, single chain antibodies or single chain Fvs (scFvs) (e.g., including monospecific, bispecific, etc.), camel antibodies, affibodies, Fab fragments, fragments F(ab'), fragments F(ab') ₂ and disulfide-linked Fv (sdFv). In certain embodiments, the antibodies described herein refer to populations of polyclonal antibodies.

Antibodies can be of any type (eg IgG, IgE, IgM, IgD, IgA or IgY), any class (eg IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 or IgA ₂ ) or any subclass (eg IgG _2a or IgG _2b ) immunoglobulin molecules. In certain embodiments, the antibodies described herein are IgG antibodies or a class (eg, human IgG1, IgG2, IgG ₃ or IgG ₄ ) or subclass thereof. In certain embodiments, the antibodies described herein are IgG1 antibodies (eg, human IgG1) or a subclass thereof. In certain embodiments, the IgG1 antibodies described herein contain one or more amino acid substitutions and/or deletions in the constant region. In certain embodiments, the antibodies described herein are IgG ₄ antibodies (eg, human IgG ₄ ) or a subclass thereof. In certain embodiments, the IgG ₄ antibodies described herein contain one or more amino acid substitutions and/or deletions in the constant region.

As used herein, the term antigen is a fragment or molecule that contains an epitope to which an antibody can specifically bind. As such, antigen

- 11 043164 also binds specifically to the antibody.

As used herein, the term epitope is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. The epitope may be a linear epitope or a conformational, non-linear or discontinuous epitope. In the case of a polypeptide antigen, for example, the epitope may be contiguous amino acids of the polypeptide (linear epitope), or the epitope may include amino acids from two or more non-contiguous regions of the polypeptide (conformational, non-linear or discontinuous epitope). One skilled in the art will appreciate that, in general, a linear epitope may or may not depend on a secondary, tertiary, or quaternary structure. As used herein, the term monoclonal antibody is a well-known term that refers to an antibody derived from a population of homogeneous or substantially homogeneous antibodies. The term monoclonal is not limited to any particular method for producing an antibody. Typically, a population of monoclonal antibodies can be obtained through cells, a population of cells, or a cell line. In specific embodiments, a monoclonal antibody as used herein is an antibody produced by a single cell or cell line, wherein the antibody immunospecifically binds to a CD47 epitope as determined, for example, by ELISA or other antigen-binding or competitive binding assay known from prior art or in the examples given in this document. In specific embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent antibody or a polyvalent (eg, divalent) antibody.

As used herein, the term non-natural amino acid refers to an amino acid that is not a proteinogenic amino acid, or a post-translationally modified variant thereof. In particular, the term refers to an amino acid that is not one of the 20 conventional amino acids, or pyrrolysine, or selenocysteine, or their post-translationally modified variants.

As used herein, the term polyclonal antibodies refers to a population of antibodies that includes a plurality of different antibodies that immunospecifically bind to the same and/or different epitopes in an antigen or antigens.

As used herein, the terms variable region or variable domain refer to the portion of an antibody, typically the light or heavy chain portion of an antibody, typically about 110 to 120 amino-terminal amino acids in the mature heavy chain and approximately 90,100 amino-terminal amino acids in the mature light chain. The variable regions contain complementarity determining regions (CDRs) flanked by framework regions (FRs). Typically, the spatial orientation of CDRs and FRs is N-terminal to C-terminal: FR1CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and epitope specificity of the antibody. In a specific embodiment, the numbering of the amino acid positions of the antibodies described herein corresponds to the EU index according to Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, the variable region is a human variable region. In some embodiments, the variable region includes murine (eg, mouse or rat) CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (eg, human or non-human primate) variable region. In some embodiments, the variable region comprises murine (eg, mouse or rat) CDRs and framework regions (FRs) (eg, human or non-human primate). As a non-limiting example, the variable region described herein is obtained by combining two or more human sequence fragments into a composite human sequence.

In certain aspects, antibody CDRs can be determined according to (i) the system according to Kabat (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme and will herein be referred to as Chothia CDRs (see, for example, Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997 , J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215( 1): 175-82 and US Pat. No. 7,709,226) or (iii) the ImMunoGeneTics (IMGT) numbering system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M .-P. et al., 1999, Nucleic Acids Res., 27:209-212 (IMGT CDRs); or (iv) MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See also, for example, Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

With respect to the Kabat numbering system, the CDRs in an antibody heavy chain molecule are usually present.

- 12 043164 exist at amino acid positions 31 to 35, which may optionally include one or two additional amino acids following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs in an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). As is well known to those skilled in the art, when using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or more amino acids due to shortening or lengthening of the FR and/or CDR, and as such, the Kabat amino acid number does not necessarily match. with its linear amino acid number. The antibodies provided herein may be of any type (eg, IgG, IgE, IgM, IgD, IgA, or IgY), any class (eg, IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgA1 or IgA ₂ ), or any subclass (eg, IgG _2a or IgG _2b or a mixture thereof) of the immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies (eg, human IgG) or a class (eg, human IgG1, IgG2, IgG ₃ or IgG ₄ ) or subclass thereof. In specific aspects, the present invention provides an antibody comprising a light chain and a heavy chain of an antibody, such as a separate light chain and heavy chain. With regard to the light chain, in a specific embodiment, the light chain of the antibody described herein is a kappa (k) light chain. In another specific embodiment, the light chain of the antibody described herein is a lambda (λ) light chain. In another embodiment, the light chain is a mixed sequence, for example, the light chain variable portion contains light chain kappa sequences and the light chain constant region contains light chain lambda sequences, or vice versa. In certain embodiments, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. Non-limiting examples of human constant region sequences have been described in the art, for example, see US Pat. No. 5,693,780 and Kabat. et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242.

In some embodiments, an anti-CD47 antibody described herein, or an antigen-binding fragment thereof, contains amino acid sequences with a certain percentage identity relative to the parent antibody.

Determination of percent identity between two sequences (eg, amino acid sequences or nucleic acid sequences) can be performed using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. sci. USA. 87:22642268, modified according to Karlin and Altschul, 1993, Proc. Natl. Acad. sci. U.S.A. 90:5873 5877. Such an algorithm is included in the NBLAST and XBLAST programs in Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with a set of nucleotide NBLAST program parameters set to, for example, alignment weight = 100, word length = 12 to find nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with a set of XBLAST program parameters set to, for example, score 50, word length = 3 to find amino acid sequences homologous to the protein molecule described herein. To obtain gapped alignments for comparison, gapped BLAST described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI-BLAST can be used to perform an iterative search that reveals long range bonds between molecules (Id.). When using the BLAST, gapped BLAST and PSI-Blast programs, you can use the default parameters from the respective programs (e.g. XBLAST and NBLAST) (see e.g. National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih. gov). Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, you can use a table of PAM120 residue substitution weights, a gap extension penalty of 12, and a gap opening penalty of 4.

The percent identity of two sequences can be determined using techniques similar to those described above that allow gaps or do not allow gaps. When calculating percent identity, as a rule, only exact matches are counted.

Anti-CD47 antibodies described herein also include monoclonal antibodies that specifically bind CD47, where the antibody does not stimulate (e.g., induce or increase) or cause a significant level of agglutination, such as erythrocyte hemagglutination (erythrocyte hemagglutination) .

Pharmaceutical compositions in accordance with the present invention may include an antibody of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the antibody, or antigen-binding fragment thereof, is an IgG isotype. In some embodiments, the antibody constant region is a human IgG1 isotype characterized by the amino acid sequence:

(SEQ ID NO: 1) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT

VPSSSLGTQT YICNVNHKPS NTKVDKKVEPEYKCKVSNKA

LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC

LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

In some embodiments, the human IgG1 constant region is modified at the amino acid Asn297 (in box, Kabat numbering) to prevent glycosylation of an antibody, such as Asn297Ala (N297A). In some embodiments, the antibody constant region is modified at the amino acid Leu235 (numbered according to Kabat) to alter interactions with the Fc receptor, such as Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the antibody constant region is modified at the amino acid Leu234 (numbered according to Kabat) to alter interactions with the Fc receptor, eg Leu234Ala (L234A). In some embodiments, the antibody constant region is changed at both amino acid 234 and 235, for example, Leu234Ala and Leu235Ala (L234A/L235A) (EU index according to Kabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the antibody constant region is a human IgG2 isotype characterized by the amino acid sequence

SEQ ID NO: 2) ASTKGPSVFP LAPCSRSTSE STAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT

VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP

APPVAGPSVF LFPPKPKDTL MISRTPEVTC

VVVDVSHEDPKVSNKGLPAP IEKTISKTKG QPREPQVYTL

PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY

KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA

LHNHYTQKSL SLSPGK

In some embodiments, the human IgG2 constant region is modified at the amino acid Asn297 (in box, Kabat numbering) to prevent glycosylation of an antibody, such as Asn297Ala (N297A). In some embodiments, the antibody constant region is a human IgG3 isotype characterized by the amino acid sequence (SEQ ID NO: 3) ASTKGPSVFP LAPCSRSTSG GTAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT

VPSSSLGTQT YTCNVNHKPS NTKVDKRVEL KTPLGDTTHT

CPRCPEPKSC DTPPPCPRCP EPKSCDTPPP CPRCPEPKSC DTPPPCPRCP

APELLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED

CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS

DGSFFLYSKL TVDKSRWQQG

In some embodiments, the human IgG3 constant region is modified at the amino acid Asn297 (in box, Kabat numbering) to prevent glycosylation of an antibody, such as Asn297Ala (N297A). In some embodiments, the human IgG3 constant region is modified at amino acid 435 to extend the half-life, for example, Arg435H is (R435H) (EU index according to Kabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the antibody constant region is a human IgG4 isotype characterized by the amino acid sequence (SEQ ID NO: 4) ASTKGPSVFP LAPCSRSTSE STAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT

- 14 043164

VPSSSLGTKT YTCNVDHKPS NTKVDKRVES CKVSNKGLPS

SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK

GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL

TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK

In some embodiments, a human IgG4 constant region is modified within the hinge region to prevent or reduce strand exchange, such as Ser228Pro (S228P). In other embodiments, the human IgG4 constant region is modified at amino acid 235 to alter interactions with the Fc receptor, eg Leu235Glu (L235E). In some embodiments, the human IgG4 constant region is modified within the hinge and amino acid 235, such as Ser228Pro and Leu235Glu (S228P/L235E). In some embodiments, the human IgG4 constant region is modified at the amino acid Asn297 (Kabat numbering) to prevent glycosylation of an antibody, such as Asn297Ala (N297A). In some embodiments, the human IgG4 constant region is modified at the amino acid positions Ser228, Leu235, and Asn297 (eg, S228P/L235E/N297A). (EU index according to Kabat et al. 1991 Sequences of Proteins of Immunological Interest). In other embodiments, the antibody is of the human IgG4 subclass and lacks glycosylation. In these embodiments, the implementation of glycosylation can be eliminated using a mutation at position 297 (numbering according to Kabat), for example N297A. In other embodiments, glycosylation can be abolished by generating an antibody in a host cell that lacks post-translational glycosylation capacity, such as in a bacterial or yeast system or a modified mammalian cell expression system.

In some embodiments, the human IgG constant region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (numbering according to Kabat, Dall'Acqua et al. 2006, J. Biol Chem Vol. 281 (33) 23514- 23524) or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al., 2010 Nature Biotech, Vol. 28 (2) 157-159). (EU index according to Kabat et al 1991 Sequences of Proteins of Immunol ogical Interest).

In some embodiments, the human IgG constant region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), e.g., modifying the amino acids described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72 ; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468; reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11. In some embodiments, a human IgG constant region is modified to induce heterodimerization. For example, the presence of an amino acid modification in the CH3 domain of Thr366 which, when replaced by a bulkier amino acid such as Try (T366W), is able to preferentially bind to a second CH3 domain having amino acid modifications with smaller amino acids at positions Thr366, Leu368, and Tyr407, such as Ser , Ala and Val, respectively (T366S/L368A/Y407V). Heterodimerization via CH3 modifications can be further stabilized by introducing a disulfide bond, for example by replacing Ser354 with Cys (S354C) and Y349 with Cys (Y349C) on opposite CH3 domains (reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15 ).

In other embodiments, implementation of the present invention, the antibody is characterized by the absence of glycosylation, but not modified at the amino acid Asn297 (numbering according to Kabat). In these embodiments, glycosylation can be abolished by generating the antibody in a host cell that lacks post-translational glycosylation capacity, such as in a bacterial or yeast system or a modified mammalian cell expression system.

4.2.2.1 Anti-CD47 antibodies for use in the methods provided in this document

In a specific aspect, antibodies that specifically bind to CD47 (eg, human CD47) are included for use in the methods provided herein. Such CD47 antibodies include all antibodies disclosed in US Patent Application Publication No. 2014/0140989, which is incorporated herein by reference in its entirety. Such CD47 antibodies also include all antibodies disclosed in International Patent Application Publication No. WO 2016/109415, which is incorporated herein by reference in its entirety.

As used herein, the terms CD47 or integrin-associated protein or IAP or ovarian cancer antigen or OA3 or Rh-related antigen or MER6 may be used interchangeably and refers to a multi-stranded transmembrane receptor belonging to the immunoglobulin superfamily. The amino acid sequence of exemplary human CD47 is shown below (GenBank accession number Q08722.1 (GI: 1171879), incorporated herein by reference). Signal sequence (amino acids 1-18) under

- 15 043164 crossed out.

MWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF

KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT

121 REGETIIELK YRWSWFSPN ENILIVIFPI FAILLFWGQF GIKTLKYRSG GMDEKTIALL

181 VAGLVITVIV IVGAILFVPG EYSLKNATGL GLIVTSTGIL ILLHYYVFST AIGLTSFVIA

241 ILVIQVIAYI LAWGLSLCI AACIPMHGPL LISGLSILAL AQLLGLVYMK FVASNQKTIQ

301 PPRKAVEEPL NAFKESKGMM NDE (SEQ ID NO: 48)

For clarity, the amino acid sequence of exemplary human CD47, excluding the signal sequence, is shown below.

QLLFNKTKSV EFTFCNDTW IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF

DGALNKSTVP 61 TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRWSWFS 121 PNENILIVIF PIFAILLFWG QFGIKTLKYR SGGMDEKTIA LLVAGLVITV IVIVGAILFV 181 PGEYSLKNAT GLGLIVTSTG ILILLHYYVF STAIGLTSFV IAILVIQVIA YILAWGLSL 241 CIAACIPMHG PLLISGLSIL ALAQLLGLVY MKFVASNQKT IQPPRKAVEE PLNAFKESKG 301 MMNDE (SEQ ID NO: 49)

The present invention also relates to pharmaceutical compositions that include one or more monoclonal antibodies that bind to CD47, or an immunologically active fragment thereof, where the antibody does not cause a significant level of erythrocyte hemagglutination after administration.

Hemagglutination is an example of a homotypic interaction, where two CD47-expressing cells exhibit aggregation or clumping when treated with a bivalent CD47-binding entity. The ability of the antibodies of the present invention to bind CD47 on the cell surface and not cause cell clumping phenomena is not limited to erythrocytes. The antibodies of the present invention have been found to uniquely bind CD47 in a manner that does not promote clumping of CD47 positive cell lines, such as Daudi cells.

In some instances, an antibody for use in the methods provided herein comprises a heavy (VH) chain variable region selected from the group consisting of SEQ ID NO: 530. An antibody optionally comprises a light (VL) chain variable region selected from the group, consisting of SEQ ID NO: 31-47. In some cases, the antibody contains a VH chain region selected from the group consisting of SEQ ID NOs: 5-30 and a VL chain region selected from the group consisting of SEQ ID NOs: 31-47. The antibodies of the present invention also include antibodies containing a heavy chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence set forth in at least one of SEQ ID NO: 530 and containing a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more identical to the sequence set forth in at least one of SEQ ID NOs: 31-47. In other aspects, the antibody comprises a VH region provided in any of SEQ ID NOs: 5, 7, 8, 11, 15-17, 20-22, and 27-30 in combination with a VL region provided in any of SEQ ID NOs: 31-39, 42, 43, 44, and 47. In another embodiment, the antibody comprises the VH region provided in any of SEQ ID NOs: 5, 7, 8, 11, 12, 15-17, 20-22, and 27- 30 in combination with a VL region provided in any of SEQ ID NOs: 31, 32, 35, 40, 41, 42, 43, 44, and 47. In another aspect, the antibody comprises a combination of a VH region chain and a VL region chain, selected from the combinations listed in the table. 1.

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Table 1. Exemplary combinations of VH/VL anti-CD47 antibodies

Antibody Heavy chain variable region (VH) Light chain variable region (VL) 2A1 SEQ ID NO: 5 SEQ ID NO: 31 2Α1-ΧΪ SEQ ID NO: 5 SEQ ID NO: 32 AB2.03 SEQ ID NO: 7 SEQ ID NO: 33 AB2.04 SEQ ID NO: 7 SEQ ID NO: 34 AB2.05 SEQ ID NO: 7 SEQ ID NO: 35 AB2.06 SEQ ID NO: 7 SEQ ID NO: 36 AB2.07 SEQ ID NO: 7 SEQ ID NO: 37 AB2.08 SEQ ID NO: 7 SEQ ID NO: 38 AB2.09 SEQ ID NO: 7 SEQ ID NO: 39 AB2.13 SEQ ID NO: 7 SEQ ID NO: 43 AB3.09 SEQ ID NO: 8 SEQ ID NO: 39 AB6.12 SEQ ID NO: 11 SEQ ID NO: 42 AB6.13 SEQ ID NO: 11 SEQ ID NO: 43 AB6.14 SEQ ID NO: 11 SEQ ID NO: 44 AB6.17 SEQ ID NO: 11 SEQ ID NO: 47 AB10.13 SEQ ID NO: 15 SEQ ID NO: 43 AB10.14 SEQ ID NO: 15 SEQ ID NO: 44 ΑΒ11Ό5 SEQ ID NO: 16 SEQ ID NO: 35 AB12.05 SEQ ID NO: 17 SEQ ID NO: 35 ΑΒ15Ό5 SEQ ID NO: 20 SEQ ID NO: 35 ΑΒ16Ό5 SEQ ID NO: 21 SEQ ID NO: 35 ΑΒ17Ό5 SEQ ID NO: 22 SEQ ID NO: 35 ΑΒ22Ό5 SEQ ID NO: 27 SEQ ID NO: 35 ΑΒ23Ό5 SEQ ID NO: 28 SEQ ID NO: 35 ΑΒ24Ό5 SEQ ID NO: 29 SEQ ID NO: 35 ΑΒ25Ό5 SEQ ID NO: 30 SEQ ID NO: 35

In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a VH sequence complementarity determining region (CDR1) as set forth in SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO : 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66 , the VH CDR2 sequence shown in SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, the VH CDR3 sequence shown in SEQ ID NO: 52 or SEQ ID NO: 77, CDR1 VL sequence shown in SEQ ID NO: 53, SEQ ID NO: 67 or SEQ ID NO: 68, CDR2 VL sequence shown in SEQ ID NO: 54, SEQ ID NO : 69, SEQ ID NO: 70 or SEQ ID NO: 71 and the CDR3 VL sequence shown in SEQ ID NO: 55. VH sequence shown in SEQ ID NO: 51, CDR3 VH sequence shown in SEQ ID NO: 52, CDR1 VL sequence shown in SEQ ID NO: 53, CDR2 VL sequence shown in SEQ ID NO: 54, and CDR3 VL sequence submitted

- 17 043164 in SEQ ID NO: 55. In another example, the antibody or its immunologically active fragment contains the CDR1 VH sequence shown in SEQ ID NO: 50, the CDR2 VH sequence shown in SEQ ID NO: 72, the CDR3 VH sequence shown in SEQ ID NO: 52, a VL CDR1 set forth in SEQ ID NO: 53, a VL CDR2 sequence set forth in SEQ ID NO: 71, and a VL CDR3 sequence set forth in SEQ ID NO: 55. In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or yield of antibody expression compared to those of the parent anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody contains one or more amino acid modifications, eg 1-15 amino acid modifications, relative to the parent anti-CD47 antibody. In a specific aspect, one or more amino acid modifications, such as 115 amino acid modifications, are within the heavy chain or VH (eg, SEQ ID NO: 6). In a specific aspect, one or more amino acid modifications, eg 1-15 amino acid modifications, are in the VH framework region (eg, SEQ ID NO: 6). In a certain aspect, this document provides an anti-CD47 antibody, which is a variant of the original anti-CD47 antibody containing the CDR (eg, CDR according to Kabat) of the original anti-CD47 antibody.

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or yield of expression of the antibody compared to that of the parent anti-CD47 antibody when expressed in a CF system, and wherein the anti-CD47 antibody contains one or more amino acid modifications, such as 1-15 amino acid modifications, relative to the parent anti-CD47 antibody. In a specific aspect, one or more amino acid modifications, such as 5 or 14 amino acid modifications, are within the heavy chain or VH (eg, SEQ ID NO: 6). In a specific aspect, one or more amino acid modifications, eg 5, 10, 13 or 14 amino acid modifications, are in the VH framework region (eg SEQ ID NO: 6). In a specific aspect, one or more amino acid modifications, eg 5, 13 or 14 amino acid modifications, are in the VH framework region (eg SEQ ID NO: 6). In a certain aspect, this document provides an anti-CD47 antibody, which is a variant of the original anti-CD47 antibody containing the CDR (eg, CDR according to Kabat) of the original anti-CD47 antibody. In certain aspects, such an anti-CD47 antibody is an IgG1, IgG2, IgG3, or IgG4 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody of the Z allotype. In certain aspects, such an anti-CD47 antibody is an IgG4 isotype antibody, such as IgG4P or IgG4PE.

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher the titer or yield of antibody expression compared to that of the original anti-CD47 antibody when expressed in the CF system. In specific embodiments, the parent anti-CD47 antibody is AB6.12 (see, for example, US Publication No. US 2014/0140989 A1, which is incorporated herein by reference in its entirety). The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the AB6.12 antibody are provided below, where the Kabat CDRs are underlined. In a specific aspect, an anti-CD47 antibody provided herein is a variant of the parent AB6.12 antibody and contains a CDR (e.g., CDR according to Kabat) of the parent AB6.12 antibody, e.g., SEQ ID NOS: 50, 72, 52, 53, 71 and 55. In certain aspects, such an anti-CD47 antibody is an IgG1, IgG2, IgG3, or IgG4 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody of the Z allotype. In certain aspects, such an anti-CD47 antibody is an IgG4 isotype antibody, such as IgG4P or IgG4PE.

Heavy chain variable region (VH) of anti-CD47 antibody AB6.12 (CDRs according to Kabat 1-3 underlined, SEQ ID NOS: 50, 72 and 52):

OMOLVOSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVROAPGOALEWMGWIDPDQGDTEYAO

KFODRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAYGSSSYPMDYWGOGTTVTV (SEQ

ID NO: 6)

Light chain variable region (VL) of anti-CD47 antibody AB6.12 (CDRs according to Kabat 1-3 underlined, SEQ ID NOS: 53, 71 and 55):

NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFOQKPGKVPKHLIYRANRLV

- 18 043164

ISGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK (SEQ ID

NO: 42)

In a specific embodiment, an anti-CD47 antibody described herein contains one or more amino acid modifications (e.g., 1-15 amino acid modifications), e.g., in the VH framework region of the parent antibody, e.g., the parent antibody selected from the anti-CD47 antibodies described in this document.

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or expression yield of the antibody compared to that of the parent anti-CD47 antibody when expressed in a CF system, and where the anti-CD47 antibody contains a VH containing the following sequence from N-terminus to C-terminus:

XiQXzQLVQSGAEVKKXsGX^SVKVSCKASGFNIKDYYLHWVRQAPGQX^LEWMGW IDPDQGDTEYAQKX6QX7RVTX8TX9DX10SX11STAYMELX12SLRSX13DTAX14YYCNA AYGSSSYPMDYWGQGTTVTV (SEQ III NO: 89), where the underlined amino acid residues for ^bL are ordered from the N-terminus to the C-terminus, where X ₁ is M, or there is no amino acid in position X ₁ , X ₂ is an amino acid with hydrophobic side chains, such as M or V, X ₃ is T or P, X ₄ is S or A, X ₅ is an acid containing aliphatic side chains, such as A or G, X ₆ is F or L, X ₇ is D or G, X ₈ is an amino acid with hydrophobic side chains such as I or M, X ₉ is R or T, X ₁₀ is R or T, X _p is M or T, X ₁₂ is S or R, X ₁₃ is a negatively charged amino acid such as E or D, and X ₁₄ is an amino acid with hydrophobic side chains such as M or V.

In specific aspects, an anti-CD47 antibody described herein comprises a VH comprising the sequence of SEQ ID NO: 89, wherein the amino acid at position X ₁ is any amino acid such as M, X ₂ is not M, X ₃ is not T, X ₄ is not S, X5 is not A, X ₆ is not F, X ₇ is not D, X ₈ is not I, X ₉ is not R, X ₁₀ is not R, X ₁₁ is not M, X ₁₂ is not is S, X ₁₃ is not E, and/or X ₁₄ is not M. In specific aspects, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of X ₁ -X ₁₄ are not these amino acids. In specific aspects, the VH amino acid sequence is not the VH amino acid sequence of the AB6.12 antibody, for example, the VH amino acid sequence is not SEQ ID NO: 6. In specific aspects, the anti-CD47 antibody described herein comprises a VH comprising the sequence of SEQ ID NO: 89, where the amino acid at position X ₇ is not G, X ₉ is not A, and/or X ₁₁ is not S. In specific aspects, any 1, 2 or 3 of X ₇ , X ₉ and X ₁₁ are not these amino acids. In specific aspects, if the amino acid at position X ₇ is G, then X ₈ is M and/or X ₁₀ is T, X ₉ is not A and/or X ₁₁ is not S.

In specific aspects, an anti-CD47 antibody described herein comprises a VH comprising the sequence of SEQ ID NO: 89, wherein the amino acid at position X ₇ is not G, X ₈ is not M, X ₉ is not E, X ₁₀ is not T and/or X ₁₁ is not T. In specific aspects, any 1, 2, 3, or 4 of X ₇ -X ₁₁ are not these amino acids. In specific aspects, if the amino acid at position X ₇ is G, then X ₈ is M, X ₁₀ is T, X ₉ is not E, and X ₁₁ is T. In a specific aspect, the anti-CD47 antibody described in herein, contains a VH containing the sequence of SEQ ID NO: 89, where the VH does not contain the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 as set forth in U.S. Patent Application Publication No. US 2014/0140989 A1, which is incorporated herein by reference in its entirety. completeness. In a specific aspect, the anti-CD47 antibody described herein comprises a VH containing the consensus sequence of SEQ ID NO: 89, wherein the VH does not contain the framework regions of the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 as specified in U.S. Patent Application Publication No. US 2014/0140989 A1, which is incorporated herein by reference in its entirety.

In specific aspects, X ₁ is M, X ₂ is V, X ₃ is P, X ₄ is A, X ₅ is G, X ₆ is L, X ₇ is G, X ₈ is M , X ₉ is T, X ₁₀ is T, X ₁₁ is T, X ₁₂ is R, X ₁₃ is D, and/or X ₁₄ is V. In specific embodiments, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of X ₁ -X ₁₄ are these amino acids.

In specific aspects, X1 is M, X2 is M, X3 is P, X4 is S, X5 is A, X6 is F, X7 is G, X ₈ is I, X ₉ is R, X ₁₀ is R, X ₁₁ is T, X ₁₂ is R, X ₁₃ is E, and/or X ₁₄ is V. In specific embodiments, any 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, or 14 of X ₁ -X ₁₄ are these amino acids.

In a specific aspect, the anti-CD47 antibody provided herein is not an AB6.12 antibody. In a specific aspect, the anti-CD47 antibody provided herein does not contain the VH (eg, SEQ ID NO: 6) and/or VL (eg, SEQ ID NO: 42) of the AB6.12 antibody. a specific aspect of the antibody to CD47 provided in this document contains one of the following amino acid sequences VH provided in table. 2.

Table 2. Amino acid sequence of VH

SEQID NO: Description Amino acid sequence of VH 89 consensuses aya X1QX2QLVQS GAEVKKX3GX4S VKVS OKASGFNIKDYYLHWVRQAPGQX5 LE WMGWIDPDQGDTE YAQKXgQXvRVTXg TX9DX10 SX11S TAYME LX12 S LRSXi^DTAXiYfYCNAAYGS S S YPMDYWGQGT TVTV 90 13m MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMG WIDPDQGDTEYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCNAA YGSSSYPMDYWGQGTTVTV 91 5m MQMQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMG WIDPDQGDTEYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCNAA YGSSSYPMDYWGQGTTVTV

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or expression yield of the antibody compared to that of the parent anti-CD47 antibody when expressed in a CF system, and wherein the anti-CD47 antibody comprises a VH comprising SEQ ID NO: 90. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody, IgG2, IgG3 or IgG4. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody of the Z allotype. In certain aspects, such an anti-CD47 antibody is an IgG4 isotype antibody, such as IgG4P or IgG4PE.

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or yield of expression of the antibody compared to that of the parent anti-CD47 antibody when expressed in a CF system, and wherein the anti-CD47 antibody comprises a VH comprising SEQ ID NO: 91. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody, IgG2, IgG3 or IgG4. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody. In certain aspects, such an anti-CD47 antibody is an IgG1 isotype antibody of the Z allotype. In certain aspects, such an anti-CD47 antibody is an IgG4 isotype antibody, such as IgG4P or IgG4PE.

In a specific aspect, the anti-CD47 antibody (IgG1-13m) provided herein comprises an IgG1 heavy chain comprising the amino acid sequence as follows:

MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMGWIDPDQGDTEYA QKLQGRVTMT TDT S T S TAYMELRS LRS DDTAVYYCNAAYGSS SYPMDYWGQGT TVTVSSAS T KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSL VKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

In a specific aspect, the anti-CD47 antibody (IgG1-13mZ) provided herein comprises an IgG1 Z allotype heavy chain comprising the amino acid sequence as follows:

SSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 82)

In a specific aspect, the anti-CD47 antibody (IgG1-5m) provided herein comprises an IgG1 heavy chain comprising the amino acid sequence as follows:

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SSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 83)

In a specific aspect, the anti-CD47 antibody (IgG4P-13m) provided herein comprises an IgG4P antibody comprising the amino acid sequence as follows:

SSWTV FYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 84)

In a specific aspect, an anti-CD47 antibody (IgG4P-5m) provided herein comprises an IgG4P heavy chain comprising the amino acid sequence as follows:

SSWTV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 85)

In a specific aspect, the anti-CD47 antibody (IgG4PE-13m) provided herein comprises an IgG4PE heavy chain comprising the amino acid sequence as follows:

SSWTV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 86)

In a specific aspect, the anti-CD47 antibody (IgG4PE-5m) provided herein comprises an IgG4PE heavy chain comprising the amino acid sequence as follows:

MQMQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDQGDTEYA QKFQGRVTITRDRS TS TAYMELRSLRSEDTAVYYCNAAYGSS SYPMDYWGQGT TVTVS SAS T KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 87)

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody that specifically binds to human CD47, wherein such anti-CD47 antibody is a variant of the parent anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or yield of antibody expression compared to that of the parent anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprises a light chain containing a kappa or lambda light chain constant region (e.g., a human kappa or lambda light chain constant region ), such as SEQ ID NO: 88.

In a specific aspect, this document provides an antibody, e.g., a monoclonal antibody, that specifically binds to human CD47, wherein such anti-CD47 antibody is

- 21 043164 is a variant of the original anti-CD47 antibody, where the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher titer or expression yield of the antibody compared to that of the original anti-CD47 antibody when expressed in a CF system, and wherein the anti-CD47 antibody comprises (i) a VH as described herein (e.g., SEQ ID NOs: 89, 90, or 91) or a heavy chain as described herein (e.g., any of SEQ ID NOs: 81-87 ), and (ii) a light chain comprising a kappa or lambda light chain constant region (e.g., human kappa or lambda light chain constant region), e.g. in SEQ ID NO: 88, e.g., as follows (anti-CD47 antibody light chain ( IgK)) or SEQ ID NO: 88 without the amino acid M at the N-terminus:

YEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 88).

In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a VL domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42, wherein the antibody specifically binds to CD47. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a VL domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 42 (for example, SEQ ID NO: 53, 71 and 55).

In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a light chain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 88, wherein the antibody specifically binds to CD47. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a light chain domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 88, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 88 (for example, SEQ ID NO: 53, 71 and 55).

In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a VH domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6, wherein the antibody specifically binds to CD47, and wherein the anti-CD47 antibody, when produced using a cell-free expression system, has a higher titer or yield of antibody expression than baseline antibody if produced in a CF expression system. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a VH domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 6 (for example, SEQ ID NO: 50, 72 and 52).

In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a light chain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 78, wherein the antibody specifically binds to CD47, and wherein the anti-CD47 antibody, when produced using a cell-free expression system, has a higher titer or yield of antibody expression than the parent antibody in the case of obtaining in the CF-expression system. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a heavy chain domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 78, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 78 (for example, SEQ ID NO: 53, 71 and 55).

In some embodiments, an antibody described herein, or an antigen thereof

- 22 043164 the binding fragment contains a light chain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence under SEQ ID NO: 79, wherein the antibody specifically binds to CD47, and wherein the anti-CD47 antibody, when produced using a cell-free expression system, has a higher antibody titer or yield than the parent antibody, when produced using a CF expression system. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a heavy chain domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 79, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 79 (for example, SEQ ID NO: 53, 71 and 55).

In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a light chain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 80, wherein the antibody specifically binds to CD47, and wherein the anti-CD47 antibody, when produced using a cell-free expression system, has a higher titer or yield of antibody expression than the parent antibody in the case of obtaining in the CF-expression system. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises a heavy chain domain characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 80, wherein the antibody specifically binds to CD47, and wherein the antibody contains CDRs (eg, CDR VL 1-3) that are identical to CDRs (eg, CDR VL 1- 3) from SEQ ID NO: 80 (for example, SEQ ID NO: 53, 71 and 55).

In table. 3 is a table of the amino acid sequences of the anti-CD47 antibody described herein. In certain embodiments, the implementation of the antibody described in this document contains any sequence of the variable region of the light chain from the table. 3 and any sequence of the variable region of the heavy chain from the table. 3.

Table 3. Amino acid sequences of the anti-CD47 antibody

SEQID NO: Description Amino acid sequence 1 IgGi ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

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LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 2 IgG2 PPSREEMT KN QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 3 IgG3 ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YTCNVNHKPS NTKVDKRVEL KTPLGDTHT CPRCPEPKSC DTPPPCPRCP EPKSCDTPPP CPRCPEPKSC DTPPPCPRCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG 4 IgG4 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK 5 Heavy chain variable region of a humanized anti-CD47 antibody EVQLQQSGAE LVRSGASVKL SCTASGFNIK DYYLHWVKQR PEQGLEWIGW IDPDNGDTEF APKFQGKATM TADTSSNTAY LQLSSLTSED TAVYYCNAAY GSSSYPMDYW GQGTSVTV 6 Anti-CD47 antibody heavy chain variable region AB6.12 QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDQGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 7 Heavy chain variable region of humanized QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV

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antibodies to CD47 8 Heavy chain variable region of a humanized anti-CD47 antibody EVQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 9 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQGRVTM TADTSSNTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 10 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQGRVTM TEDTSTDTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV eleven Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDQGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 12 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDYGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 13 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDSGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV

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14 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNADTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 15 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNTDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 16 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSPYPMDYW GQGTTVTV* 17 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGYTFT YYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 18 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFTFT YYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 19 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGYNFT YYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 20 Variable region of severe QMQLVQSGAE VKKTGSSVKV SCKASGYTIT YYYLHWVRQA

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humanized anti-CD47 antibody chains PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 21 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGYTFK YYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 22 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGYTFT DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 23 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFTFT DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 24 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFTIT DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 25 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGYTFK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 26 Heavy chain variable region of humanized QMQLVQSGAE VKKTGSSVKV SCKASGFTFK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV

- 27 043164

antibodies to CD47 27 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY LQLSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 28 Heavy chain variable region of a humanized anti-CD47 antibody QMQLVQSGAE VKKTGSSVKV SCKASGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLTSED TAVYYCNAAY GSSSYPMDYW GQGTTVTV 29 Heavy chain variable region of a humanized anti-CD47 antibody EVQLVQSGAE VKKPGATVKI SCKVSGFNIK DYYLHWVRQA PGQALEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV thirty Heavy chain variable region of a humanized anti-CD47 antibody EVQLVQSGAE VKKPGATVKI SCKVSGFNIK DYYLHWVQQA PGKGLEWMGW IDPDNGDTEY AQKFQDRVTI TRDRSMSTAY MELSSLRSED TAMYYCNAAY GSSSYPMDYW GQGTTVTV 31 Light chain variable region of a humanized anti-CD47 antibody DIKMTQSPSS LYASLGERVT ITCKASQDIH RYLSWFQQKP GKSPKILIYR ANRLVDGVPS RFSGSGSGQD YSLTISSLEY EDMGIYYCLQ YDEFPYTFGG GTKLEMK 32 Light chain variable region of a humanized anti-CD47 antibody DIKMTQSPSS LYASLGERVT ITCKASQDIH RYLSWFQQKP GKSPKILIYR ANRLVDGVPS RFSGSGSGQD YSLTISSLEY EDMGIYYCLQ YDEFPYTFGG GTKLEIK

- 28 043164

33 Light chain variable region of a humanized anti-CD47 antibody DIQMTQSPSS LSASVGDRVT ITCKASQDIH RYLSWYQQKP GKAPKLLIYR ANRLVDGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCLQ YDEFPYTFGG GTKVEIK 34 Light chain variable region of a humanized anti-CD47 antibody DIQMTQSPSS LSASVGDRVT ITCKASQDIH RYLSWFQQKP GKAPKSLIYR ANRLVDGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 35 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP GKVPKHLIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 36 Light chain variable region of a humanized anti-CD47 antibody DIQMTQSPSS LSASVGDRVT ITCKASQDIH RYLSWYQQKP GKAPKRLIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 37 Light chain variable region of a humanized anti-CD47 antibody DIQMTQSPSS LSASVGDRVT ITCRASQDIH RYLAWYQQKP GKVPKLLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCLQ YDEFPYTFGQ GTKVEIK 38 Light chain variable region of a humanized anti-CD47 antibody EIVLTQSPAT LSLSPGERAT LSCRASQDIH RYLAWYQQKP GQAPRLLIYR ANRRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCLQ YDEFPYTGFQ GTRLEIK 39 Variable region of the lung DIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP

- 29 043164

humanized anti-CD47 antibody chains GKVPKHLIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 40 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCRARQGIH RYLSWFQQKP GKVPKHLIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 41 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP GKVPKILIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 42 Anti-CD47 antibody light chain variable region AB6.12 NIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP GKVPKHLIYR ANRLVSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 43 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCRARQGIH RYLSWFQQKP GKVPKILIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 44 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCRARQGIH RYLSWFQQKP GKVPKHLIYR ANRLVSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 45 Light chain variable region of humanized NIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP GKVPKLLIYR ANRLVDGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK

- 30 043164

antibodies to CD47 46 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCKASQDIH RYLSWFQQKP GKVPKLLIYR ANRLVSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 47 Light chain variable region of a humanized anti-CD47 antibody NIQMTQSPSA MSASVGDRVT ITCRARQGIH RYLSWFQQKP GKVPKLLIYR ANRLVSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ YDEFPYTFGG GTKVEIK 48 ILLUSTRATI IN NAYA CONSEQUENT ELVNOSTV CD47 HUMAN MWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT REGETIIELK YRWSWFSPN ENILIVIFPI FAILLFWGQF GIKTLKYRSG GMDEKTIALL VAGLVITV IV IVGAILFVPG EYSLKNATGL GLIVTSTGIL ILLHYYVFST AIGLTSFVIA ILVIQVIAYI LAWGLSLCI AACIPMHGPL LISGLSILAL AQLLGLVYMK FVASNQKTIQ PPRKAVEEPL NAFKESKGMM NDE 49 ILLUSTRATI VNA CONSEQUENT FIRST CD47 HUMAN WITHOUT SIGNAL CONSEQUENT ELBNOST QLLFNKTKSV EFTFCNDTW IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSWFS PNENILIVIF PIFAILLFWG QFGIKTLKYR SGGMDEKTIA LLVAGLVITV IVIVGAILFV PGEYSLKNAT GLGLIVTSTG ILILLHYYVF STAIGLTSFV IAILVIQVIA YILAWGLSL CIAACIPMHG PLLISGLSIL ALAQLLGLVY MKFVASNQKT IQPPRKAVEE PLNAFKESKG MMNDE 50 Amino acid sequence of CDR1 VH GFNIKDYYLH 51 Amino acid WIDPDNGDTE

- 31 043164

sequence CDR2 VH 52 Amino acid sequence of CDR3 VH NAAYGSSSYPMDY 53 Amino acid sequence of CDR1 VL KASQDIHRYLS 54 Amino acid sequence of CDR2 VL RANRLVD 55 Amino acid sequence of CDR3 VL LQYDEFPYT 56 Epitope CD47 KGRD 57 Amino acid sequence of CDR1 VH GYTFTYYYLH 58 Amino acid sequence of CDR1 VH GFTFTYYYLH 59 Amino acid sequence of CDR1 VH GYNFTYYYLH 60 Amino acid sequence of CDR1 VH GYTITYYYLH 61 Amino acid sequence of CDR1 VH GYTFKYYYLH

- 32 043164

62 Amino acid sequence of CDR1 VH GYTFTDYYLH 63 Amino acid sequence of CDR1 VH GFTFTDYYLH 64 Amino acid sequence of CDR1 VH GFTITDYYLH 65 Amino acid sequence of CDR1 VH GYTFKDYYLH 66 Amino acid sequence of CDR1 VH GFTFKDYYLH 67 Amino acid sequence of CDR1 VL RASQDIHRYLA 68 Amino acid sequence of CDR1 VL RARQGIHRYLS 69 Amino acid sequence of CDR2 VL RANRLQS 70 Amino acid sequence of CDR2 VL RANRRAT 71 Amino acid sequence of CDR2 VL RANRLVS

- 33 043164

72 Amino acid sequence of CDR2 VH WIDPDQGDTE 73 Amino acid sequence of CDR2 VH WIDPDYGDTE 74 Amino acid sequence of CDR2 VH WIDPDSGDTE 75 Amino acid sequence of CDR2 VH WIDPDNADTE 76 Amino acid sequence of CDR2 VH WIDPDNTDTE 77 Amino acid sequence of CDR3 VH NAAYGSSPYPMDY 78 Anti-CD47 heavy chain IgGl MQMQLVQSGA EVKKTGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQDRVT ITRDRSMSTA YMELSSLRSE DTAMYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP

- 34 043164

VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 79 Anti-CD47 heavy chain IgG4P MQMQLVQSGA EVKKTGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQDRVT ITRDRSMSTA YMELSSLRSE DTAMYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 80 Anti-CD47 IgG4PE heavy chain (containing substitutions S228P and L235E) MQMQLVQSGA EVKKTGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQDRVT ITRDRSMSTA YMELSSLRSE DTAMYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFEGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 81 Anti-CD47 heavy chain IgGl13m MQVQLVQSGA EVKKPGASVK VSCKASGFNI KDYYLHWVRQ APGQGLEWMG WIDPDQGDTE YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQ S SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY

- 35 043164

K 82 Anti-CD47 heavy chain IgGl13mZ MQVQLVQSGA EVKKPGASVK VSCKASGFNI KDYYLHWVRQ APGQGLEWMG WIDPDQGDTE YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQ S SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPS RE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK 83 Anti-CD47 heavy chain IgGl5m MQMQLVQSGA EVKKPGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQGRVT ITRDRSTSTA YMELRSLRSE DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQ S SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPS RD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 84 Anti-CD47 heavy chain IgG4P-13m MQVQLVQSGA EVKKPGASVK VSCKASGFNI KDYYLHWVRQ APGQGLEWMG WIDPDQGDTE YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQ EEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

- 36 043164

85 Anti-CD47 heavy chain IgG4P-5m MQMQLVQSGA EVKKPGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQGRVT ITRDRSTSTA YMELRSLRSE DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQ EEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 86 Anti-CD47 heavy chain IgG4PE-13m MQVQLVQSGA EVKKPGASVK VSCKASGFNI KDYYLHWVRQ APGQGLEWMG WIDPDQGDTE YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFEGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEE MT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 87 Anti-CD47 heavy chain IgG4PE-5m MQMQLVQSGA EVKKPGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKFQGRVT ITRDRSTSTA YMELRSLRSE DTAVYYCNAA YGSSSYPMDY WGQGTTVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFEGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEE MT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 88 Light chain (Ig kappa) MNIQMTQSPS AMSASVGDRV TITCKASQDI HRYLSWFQQK PGKVPKHLIY RANRLVSGVP SRFSGSGSGT EFTLTISSLQ PEDFATYYCL QYDEFPYTFG GGTKVEIKRT VAAPSVEIEP PSDEQLKSGT ASWCLLNNF YPREAKVQWK 89 90 Anti-CD47 Heavy Chain Variable Region Consensus Sequence VH - 13m anti-CD47 antibodies VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF _NRGEC XiQXsQLVQS GAE VKKX3GX4S VKVS CKAS G ΕΝ I KDYYLHWVRQ APGQX ₅ LEWMGWIDPDQGDTEYAQKX ₆ QX ₇ RVTX ₈ TX ₉ DX ₁₀ SXi ₁ STAYMELX ₁₂ SLRSX ₁₃ DT7OC YWG QGTTVTV 91 VH - 5m antibodies to CD47 MQMQLVQSGA EVKKPGSSVK VSCKASGFNI KDYYLHWVRQ APGQALEWMG WIDPDQGDTE YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCNAA YGSSSYPMDY WGQGTTVTV

In some embodiments, the anti-CD47 antibodies described herein are used in combination with one or more additional agents, or a combination of additional agents. Suitable additional agents include modern pharmaceutical and/or surgical therapies for the intended use, such as cancer. For example, anti-CD47 antibodies can be used in combination with one or more additional chemotherapeutic or anticancer agents. Alternatively, the additional chemotherapeutic agent is radiation therapy. In some embodiments, the chemotherapeutic agent is a cell death inducing agent. In some embodiments, the chemotherapeutic agent induces a loss of phospholipid asymmetry within the plasma membrane, for example, causes phosphatidylserine (PS) to act on the cell surface. In some embodiments, the chemotherapeutic agent induces endoplasmic reticulum (ER) stress. In some embodiments, the chemotherapeutic agent is a proteasome inhibitor. In some embodiments, the chemotherapeutic agent induces translocation of ER proteins to the cell surface. In not

- 37 043164 in which embodiments the chemotherapeutic agent induces translocation and the effect of calreticulin on the cell surface. In some embodiments, the anti-CD47 antibody and the additional agent are formulated in a single therapeutic composition, and the anti-CD47 antibody and the additional agent are administered simultaneously. Alternatively, the anti-CD47 antibody and the additional agent are separated from each other, e.g., each is formulated into a separate therapeutic composition, and the anti-CD47 antibody and additional agent are administered simultaneously, or the anti-CD47 antibody and additional agent are administered at different times during the treatment regimen. . For example, an anti-CD47 antibody is administered before the addition agent, an anti-CD47 antibody is administered after the addition, or an anti-CD47 antibody and the additional agent are administered in turn. As described herein, the anti-CD47 antibody and the additional agent are administered in single or multiple doses.

In specific aspects, the anti-CD47 antibodies provided herein comprise one or more unnatural amino acid residues at site-specific positions. See, for example, US Patent Application Publication No. 2014/0046030 A1, which is incorporated herein by reference in its entirety. In specific aspects, non-natural amino acid residues at site-specific positions are advantageous in terms of antibody yield, solubility, binding affinity, and/or activity. Non-limiting examples of non-natural amino acids are described, see, for example, US Application Publication No. US 2014/0066598 A1.

In a specific aspect, provided herein are anti-CD47 antibodies conjugated to a conjugating moiety or agent, such as a label or toxin. The conjugating fragment may be any conjugating fragment that is considered applicable to a person skilled in the art. For example, the conjugating moiety may be a polymer, such as polyethylene glycol, which may improve the in vitro or in vivo stability of the antibody. The conjugating moiety may have therapeutic activity, resulting in an antibody-drug conjugate. The conjugating moiety may be a molecular burden that is detrimental to target cells. The conjugating fragment may be a label useful for detection or diagnosis. In certain aspects, the conjugating fragment is associated with the antibody through a direct covalent bond. In certain aspects, the conjugating fragment is linked to the antibody via a linker. In specific aspects, the conjugating fragment or linker is attached via one of the non-natural amino acids of the anti-CD47 antibody. Exemplary conjugating fragments and linkers are described, for example, in US Application Publication No. US2014/0046030 A1, which is incorporated herein by reference in its entirety.

Antibodies or antigen-binding fragments described herein that immunospecifically bind to CD47 (eg, human ECD CD47) can be obtained by any method known in the art. Antibodies described herein may, for example, include chimeric antibodies. A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:797-202 and US Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

Antibodies or antigen-binding fragments obtained using techniques such as those described herein can be isolated using standard, well-known techniques. For example, antibodies or antigen-binding fragments can be suitably separated from, for example, culture medium, ascitic fluid, serum, cell lysate, synthesis reaction material, or the like, using conventional immunoglobulin purification procedures such as, for example, protein A- sepharose,_hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. As used herein, an isolated or purified antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals in the case of chemical synthesis, or from components of the CF expression system, used to make antibodies. Antibodies described herein include antibody fragments that recognize specific CD47 antigens and can be obtained by any technique known to those skilled in the art. For example, the Fab and F(ab') ₂ fragments described herein can be prepared by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab') ₂ fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region. Alternatively, the antibody fragments described herein can be produced in a conventional manner using well known recombinant expression techniques. Cm.,

- 38 043164 for example, PCT Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869;

Sawai et al., 1995, AJRI 34:26-34 and Better et al., 1988, Science 240:1041-1043.

Antibodies described herein may, for example, include humanized antibodies, such as deimmunized or compound human antibodies. The humanized antibody may contain human constant region sequences. In certain embodiments, the humanized antibody can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG ₂ , IgG ₃ , and IgG4. In certain embodiments, the humanized antibody may comprise kappa or lambda light chain constant sequences. Humanized antibodies can be obtained using various methods known in the art, including, but not limited to, CDR grafting (European patent EP No. 239400; International Publication No. WO 91/09967 and US patents Nos. surface changes (EP 592106 and EP 519596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814 and Roguska et al., 1994, PNAS 91:969-973), strand shuffling (US Pat. No. 5,565,332), as well as the techniques described, for example, in US Pat. No. 6,407,213; Pat. US No. 5766886, WO 9317105, Ta et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16): 10678-84 (1997), Roguska et al, Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al, Cancer Res. 55(8): 1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994) and Pedersen et al, J. Mol. Biol. 235(3):959-73 (1994). See also the publication of US Pat. US No. 2005/0042664 Al (February 24, 2005), each of which is incorporated herein by reference in its entirety.

Antibodies described herein may, for example, be polyspecific, eg, bispecific antibodies. Methods for producing polyspecific (e.g., bispecific) antibodies are described, see, for example, US Pat.

Single domain antibodies, eg, antibodies lacking light chains, can be prepared using methods well known in the art. See Riechmann et al., 1999, J. Immunol. 231:25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302; US patent No. 6005079 and international publications No. WO 94/04678, WO 94/25591 and WO 01/44301. Human antibodies can be obtained using any method known in the art. For example, well-known transgenic mice that are not capable of expressing functional endogenous mouse immunoglobulins but that can express human immunoglobulin genes can be used. As an alternative, for example, the phage display techniques described above can be used. In addition, in some embodiments, human antibodies can, for example, be generated using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that immunospecifically bind to the target antigen (eg human ECD CD47). Such methods are known and described in the art, see, for example, Shinmoto et al., Cytotechnology, 2004, 46:19-23; Naganawa et al., Human Antibodies, 2005, 14:27-31.

Antibody variable domains with the desired binding specificity (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably occurs with an immunoglobulin heavy chain constant domain containing at least a portion of a hinge of the CH2 and CH3 regions. It is preferred that a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of the fusions. DNA encoding immunoglobulin heavy chain and, if desired, immunoglobulin light chain fusions are inserted into separate expression vectors and co-transfected into a suitable host organism. For additional details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

In accordance with another approach, described in WO 96/27011, the interface between a pair of antibody molecules can be designed in such a way as to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred interface contains at least a portion of the CH3 region of the constant domain of the antibody. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). Compensation cavities having the same or similar size to the larger side chain(s) are formed at the interface portion of the second antibody molecule by replacing large amino acid side chains with smaller chains (eg, alanine or threonine). This provides a mechanism to increase the yield of the heterodimer over other undesirable end products such as homodimers.

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Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F(ab')2 bispecific antibodies). Methods for obtaining bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be generated using a chemical bond. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to form F(ab') ₂ fragments. These fragments are reduced in the presence of a dithiol complexing agent based on sodium arsenite to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfides. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The obtained bispecific antibodies can be used as agents for selective immobilization of enzymes.

4.2.3 Pharmaceutical compositions and kits

Provided herein are compositions, pharmaceutical compositions, and kits containing one or more of the proteinaceous therapeutic agents described herein. Also provided herein are compositions, pharmaceutical compositions, and kits containing an anti-CD20 antibody, such as rituximab, alone or in combination with the protein therapeutic agents described herein. In particular, provided herein are compositions, pharmaceutical compositions, and kits comprising one or more of the antibodies (eg, anti-CD47 antibodies) described herein, or antigen-binding fragments or conjugates thereof. In certain aspects, the compositions (eg, pharmaceutical compositions) described herein can be used in vitro, in vivo, or ex vivo. Non-limiting examples of uses include uses to reduce immunogenicity, uses to modulate (eg, inhibit or induce/enhance) CD47 activity, and uses to control or treat a disorder, such as cancer. In specific embodiments, provided herein is a pharmaceutical composition comprising an antibody (eg, a humanized antibody) described herein (or an antigen-binding fragment thereof) and a pharmaceutically acceptable carrier or excipient.

As used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the US federal or state government, or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more specifically in humans.

Compositions containing one or more of the antibodies provided herein, or an antigen-binding fragment thereof, can be prepared for storage by mixing an antibody having the desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA; Remington: The Science and Practice of Pharmacy, 21st ed. (2006) Lippincott Williams & Wilkins, Baltimore, MD). Such formulations may, for example, be in the form of, for example, lyophilized formulations or aqueous solutions. Suitable pharmaceutical carriers for administering the antibodies provided herein include any such carriers known to those skilled in the art that are suitable for a particular route of administration. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Formulations used for in vivo administration may be sterile. This is easily achieved, for example, by filtration through, for example, sterile filter membranes.

In specific aspects, pharmaceutical compositions for use in the methods provided herein comprise therapeutically effective amounts of one or more of the proteinaceous therapeutic agents provided herein in a pharmaceutically acceptable carrier. In specific aspects, pharmaceutical compositions for use in the methods provided herein comprise a therapeutically effective amount of an anti-CD20 antibody, such as rituximab, alone or in combination with one or more of the protein therapeutic agents provided herein, in a pharmaceutically acceptable carrier. In specific aspects, pharmaceutical compositions for use in the methods provided herein contain a therapeutically effective amount of one or more antibodies or antigen-binding fragments provided herein, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, control, or amelioration of the condition or disorder described herein, or one or more of their symptoms.

Compositions for use in the methods provided herein may contain one or more of the protein therapeutic agents provided herein. compo

- 40 043164 positions for use in the methods provided herein may contain rituximab, alone or in combination with one or more of the protein therapeutic agents presented herein. Compositions for use in the methods provided herein may contain one or more of the antibodies provided herein, or an antigen-binding fragment thereof. In one embodiment, compositions are provided wherein the antibodies or antigen-binding fragments described herein are formulated into suitable pharmaceutical preparations such as solutions, suspensions, powders, sustained release formulations, or elixirs in sterile solutions or suspensions for parenteral administration or as a formulation. for transdermal administration and dry powder inhalers.

In one embodiment, compositions for use in the methods provided herein are formulated for single administration. To formulate a composition, a mass fraction of a compound is dissolved, suspended, dispersed, or otherwise mixed in a carrier of choice at an effective concentration such that the condition being treated relieves, prevents, or ameliorates one or more symptoms.

In certain aspects, an anti-CD20 antibody, such as rituximab, is included in a pharmaceutically acceptable carrier in an effective amount sufficient to provide a therapeutically beneficial effect with no or minimal or minor unwanted side effects to the patient being treated.

In certain aspects, proteinaceous drugs for use in the methods provided herein are included in a pharmaceutically acceptable carrier in an effective amount sufficient to provide a therapeutically beneficial effect with no or minimal or minor unwanted side effects to the patient being treated.

In certain aspects, antibodies for use in the methods provided herein are included in a pharmaceutically acceptable carrier in an effective amount sufficient to provide a therapeutically beneficial effect with no or minimal or minor unwanted side effects in the patient being treated.

The concentrations of a proteinaceous therapeutic agent, such as an anti-CD47 antibody, in a pharmaceutical composition for use in the methods provided herein will depend, for example, on the physicochemical characteristics of the antibody, dosage regimen and amount administered, as well as other factors. Pharmaceutical compositions for use in the methods described herein are intended for administration to humans or animals (eg, mammals) in unit dosage forms, such as sterile solutions or suspensions for parenteral (eg, intravenous) administration, containing suitable amounts of the compounds or their pharmaceutically acceptable derivatives. . Pharmaceutical compositions are also intended to be administered to humans and animals in unit dosage form such as tablets, capsules, pills, powders, granules and oral or nasal solutions or suspensions, as well as oil-in-water emulsions, containing suitable amounts of the proteinaceous therapeutic agent or a pharmaceutically acceptable agent thereof. derivatives. The protein therapeutic agent in one embodiment is formulated and administered in unit dosage forms or multiple dosage forms. As used herein, unit dosage forms refer to physically discrete units suitable for humans or animals (eg, mammals) and packaged individually. Each unit dose contains a predetermined amount of proteinaceous therapeutic agent sufficient to produce the desired therapeutic effect, in combination with the necessary pharmaceutical carrier, binder or diluent. Examples of unit dosage forms include ampoules and syringes, as well as individually packaged tablets or capsules. Standard dosage forms can be entered in the form of fractional parts or multiples of them. A multiple dosage form is a plurality of unit dosage forms packaged in a single container for administration in the form of separate unit dosage forms. Examples of multiple dosage forms include vials, tablet bottles or capsules or bottles. Therefore, in specific aspects, a multiple dosage form is a plurality of unit dosage forms that are not separated in the package.

In certain embodiments, one or more protein therapeutic agents for use in the methods described herein are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing antibodies and optional pharmaceutical excipients in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, with thus forming a solution or suspension. In some embodiments, the pharmaceutical composition for administration provided herein may also contain minor amounts of non-toxic auxiliary materials such as wetting agents, emulsifying agents, solubilizing agents and pH buffering agents, and the like.

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Methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see for example Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA; Remington: The Science and Practice of Pharmacy, 21st ed. (2006) Lippincott Williams & Wilkins, Baltimore, MD.

In one embodiment, parenteral administration involves injection, either subcutaneously, intramuscularly, or intravenously. Injectable preparations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectable preparations, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. Other routes of administration may include enteral administration, intracerebral administration, nasal administration, intra-arterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.

Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products , ready to be combined with a binder immediately before use, and sterile emulsions. Solutions may be aqueous or non-aqueous.

When administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable agents.

Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

In some embodiments, intravenous or intra-arterial infusion of a sterile aqueous solution containing a proteinaceous therapeutic agent for use in the methods described herein is an effective route of administration. In another embodiment, a sterile aqueous or oily solution or suspension containing a proteinaceous therapeutic agent is provided for use in the methods described herein, administered as needed to obtain the desired pharmacological effect.

In specific embodiments, the protein therapeutic agent for use in the methods described herein may be suspended in a micronized or other suitable form. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen carrier or vehicle.

In other embodiments, the pharmaceutical formulations are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

A lyophilized powder can, for example, be prepared by dissolving a protein therapeutic agent for use in the methods provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterile. Suitable solvents may contain a stability aid or other pharmacological component of the powder or reconstituted solution derived from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. A suitable solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment at approximately neutral pH. Further sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides an example of a formulation. In one embodiment, the resulting solution will be dispensed into lyophilization vials. The lyophilized powder can be stored under suitable conditions, for example at a temperature of from about 4° C. to room temperature.

Reconstitution of such a lyophilized powder with water for injection provides a formulation for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable vehicle.

In certain aspects, a protein therapeutic agent for use in the methods provided herein may be formulated for topical administration or topical application,

- 42 043164 such as for topical application on the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions and for application to the eye or for intracisternal or intraspinal use. Topical administration is intended for transdermal delivery, as well as for administration to the eyes or mucous membranes, or for inhalation therapies. It is also possible to administer nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients.

Anti-CD47 antibodies, anti-CD20 antibodies such as rituximab, and other proteinaceous therapeutic agents for use in the methods provided herein can also be formulated to target the particular tissue, organ, or other body site of the subject to be treated. Many such methods for targeting are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of this invention. For non-limiting examples of methods for targeting, see, for example, US Pat. , 6004534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5,709,874. In some embodiments, the anti-CD47 antibodies described herein are targeted (or otherwise administered) to the eyes, bone marrow, gastrointestinal tract, lungs, brain, or joints. In specific embodiments, an anti-CD47 antibody described herein is capable of crossing the blood-brain barrier.

5. Examples

The examples in this section (ie, section 5) are provided for illustration and not limitation.

5.1 Example 1 Co-dosing of rituximab and anti-CD47 cynomolgus macaque antibody

The aim of the study was to monitor the immunogenicity of a humanized anti-CD47 antibody containing the heavy chain variable region CDRs from SEQ ID NOs: 50, 72 and 52 and the light chain variable region CDRs from SEQ ID NOs: 53, 71 and 55 (hereinafter anti-CD47 antibody) at co-administration with rituximab or rituximab and methotrexate and evaluation of its effect on pharmacokinetics after intravenous administration to cynomolgus monkeys. In the first part of this study, anti-CD47 antibody was administered as four intravenous injections (IV) to 3 groups of cynomolgus monkeys (5 animals/group, total of 15 animals) on study days 1,8, 15 and 22, group 1 and study days 15, 22 , 29 and 36 groups 2 and 3 at doses of 20 mg/kg (doses 1-4). Rituximab was administered as four intravenous injections to Group 2 and Group 3 animals on study days 1, 8, 15 and 22 at doses of 10 mg/kg. Methotrexate was administered as three subcutaneous injections to Group 3 animals on study days 15, 16 and 18 at doses of 0.4 mg/kg.

In the second part of this study, anti-CD47 antibody was administered as a single intravenous injection to Group 1 and Group 3 animals on study day 78 at a dose of 20 mg/kg (dose 5). Methotrexate was administered as multiple subcutaneous injections to Group 3 animals on study days 71, 72, 74, 78, 79, 81, 88, 95, 102 and 109 at a dose of 0.4 mg/kg.

Concentration data after the first, fourth and fifth doses of anti-cynomolgus CD47 antibody were used for pharmacokinetic (PK) evaluations to understand the effect of co-dosed drugs on the PK of anti-CD47 antibody. Anti-CD47 antibody immunogenicity was assessed by serum anti-drug (ADA) titer assessment (with anti-CD47 drug) weekly throughout the study. The number of B cells was monitored weekly throughout the study. _All animals treated with anti-CD47 antibody alone (Group 1) became ADA positive before dosing _. 4, serum concentrations of anti-CD47 antibody for five animals from group 1 ranged from 3-37% AUC ₀ _ ₁₆₈ after dose 1. This decrease in AUC after seroconversion was associated with an ADA-mediated decrease in exposure to free anti-CD47 antibody. The number of CD20+ B cells in these animals was within the normal range. The number of peripheral CD20+ B cells for all animals that received rituximab (groups 2 and 3) decreased rapidly and was <100 cells/µl within 24 hours after receiving the first dose of rituximab. The number of B cells began to recover around day 15 after the first dose of rituximab in 8 of 10 animals in groups 2 and 3. This recovery of B cells is associated with antibodies to rituximab, since all animals had detectable antibodies to rituximab by day 15 with corresponding decrease in serum rituximab concentrations. The 8 animals that showed recovery in B cells were positive for the presence of anti-drug antibodies (ADA) by day 36 with varying titers from 5 to 625 and varying levels of reduction in exposure in all 8 animals ranging from 2-53% AUC ₀ _ ₁₆₈ after dose 1.

In animals 11 and 15, where significant concentrations of rituximab were maintained, peripheral CD20+ B cell counts remained negligible until day 36 when these animals were given a fourth dose of anti-CD47 antibody. These two animals, which received all 4 doses of anti-CD47 antibody in the context of effective B-cell depletion, remained ADA-negative for an extended period of time until day 71 and the end of the study, respectively. Anti-CD47 antibody concentrations

- 43 043164 after dose 4 were similar to the concentrations observed after dose 1, with AUC ₀ - ₁₆₈ at week 4 being 115 and 83% of the mean AUC at week 1 in animals 11 and 15, respectively. Monkeys treated with anti-CD47 antibody alone (Group 1) or a combination of all three (Group 3) were then re-introduced with anti-CD47 antibody 12 weeks later (Day 78). In two animals 11 and 15, where ADA development was reduced by effective B cell depletion, PK profiles at week 12 were comparable to PK profiles after 1 dose of anti-CD47 antibody, and AUC _0-168 at week 12 was 127 and 120% of mean AUC _{of 0-168} after the first dose, confirming the absence of ADA-mediated loss of exposure in these animals. In addition, serum levels of anti-drug antibodies in these two animals were maintained at a low level, with detectable drug present for three weeks after the last dose. Anti-CD47 antibody concentrations in ADA-positive animals were below the limit of quantitation for 4 days after the last dose. These data indicate that depletion of B cells by rituximab reduces the immune response to anti-CD47 antibodies and increases drug exposure over extended periods of time. In addition, no toxicity was observed in monkeys with anti-CD47 plus rituximab or with anti-CD47 plus rituximab plus methotrexate.

5.2 Example 2 Clinical Trial of Anti-CD47 Antibodies in Combination with Rituximab

This document describes an open-label Phase 1 dose escalation and expansion study of anti-CD47 antibody administered by intravenous (IV) infusion in subjects with advanced resistant solid and hematologic cancer. The study consisted of 2 parts. Part A is a dose escalation phase in which increasing doses of anti-CD47 antibody are used, and part B is an escalation and expansion phase in which anti-CD47 antibody in combination with rituximab is administered to subjects with CD20-positive non-Hodgkin's lymphoma (NHL). Expansion may occur at the maximum tolerated dose (MTD) set in the dose escalation phase and/or at a lower dose, or at an alternative allowable dosing regimen based on the review of safety data, PK and pharmacodynamics from Part A. Modified dose escalation schedule 3+3 is used to determine the initial toxicity of the anti-CD47 antibody. Cohorts of 3 to 6 evaluable subjects were treated with anti-CD47 antibody at specific dosing regimens, and in the case of dose-limiting toxicity (DLT) in one subject, the cohorts were expanded to a full cohort of 6 evaluable subjects. In this dose cohort, subjects 4 to 6 could be enrolled before the first 3 subjects complete Cycle 1 to obtain additional safety information and ensure that there are enough evaluable patients to evaluate DLT. No more than one subject per day was included in this dose escalation cohort.

Doses included 0.3 mg/kg IV once a week (QW) and 1, 2, 4, 8, 15, and 20 mg/kg once every 2 weeks (Q2W). During dose escalation, the decision was made to evaluate the lower dose cohort, the intermediate dose cohort, an alternative dosing interval, or to declare an MTD based on an analysis of clinical and laboratory safety data for the previous dose cohorts. All treatments were administered in 28 day cycles and anti-CD47 antibody was administered at D1 and D15. After the first dose in any cohort, subjects were observed for at least 28 days (cycle 1, DLT window) before starting the next higher protocol-defined dose of the cohort. The starting dose for group 1 was 0.3 mg/kg on day 1 of cycle 1 followed by 1 mg/kg QW.

For Part B, in subjects with relapsed or refractory CD20-positive non-Hodgkin's lymphoma who had previously received rituximab, rituximab 375 mg/m ² once weekly dosing was started two weeks prior to anti-CD47 antibody administration. Rituximab was dosed at four weekly doses before and during cycle 1 (D-15, D-8, Dl, D8), once every cycle for cycles 2-6 (at D8) and, if responded, every two thereafter. months up to 12 cycles (cycles 8, 10 and 12). The serial number of B cells and PK was assessed. In addition, PK, PD, and ADA scores were performed in the same way as for the part

Inclusion Criteria

1. Men and women 18 years of age or older with advanced, recurrent, or refractory solid tumors, multiple myeloma (MM), or non-Hodgkin's lymphoma (NHL) in Part A.

In Part B, only subjects with relapsed or refractory CD20-positive NHL.

2. At least one site of measurable disease in subjects with solid tumors and NHL.

3. Eastern Joint Cancer Group (ECOG PS) General Status 0 or 1.

4. Subjects must have adequate hematopoietic, hepatic, renal, and coagulation function according to specific laboratory criteria.

5. Women and men must consent to methods of contraception and avoid conception throughout the study and up to 8 weeks after the last dose of CD47 antibody. In Part B, women of childbearing potential must continue to use effective contraception for 12 months after treatment with rituximab.

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Exclusion Criteria

1. Lymphomas of a high degree (Burkitt or lymphoblastic), plasmatic leukemia.

2. Rapidly progressive high-grade solid tumors (eg, small cell lung cancer, germ cell tumors, neuroblastoma) with extensive tumor burden.

3. Symptomatic damage to the central nervous system.

4. Impaired cardiac function or clinically significant heart disease.

5. Prior RBC transfusion less than 3 months prior to anti-CD47 antibody administration.

6. Prior autologous stem cell transplant < 3 months prior to anti-CD47 antibody administration.

7. Prior allogeneic stem cell transplant with standard or reduced intensity conditioning < 6 months prior to anti-CD47 antibody administration.

8. Previous systemic cancer treatment or test methods < 5 half-lives or 4 weeks prior to anti-CD47 antibody administration, whichever is shorter.

9. Major surgery < 2 weeks prior to anti-CD47 antibody administration.

10. Pregnant or lactating women.

11. Known HIV infection.

12. Known chronic hepatitis B or C (HBV/HCV) infection.

13. Permanent treatment with long-term therapeutic dosing of anticoagulants.

14. History of autoimmune hemolytic anemia or autoimmune thrombocytopenia.

15. History of concomitant secondary cancers requiring active permanent systemic treatment.

All references (e.g., publications, or patents, or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication, or patent or patent application) has been specifically and individually set forth for inclusion by reference in its entirety for all purposes.

Other embodiments are within the scope of the following claims.

Claims (16)

1. The use of a combination for the treatment of cancer, where the combination includes rituximab and a composition containing an antibody that binds to CD47, or an antigen-binding fragment containing (CDR) 1 heavy chain variable region (VH), containing SEQ ID NO: 50, CDR2 VH , containing SEQ ID NO: 72, a VH CDR3 sequence containing SEQ ID NO: 52, a light chain variable region (VL) CDR1 containing SEQ ID NO: 53, a VL CDR2 containing SEQ ID NO: 71, and a VL CDR3 containing SEQ ID NO: 55, wherein the antibody that binds to CD47 or its antigen-binding fragment is an IgG isotype. 2. Use according to claim 1, wherein the CD47-binding antibody or antigen-binding fragment thereof comprises a VH containing a sequence selected from the group consisting of SEQ ID NOs: 6 and 11. 3. Use according to claim 1 or 2, wherein the CD47-binding antibody or antigen-binding fragment thereof comprises a VL containing a sequence selected from the group consisting of SEQ ID NOs: 42 and 46. 4. Use according to any one of claims 1 to 3, wherein the antibody that binds to CD47, or an antigen-binding fragment thereof, is an IgG isotype selected from the group consisting of an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, and an IgG4 isotype. 5. Use according to any one of claims 1 to 3, wherein the antibody that binds to CD47 or its antigen-binding fragment is an IgG isotype selected from IgG4P and IgG4PE. 6. Use according to any one of claims 1 to 5, wherein the composition is a pharmaceutical composition containing a pharmaceutically acceptable carrier. 7. Use according to any one of claims 1 to 6, wherein administration of the composition and administration of rituximab occur serially, sequentially, simultaneously or in parallel. 8. Use according to any one of claims 1 to 7, where the antibody or its antigen-binding fragment is administered to the subject at a dose of 0.3; 1; 2; 4; 8; 15 or 20 mg/kg. 9. Use according to any one of claims 1 to 8, wherein rituximab is administered to the subject at a dose of 300, 325, 350, 375, 400, 425, 450, or 500 mg/m ² . 10. Use according to any one of claims 1 to 9, further comprising another anti-cancer agent. 11. Use according to any one of claims 1 to 10, wherein the cancer is a hematological cancer. 12. Use according to claim 11 wherein the hematologic cancer is multiple myeloma, non-Hodgkin's lymphoma, or acute myeloid leukemia (AML). 13. Use according to any one of claims 1-12, wherein the composition does not include administering an inhibitor to the subject - 45 043164 proteasomes. 14. Use according to claim 13 wherein the proteasome inhibitor is bortezomib. 15. Use according to any one of claims 1-14, wherein the composition does not include administering methotrexate to the subject. 16. A method of reducing immunogenicity in a subject, comprising administering to the subject rituximab in combination with an antibody that binds to CD47, or an antigen-binding fragment thereof, wherein the antibody that binds to CD47, or an antigen-binding fragment thereof, comprises a complementarity-determining region (CDR) 1 of the variable region of severe chain (VH) containing SEQ ID NO: 50, CDR2 VH containing SEQ ID NO: 72, CDR3 VH sequence containing SEQ ID NO: 52, CDR1 light chain variable region (VL) containing SEQ ID NO: 53, CDR2 VL containing SEQ ID NO: 71 and CDR3 VL containing SEQ ID NO: 55; or CDR1 VH containing SEQ ID NO: 50, CDR2 VH containing SEQ ID NO: 51, CDR3 VH containing SEQ ID NO: 52, CDR1 VL containing SEQ ID NO: 53, CDR2 VL containing SEQ ID NO: 54, and CDR3 VL containing SEQ ID NO: 55.