WO2023159220A1 - Anticorps anti-cd47 - Google Patents

Anticorps anti-cd47 Download PDF

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Publication number
WO2023159220A1
WO2023159220A1 PCT/US2023/062875 US2023062875W WO2023159220A1 WO 2023159220 A1 WO2023159220 A1 WO 2023159220A1 US 2023062875 W US2023062875 W US 2023062875W WO 2023159220 A1 WO2023159220 A1 WO 2023159220A1
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antibody
antigen
cancer
subject
binding
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PCT/US2023/062875
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English (en)
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William Robert ARATHOON
Raffaella BRIANTE
Cindy TAN
Qianting ZHAI
Pingping Zhang
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Kenjockety Biotechnology, Inc.
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Publication of WO2023159220A1 publication Critical patent/WO2023159220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention concerns antibodies that bind to Cluster of Differentiation 47 (CD47).
  • CD47 Cluster of Differentiation 47
  • the invention further concerns methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders that are characterized by or associated with the expression of CD47.
  • CD47 Cluster of Differentiation 47
  • IAP integrin-associated protein
  • CD47 is overexpressed on numerous malignant tumors, including hematological malignancies and solid tumors, such as, for example, acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), myelodysplastic syndromes, multiple myeloma (MM), chronic myeloid leukemia (CML), hepatocellular carcinoma (HCC), gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, bladder cancer and brain tumors.
  • AML acute myeloid leukemia
  • NHL non-Hodgkin’s lymphoma
  • MM multiple myeloma
  • CML chronic myeloid leukemia
  • HCC hepatocellular carcinoma
  • gastric cancer non-small cell lung cancer
  • NSC non-small cell lung cancer
  • ovarian cancer breast cancer
  • head and neck cancer bladder cancer and brain tumors.
  • Anti-CD47 monoclonal antibodies have been in clinical development and various stages of clinical
  • Drug resistance a well-known phenomenon that results when diseases become tolerant to pharmaceutical treatments, is a major and increasing challenge in various fields of medicine, including oncology. Some methods of drug resistance are disease-specific, while others, such as drug efflux, which is observed in microbes and human drug-resistant cancers, are evolutionarily conserved. Although many types of cancers are initially susceptible to chemotherapy, over time they can develop resistance through these and other mechanisms, such as DNA mutations and metabolic changes that promote drug inhibition, degradation, and enhanced efflux.
  • Efflux pumps are proteins expressed by virtually all living cells and have evolved to naturally expel various compounds from the cells.
  • Members of the ATP-binding cassette (ABC) transporter family proteins are examples of EPs that enable drug efflux and are important, well-studied regulators at the plasma membranes of healthy cells.
  • ABSC ATP-binding cassette
  • a transporter’s structure varies from protein to protein (e.g., there are 49 known members of the ABC family in humans), they are all classified by the presence of two distinct domains — a highly conserved nucleotide -binding domain and a more variable transmembrane domain.
  • Multidrug resistance protein 1 encoded by the ATP Binding Cassette Subfamily B Member 1 (MDR1, ABCB1, P-glycoprotein) gene, was the first of these to be identified and has been studied extensively. Normal expression of ABCB 1 is increased in certain tissues (e.g., colon, liver, and kidney) when these tissues become neoplastic and increased expression in response to treatment with certain chemotherapeutics demonstrates that both intrinsic and extrinsic mechanisms of ABCB1 overexpression are at play.
  • MDR1 ATP Binding Cassette Subfamily B Member 1
  • EPs enable cells and tumors to develop resistance to chemotherapeutic agents. Such resistance is frequently associated with enhanced efflux of the treatment molecules from the resistant cells.
  • This chemotherapy resistance is termed multi-drug resistance (MDR) when it applies to more than one chemotherapeutic agent.
  • MDR multi-drug resistance
  • Various small molecule inhibitors have been developed that target and inhibit EPs, but none have been successful in the human clinical setting for a variety of reasons, among which is their tendency to penetrate into and affect all cells in the body, including healthy cells that employ EPs for efflux of naturally-occurring cellular toxins, regardless of the function of the cells or their efflux pumps.
  • Multi-specific antibodies binding to the efflux pump ABCB1 and a tumor-associated antigen are disclosed in W02020206033 and WO2021155028, multi-specific antibodies binding to the efflux pump ABCG2 and a tumor-associated antigen are disclosed in WO 2021247426 and WO2021247423, the entire disclosures of which are hereby expressly incorporated by reference herein.
  • aspects of the invention include isolated anti-CD47 monoclonal antibodies, or antigen-binding fragments thereof, comprising an antigen-binding site for CD47, wherein said antigen-binding site comprises: a heavy chain variable region (VH) in which a full set of CDRHsl-3, combined, has greater than or equal to 85% sequence identity at the amino acid level to the CDRHsl-3, combined, of the antibodies whose CDR sequences are provided in Table 2; and/or a light chain variable region (VL) in which a full set of CDRLsl-3, combined, has greater than or equal to 85% sequence identity at the amino acid level to the CDRLsl-3, combined, of the antibodies whose CDR sequences are provided in Table 2.
  • VH heavy chain variable region
  • VL light chain variable region
  • aspects of the invention include isolated anti-CD47 monoclonal antibodies comprising an antigen-binding site for CD47, wherein said antigen-binding site comprises CDRHsl-3 or CDRLsl-3 of an antibody shown in Table 2, or an antigen-binding fragment thereof.
  • said antigen-binding site comprises CDRHsl-3 and CDRLsl-3 of an antibody shown in Table 2, or an antigen-binding fragment thereof. In some embodiments, said antigenbinding site comprises a heavy chain variable region or a light chain variable region of an antibody shown in Table 2, or an antigen-binding fragment thereof. In some embodiments, said antigen-binding site comprises a heavy chain variable region and a light chain variable region of an antibody shown in Table 2, or an antigen-binding fragment thereof.
  • an anti-CD47 antibody is an IgG antibody, or an antigen-binding fragment thereof. In some embodiments, an anti-CD47 antibody is an IgGl or IgG4 antibody, or an antigen-binding fragment thereof. In some embodiments, an anti-CD47 antibody is an IgGl antibody, or an antigen-binding fragment thereof.
  • the antigen-binding fragment is selected from the group consisting of Fab, scFab, Fab’, F(ab’)2, Fd, Fv, diabody, triabody, minibody, scFv, and scFv-Fc fragments.
  • the antigen-binding fragment is a Fab, Fab', Fv, (Fab')2, or scFv fragment.
  • an anti-CD47 antibody, or an antigen-binding fragment thereof comprises consensus framework sequences.
  • the consensus framework sequences comprise sequences of a human consensus framework.
  • the CD47 is human.
  • the anti-CD47 antibody, or antigen-binding fragment thereof is chimeric, humanized or human.
  • the anti- CD47 antibody, or antigen-binding fragment thereof exhibits one or more effector functions selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and Clq binding against CD47, or an antigen-binding fragment thereof.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • aspects of the invention include isolated anti-CD47 monoclonal antibodies, or antigen-binding fragments thereof, that compete with an antibody described herein to bind to CD47, or that antagonize or block the binding of an antibody described herein to CD47.
  • the anti-CD47 antibody, or antigen-binding fragment thereof is multispecific. In some embodiments, the anti-CD47 antibody, or antigen-binding fragment thereof, is bispecific. In some embodiments, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises an antigen-binding site for an ABC transporter. In some embodiments, the ABC transporter is ABCB1, ABCG2, ABCC1 or ABCC4.
  • the anti-CD47 antibody, or antigen-binding fragment thereof comprises an antigen-binding site for a tumor antigen other than CD47.
  • the tumor antigen is selected from the group consisting of EGFR, HER2, PD-L1, CD19, CD20, CD38, and CD40.
  • the tumor antigen is selected from the group consisting of AXL, LIV1, MET, CD73, CD70, LY6E, and CD44.
  • compositions comprising an antibody or antigen-binding fragment thereof as described herein.
  • the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • aspects of the invention include methods of treating a disease or condition associated with overexpression of CD47 or with a CD47-related signaling pathway, comprising administering to a subject in need an antibody or an antigen-binding fragment thereof as described herein, or a composition as described herein.
  • the CD47-related signaling pathway is the CD47/SIRPalpha or CD47/TSP-1 pathway.
  • the disease or condition is a cancer.
  • the cancer is a hematologic tumor or solid tumor.
  • the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), lymphoma, breast cancer, head and neck cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, liver cancer, renal cancer, pancreatic cancer, bladder cancer, colorectal cancer, neuroglioma, melanoma, and hepatocellular carcinoma (HCC).
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • NHL non-Hodgkin's lymphoma
  • MM multiple myeloma
  • breast cancer breast cancer
  • head and neck cancer gastric cancer
  • the cancer is selected from the group consisting of AML, CML, ALL, CLL, NHL, ML, and MM. In some embodiments, the cancer is selected from the group consisting of gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, and bladder cancer.
  • NSC non-small cell lung cancer
  • the antibody or antigen-binding fragment thereof is administered intravenously or subcutaneously.
  • the methods comprise administering the antibody or antigen-binding fragment thereof in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an immunotherapeutic agent, an inhibitor of a multidrug resistance transporter, or a combination thereof.
  • the at least one additional active agent is a chemotherapeutic agent.
  • the chemotherapeutic agent is a taxane, a vinca alkaloid, or an anthracycline.
  • the chemotherapeutic agent is an alkylating agent, a nitrosourea, an anti-metabolite, an antitumor antibiotic, or a steroid hormone.
  • the chemotherapeutic agent is Paclitaxel, Colchicine, Verapamil, Vinblastine, Topotecan, Doxorubicin, Daunorubicin, Etoposide, or Nilotinib.
  • the at least one additional active agent is an immunotherapeutic agent.
  • the immunotherapeutic agent is a therapeutic antibody.
  • the therapeutic antibody is an anti-CD20, anti- HER2, or anti-PDL-1 antibody.
  • the therapeutic antibody binds to an immune check point marker.
  • the immune check point marker is selected from the group consisting of: CTLA4, LAG-3 and TIM-3.
  • aspects of the invention include use of an antibody or an antigen-binding fragment described herein, or a composition described herein, in the preparation of a medicament for the treatment of a disease or condition associated with overexpression of CD47 or with a CD47-related signaling pathway.
  • aspects of the invention include an antibody or an antigen-binding fragment described herein, or a composition described herein, for use in the treatment of a disease or condition associated with overexpression of CD47 or with a CD47-related signaling pathway.
  • aspects of the invention include one or more nucleic acids comprising one or more sequences encoding an antibody or antigen-binding fragment as described herein.
  • the one or more nucleic acids are operably linked to a promoter.
  • aspects of the invention include one or more expression vectors comprising one or more nucleic acids as described herein.
  • aspects of the invention include mammalian cells genetically modified with the one or more expression vectors described herein.
  • the mammalian cell is an immune cell.
  • kits comprising an antibody or antigen-binding fragment as described herein.
  • the kit further comprises at least one additional active agent.
  • the additional active agent comprises a chemotherapeutic agent, an immunotherapeutic agent, or an inhibitor of a multidrug resistance transporter.
  • the kit further comprises instructions for use.
  • aspects of the invention include diagnostic methods for determining whether a subject has or is at risk of developing a disorder characterized by expression of CD47, the methods comprising: (a) contacting a biological test sample from the subject with an anti-CD47 antibody, or an antigen-binding fragment thereof, as described herein, to generate a CD47-antibody complex; (b) detecting a concentration of the CD47-antibody complex in the biological test sample; and (c) comparing the concentration of the CD47-antibody complex to a reference value to determine whether the subject has or is at risk of developing the disorder.
  • aspects of the invention include diagnostic methods for determining whether a subject has or is at risk of developing a disorder characterized by expression of CD47 and an ATP -binding cassette (ABC) transporter, the methods comprising: (a) contacting a biological test sample from the subject with a multispecific antibody, or a multispecific antigen-binding fragment thereof, comprising an antigen binding site for CD47 as described herein and an antigen binding site for an ABC transporter to generate a CD47-ABC transporter-antibody complex; (b) detecting a concentration of the CD47- ABC transporter-antibody complex in the biological test sample; and (c) comparing the concentration of the CD47-ABC transporter-antibody complex to a reference value to determine whether the subject has or is at risk of developing the disorder.
  • aspects of the invention include diagnostic methods for determining whether a subject has or is at risk of developing a disorder characterized by expression of CD47 and a tumor associated antigen (TAA) other than an ABC transporter, the methods comprising: (a) contacting a biological test sample from the subject with a multispecific antibody, or a multispecific antigen-binding fragment thereof, comprising an antigen binding site for CD47 as described herein and the TAA to generate a CD47- TAA-antibody complex; (b) detecting a concentration of the CD47-TAA-antibody complex in the biological test sample; and (c) comparing the concentration of the CD47-TAA-antibody complex to a reference value to determine whether the subject has or is at risk of developing the disorder.
  • TAA tumor associated antigen
  • the TAA is selected from the group consisting of: EGFR, HER2, PD- Ll, CD 19, CD20, CD38, and CD40. In some embodiments, the TAA is selected from the group consisting of: AXL, LIV1, MET, CD73, CD70, LY6E, and CD44. In some embodiments, the antibody comprises a detectable label.
  • FIG. 1 Panels A and B schematically depict a bispecific monoclonal antibody structure comprising an anti-CD47 antibody described herein.
  • Panel A illustrates a bispecific antibody with optional mutations (HC DD/HC KK) in the heavy chain Fc regions leading to electrostatic steering effects and a common light chain.
  • Panel B illustrates a bispecific antibody with optional knob-into- hole mutations in the heavy chain Fc regions (HC HIR/HC KbR) and a common light chain.
  • FIGs. 2A and 2B, Panels A-D illustrate various alternative IgG-based bispecific antibody structures within the scope herein.
  • Panel A of FIG. 2A illustrates a bispecific antibody with two additional scfv fragments targeting Target 2 linked to the N-terminal of the heavy chains of the antibody targeting Target 1.
  • Panel B of FIG. 2A illustrates a bispecific antibody with optional mutations (HC DD/HC KK) in the heavy chain Fc regions leading to electrostatic steering effects consisting of a heavy chain/light chain pair targeting Target 1 and a FC-scfv moiety targeting Target 2.
  • optional K- i-H mutations are not always shown, they might be present in any of the structures illustrated.
  • (GGGGS)n SEQ ID NO: 1
  • GSTGGGS(GGGGS)n are flexible peptide linkers commonly used in protein engineering.
  • Panel C of FIG. 2B illustrates a bispecific antibody with optional mutations (HC DD/HC KK) in the heavy chain Fc regions leading to electrostatic steering effects consisting of two FC-scfv moieties, one targeting Target 1 and the other targeting Target 2. Both scfvs are linked to the N-termini of the heavy chains.
  • Panel D of FIG. 2B illustrates a bispecific antibody with optional mutations (HC DD/HC KK) in the heavy chain Fc regions leading to electrostatic steering effects consisting of an antibody targeting target 1 and a scfv moiety linked to the N-terminal of one of the heavy chains targeting Target 2.
  • optional K-i-H mutations are not shown, they might be present in any of the structures illustrated.
  • FIGs. 2A and 2B disclose “(GGGGS)n” as SEQ ID NO: 220 and “GSTGGGS(GGGGS)nS” as SEQ ID NO: 221.
  • composition/method/kit By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim.
  • CD47 is used herein to refer to Cluster Differentiation 47 protein, also known as integrin associated protein (IAP), including all native human and non-human CD47 proteins. Specifically included within the definition are the full length native human CD47 (NP_001768) and the full length native Macaca mulatta leukocyte surface antigen CD47 (NP_001253446.1) sequences, CD47 of other non-human mammalian species, as well as all naturally occurring variants, such as alternatively spliced isoforms, of such proteins.
  • IAP integrin associated protein
  • MDR1 MDR1
  • ABSCB1 full length native human ABCB1 protein
  • Pgp full length native Macaca mulatta ABCB1
  • KPB1 KPB1
  • tumor-associated antigen or “TAA” is used herein in the broadest sense and includes tumor-specific antigens (TSA) that are exclusively expressed by tumor cells and antigens that are preferentially expressed by tumor cells but are also found in normal cells.
  • TSA tumor-specific antigens
  • antibody and “immunoglobulin” and their grammatical variants include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fc fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, including antibodies comprising only heavy chains (e.g. VHH camelid antibodies), bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like.
  • An antibody may be monovalent or bivalent.
  • the antibodies used herein may be used to assay expression of a target antigens(s) on a cell surface, e.g., in a cell sample or a tissue sample from a patient.
  • the term “antibody” encompasses, but is not limited to, a tetramer of two heavy and two light chains, wherein the heavy and light chains are interconnected by, for example, disulphide bonds.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable regions of the heavy and light chains comprise binding regions that interact with antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system.
  • the term “antibody” includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof.
  • a subject antibody is an IgG isotype, i.e., IgGl, IgG2, IgG3, or IgG4, e.g., IgGl.
  • immunoglobulin refers to a protein including one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes; and numerous immunoglobulin variable region genes.
  • Full- length immunoglobulin light chains (about 25 kD or 214 amino acids) are encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus.
  • Full-length immunoglobulin heavy chains (about 50 kD or 446 amino acids) are encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other aforementioned constant region genes at the C-terminus, e.g., gamma (encoding about 330 amino acids).
  • a subject antibody comprises a whole immunoglobulin comprising full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.
  • multispecific antibody is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity.
  • Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH-VL unit has polyepitopic specificity, antibodies having two or more VL and VH domains with each VH-VL unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently.
  • Polyepitopic specificity refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). “Monospecific” refers to the ability to bind only one epitope. The term “multispecific” specifically includes “bispecific.”
  • the multi-specific antibodies are bispecific, such as bispecific IgG, e.g., bispecific IgGl or IgG4, especially IgGl antibodies.
  • bispecific antibody formats are illustrated in FIG. 1 and FIGs. 2A and 2B. These figures are for illustration purposes only and are not intended to limit the scope of possible bispecific antibody structures encompassed herein.
  • the IgG (e.g., IgGl) constant region additionally includes a KKDD enhancing heterodimer formation through electrostatic steering effects.
  • the human IgGl heavy chain constant region present in such bispecific antibodies may include additional mutations, e.g., substitutions to modulate Fc function.
  • the LALAPG effector function mutations L234A, L235A, and P329G
  • the N297A mutation may be introduced to reduce antibody dependent cellular cytotoxicity (ADCC).
  • the numbering of the substitutions is based on the EU numbering system.
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • the bispecific anti-CD47 antibodies herein may include knob-into-hole (K-i-H) mutations, to improve heterodimer formation and/or other properties of the bispecific antibodies.
  • binding fragment refers to one or more fragments of a full-length antibody that are capable of specifically binding to an antigen.
  • binding fragments include, without limitation, (i) a Fab fragment (a monovalent fragment including, e.g., consisting of, the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment (including, e.g., consisting of, the VH and CHI domains); (iv) a Fv fragment (including, e.g., consisting of, the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (including, e.g., consisting of, the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (including, e
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “human consensus framework” is a framework (FR) which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin variable light chain (VL) or variable heavy chain (VH) framework sequences.
  • VL variable light chain
  • VH variable heavy chain
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup III as in Kabat et al., supra.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human frameworks (FRs). At least a portion of a humanized antibody constant region is derived from a human antibody.
  • the constant regions are from a human IgG antibody, such as, human IgGl, IgG2, IgG3, or IgG4, such as human IgGl or IgG4, preferably human IgGl .
  • the antibodies herein comprise the human IgGl constant region having the amino acid sequence set forth in UniProt: P01857-1, version 1.
  • the human light chain constant region is a human kappa light chain constant region.
  • a “humanized form” of an antibody e.g., a non-human antibody, refers to an antibody that has undergone humanization.
  • epitope refers to a region of an antigen that is recognized by the immune system, for example by antibodies, B cells, or T cells.
  • the epitope is the specific region of the antigen to which an antibody binds.
  • An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. In some instances, isolated antibody will be prepared by at least one purification step.
  • the anti-CD47 antibodies herein are generally purified to substantial homogeneity.
  • the terms “substantial homogeneity,” “substantially homogeneous,” and “substantially homogeneous form” are used to indicate that the product is substantially devoid of by-products originated from undesired polypeptide combinations (e.g., homodimers or homo-multimers). Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2% or 1% by weight or is less than 1% by weight. In one embodiment, the by-product is below 5% by weight.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules, including antibodies comprising only heavy chains (e.g. VHH camelid antibodies); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • ‘Fv” is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen and can form the antigen binding site, although at a lower affinity than the entire binding site comprising the three CDRs of each variable domain.
  • the “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Single-chain Fv Single-chain Fv
  • sFv single-chain Fv
  • scFv single-chain Fv
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • Fc region generally refers to a dimer complex comprising the C- terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody.
  • the Fc region may comprise native or variant Fc sequences.
  • the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl terminus of the Fc sequence.
  • Fc polypeptide herein is meant one of the polypeptides that make up an Fc region, e.g., a monomeric Fc.
  • An Fc polypeptide may be obtained from any suitable immunoglobulin, such as IgGl, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
  • the effector functions of antibodies are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fc receptors FcR
  • an Fc polypeptide comprises part or all of a wild-type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence.
  • a “native sequence Fc region” or “wild-type Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • the anti-CD47 antibody herein comprises an IgG Fc region, preferably derived from a wild-type human IgGl Fc region.
  • wild-type human IgG Fc it is meant a sequence of amino acids that occurs naturally within the human population. The Fc sequence may vary slightly between individuals and such variations are still included within the definition of “wild-type” human IgG Fc.
  • the Fc region may contain additional alterations that are not related to the present invention, such as a mutation in a glycosylation site or inclusion of an unnatural amino acid.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology therewith.
  • the term “CH2 domain” of a human IgG Fc region usually extends from about residues 231 to about 340 of the IgG according to the EU numbering system.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Molec. Immunol. 22: 161-206 (1985)).
  • Fc-region-comprising antibody refers to an antibody that comprises an Fc region.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody.
  • an antibody having an Fc region in accordance with embodiments of the invention can comprise an antibody with or without K447.
  • K-i-H The “knob-into-hole” or “K-i-H” technology directs the pairing of two polypeptides in vitro or in vivo by introducing a pertuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact.
  • K-i-Hs have been introduced in the Fc:Fc binding interfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see, e.g., Zhu et al., 1997 Protein Science 6:781-788; WO 96/027011, and WO 98/050431).
  • Multi-specific antibodies having K-i-H modifications in their Fc regions can further comprise single variable domains linked to each Fc region or can further comprise different heavy chain variable domains that pair with similar or different light chain variable domains.
  • knob, hole or knob/hole wild-type is meant to refer to the protein sequence without artificially introduced mutations, but otherwise to include all sequences that occur naturally within the human population.
  • a “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the hetero-multimer, and thereby favor hetero-multimer formation over homo-multimer formation, for example.
  • the protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance.
  • the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue.
  • the upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.
  • the preferred import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine.
  • the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.
  • a “cavity” refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide.
  • the cavity may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue.
  • the upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide.
  • the preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine.
  • the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.
  • the protuberance or cavity can be “introduced” into the interface of a first or second polypeptide by synthetic means, e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these and other techniques known in the art.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • effector functions include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • VH-VL polypeptide chain
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).
  • Kd dissociation constant
  • Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5 -fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60- fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
  • Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0. 1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
  • nM nanomolar
  • pM picomolar
  • fM femtomolar
  • the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • the terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • a MDRl-specific antibody binds specifically to an epitope within a MDR1 polypeptide.
  • Non-specific binding would refer to binding with an affinity of less than about IO -7 M, e.g., binding with an affinity of 10’ 6 M, 10’ 5 M, 10’ 4 M, etc.
  • CDR complementarity determining region
  • CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol.
  • CDR means a complementary determining region of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999).
  • Framework Region or “FR” residues are those variable domain residues other than the hypervariable region/CDR residues as herein defined. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein.
  • the amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison.
  • variable region when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody.
  • a variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs.
  • framework region is intended to mean each domain of the framework that is separated by the CDRs.
  • a VH chain can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a VL chain can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • chimeric antigen receptor or “CAR” is used herein in the broadest sense to refer to an engineered receptor, which grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to membrane -spanning and intracellular-signaling domains.
  • a desired binding specificity e.g., the antigen-binding region of a monoclonal antibody or other ligand
  • the receptor is used to graft the specificity of a monoclonal antibody onto a T-cell to create a chimeric antigen receptors (CAR).
  • CAR-T cells are T-cells that have been genetically engineered to produce an artificial T-cell receptor for use in immunotherapy.
  • “CAR-T cell” means a therapeutic T-cell expressing a transgene encoding one or more chimeric antigen receptors comprised minimally of an extracellular domain, a transmembrane domain, and at least one cytosolic domain.
  • one or more portions of an antibody described herein e.g., a VH region sequence
  • a VH region sequence of an antibody described herein is used as part of a CAR-T structure, e.g., as part of a targeting domain of the CAR-T structure .
  • aspects of the invention include one or more antibody sequences, as described herein, which are in a CAR-T format, for use as one or more binding domains that provide antigen specificity to a CAR-T cell.
  • antibody-encoding nucleic acid molecule refers to a nucleic acid molecule, e.g., an mRNA molecule or a DNA molecule, that encodes one or more polypeptide subunits of an antibody, and which can be delivered to a cell and expressed and/or transcribed to facilitate production of the encoded antibody.
  • a nucleic acid molecule e.g., an mRNA molecule or a DNA molecule
  • Such techniques are described, for example, in Van Hoecke, L., Roose, K. How mRNA therapeutics are entering the monoclonal antibody field. J Transl Med 17, 54 (2019). https://doi.org/10.1186/sl2967-019-1804-8 and EP2101823B1, the disclosures of which are incorporated herein by reference in their entireties.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • a “chemotherapeutic agent,” also referred to an “antineoplastic agent,” can be a cytotoxic agent which is used for treating a cancer or other disease or disorder.
  • cancer and “cancerous” refer to a physiological condition in mammals, including humans, that is typically characterized by unregulated cell growth/proliferation. Included in this definition are benign and malignant cancers. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer (hepatocellular carcinoma; HCC), gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cell carcinoma), liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, and various types of head and neck cancer.
  • hepatocellular cancer hepatocellular carcinoma
  • gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer,
  • cancer specifically includes hematological malignancies and solid tumors, such as, for example, acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), myelodysplastic syndromes, multiple myeloma (MM), chronic myeloid leukemia (CML), gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, bladder cancer and brain tumors.
  • AML acute myeloid leukemia
  • NHL non-Hodgkin’s lymphoma
  • MM multiple myeloma
  • CML chronic myeloid leukemia
  • gastric cancer non-small cell lung cancer
  • NSCC non-small cell lung cancer
  • “early -stage cancer” is meant a cancer that is not invasive or metastatic or is classified as a
  • precancerous refers to a condition or a growth that typically precedes or develops into a cancer.
  • non-metastatic is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or into tissues other than the primary site.
  • a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, including in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
  • a “therapeutically effective amount” or “efficacious amount” refers to the amount of a targetspecific antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the antibody, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the therapeutically effective amount of an anti-CD47 antibody herein may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the antibody or antibody fragment may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
  • “Reduce” or “inhibit” can refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of the blood vessels in angiogenic disorders.
  • refractory refers to a disease or condition that does not respond to treatment.
  • refractory cancer refers to cancer that does not respond to treatment.
  • a refractory cancer may be resistant at the beginning of treatment or it may become resistant during treatment. Refractory cancer may also be called resistant cancer.
  • a “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides.
  • the term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • Conservative amino acid substitutions refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.
  • Conservative amino acid substitutions may preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.
  • expression vector or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
  • An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR.
  • a stimulatory molecule e.g., a TCR/CD3 complex or CAR
  • its cognate ligand or tumor antigen in the case of a CAR
  • Stimulation can mediate altered expression of certain molecules.
  • the term “stimulatory molecule,” refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the immune cell signaling pathway.
  • the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an ITAM containing cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP 10, and DAP 12.
  • costimulatory molecule refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are contribute to an efficient immune response.
  • Costimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la/CD18), ICOS (CD278), and 4-1BB (CD137).
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell.
  • immune effector function e.g., in a CAR-T cell
  • examples of immune effector function, e.g., in a CAR-T cell include cytolytic activity and helper activity, including the secretion of cytokines.
  • ‘Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T cells e.g., alpha/beta T cells and gamma/delta T cells
  • B cells natural killer (NK) cells
  • natural killer T (NKT) cells e.g., myeloid-derived phagocytes.
  • Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of proteins from different species or from a consensus sequence based on a plurality of proteins having the same or similar function.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
  • a “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores.
  • a “frozen” formulation is one at a temperature below 0 °C.
  • a “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected time period.
  • Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc.
  • aggregate formation for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection
  • icIEF image capillary isoelectric focusing
  • capillary zone electrophoresis amino-terminal or carboxy-terminal sequence analysis
  • mass spectrometric analysis SDS-PAGE analysis to compare reduced and intact antibody
  • peptide map for example tryp
  • Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc. II. Detailed Description
  • the present invention concerns anti-CD47 antibodies, as well as methods of making and using such antibodies, and compositions comprising such antibodies.
  • CD47 has been shown to be associated with a variety of malignant cancers, including hematological malignancies, such as acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), myelodysplastic syndromes, multiple myeloma (MM), chronic myeloid leukemia (CML), and solid tumors, such as gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, bladder cancer and brain tumors.
  • AML acute myeloid leukemia
  • NHL non-Hodgkin’s lymphoma
  • MM multiple myeloma
  • CML chronic myeloid leukemia
  • solid tumors such as gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, bladder cancer and brain tumors.
  • the present disclosure concerns antibodies comprising an antigen-binding site for CD47.
  • Heavy and light chain variable region sequences and CDRs of such antibodies are shown in the following Table 2.
  • Table 2 From left to right, 1st column: Anti-CD47 antibody name, 2nd column: VH region, 3rd column: HCDR1, 4th column: HCDR2, 5th column: HCDR3, 6th column: VL region, 7th column: LCDR1, 8th column: LCDR2, 9th column: LCDR3.
  • a suitable antibody may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific antibody, or as part of a CAR- T structure.
  • an anti-CD47 antibody herein comprises an antigen-binding site, comprising the heavy or light chain CDRs (CDRHsl-3 and/or CDRLsl-3) of an antibody listed in Table 2.
  • the antibody herein comprises an antigen-binding site, comprising the heavy and light chain CDRs (CDRHsl-3 and CDRLsl-3) of an antibody listed in Table 2.
  • an anti-CD47 antibody herein comprises a heavy or light chain variable region sequence shown in Table 2.
  • an anti-CD47 antibody herein comprises a heavy and light chain variable region sequence shown in Table 2.
  • antigen-binding fragments of such full-length anti- CD47 antibodies such as, without limitation, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, an isolated CDR, a single chain Fv (scFv), heavy chain antibodies and diabodies.
  • the antibody is a full-length antibody or an antigen-binding fragment thereof.
  • a CDR sequence in an anti-CD47 antibody of the invention comprises one or two amino acid substitutions relative to a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequence, or a set of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences in any one of the CDRH and/or CDRL sequences in Table 2.
  • an anti-CD47 antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRHs 1, 2, and 3 (combined) has greater than or equal to eighty- five percent (85%) sequence identity at the amino acid level to the CDRHs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 2, and binds to CD47.
  • VH heavy chain variable domain
  • an anti-CD47 antibody preferably comprises a light chain variable domain (VL) in which the full set of CDRLs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level to the CDRLs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 2, and binds to CD47.
  • VL light chain variable domain
  • an anti-CD47 antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identify, or at least 99% identity to any of the heavy chain variable region sequences Table 2, and binds to CD47.
  • an anti-CD47 antibody comprises a light chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identify, or at least 99% identity to any of the light chain variable region sequences Table 2, and binds to CD47.
  • the anti-CD47 antibodies disclosed herein may be of any isotype, including IgA, IgG, IgE, IgD, IgM and subtypes thereof.
  • a subject antibody is an IgG isotype, i.e., IgGl, IgG2, IgG3, IgG4, e.g., IgGl or IgG4.
  • the subject anti-CD47 antibodies may have a human consensus framework (FR).
  • FR human consensus framework
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup III as in Kabat et al., supra.
  • the CD47-binding domain of the subject antibodies exhibits high affinity binding to CD47.
  • a subject CD47-binding domain binds to CD47 with an affinity of at least about IO -7 M, at least about IO -8 M, at least about 10’ 9 M, at least about 10’ 10 M, at least about I0’ 11 M, or at least about 10’ 12 M, or greater than 10’ 12 M.
  • a subject CD47-binding domain binds to an epitope present on CD47 with an affinity of from about 10’ 7 M to about 10’ 8 M, from about 10’ 8 M to about IO -9 M, from about IO -9 M to about IO -10 M, from about IO -10 M to about IO’ 11 M, or from about 10 11 M to about IO -12 M, or greater than 10 12 M.
  • a subject CD47-binding domain exhibits substantially no binding to any epitopes formed by amino acids within other related, but sequence dissimilar, proteins such as related but sequence dissimilar EPs. Any binding of a subject CD47-binding domain to an epitope formed by amino acids within a related, but sequence dissimilar, protein is generally non-specific binding of a substantially lower affinity than the specific binding of the CD47-binding domain to the epitope on CD47.
  • a substantially lower affinity is generally at least a two-fold, three-fold, five-fold, 10-fold, 50-fold, 100- fold, 500-fold, or 1000-fold lower affinity.
  • the anti-CD47 antibodies herein include multi-specific, including bispecific, antibodies, including at least one further binding specificity.
  • an anti-CD47 antibody, or antigen-binding fragment thereof is multispecific (e.g., bispecific) and comprises an antigen-binding site for a tumor antigen other than an ABC transporter.
  • the further binding specificity is directed to another tumor antigen, such as, for example, EGFR, HER2, PD-L1, CD19, CD20, CD38, or CD40.
  • the further binding specificity is directed to another tumor antigen, such as, for example, AXL, LIV1, MET, CD73, CD70, LY6E, CD44.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for EGFR. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for HER2. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen -binding site for CD47 and an antigen-binding site for PD-L1.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD 19.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigenbinding site for CD47 and an antigen-binding site for CD20.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD38.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD40. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for AXL. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen -binding site for CD47 and an antigen-binding site for LIV 1.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigenbinding site for CD47 and an antigen-binding site for MET. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD73. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD70.
  • a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for LY6E. In one embodiment, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof comprises an antigen-binding site for CD47 and an antigen-binding site for CD44.
  • the further binding specificity is directed to an ABC transporter, such as, for example, ABCB1, ABCG2, ABCC1, ABCC2, ABCC3, and/or ABCC4, especially to ABCB1 (MDR1).
  • the multi-specific antibodies comprise an antigen-binding site for ABCB1.
  • Such ABCB1 antigen-binding sites may, for example, be obtained from anti-ABCBl antibodies disclosed in W02020206033 or WO2021155028.
  • the multi-specific antibodies herein comprise an antigen-binding site for ABCG2.
  • Such ABCG2 antigen-binding sites may, for example, be obtained from the anti-ABCG2 antibodies disclosed in WO 2021247426 or WO2021247423.
  • the anti-CD47 antibodies described herein, including multi-specific antibodies, may be chimeric, human or humanized.
  • a chimeric antibody a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, such as a murine (mouse or rat) antibody.
  • a human antibody anti-CD47 antibody possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody- encoding sequences.
  • a humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human frameworks (FRs). At least a portion of a humanized antibody constant region is derived from a human antibody.
  • the two constant regions are from a human IgG antibody, such as, human IgGl.
  • the bispecific antibody molecules disclosed herein include a first and a second heavy chain each comprising a heavy chain variable region as provided herein and a human IgGl constant region having the amino acid sequence set forth in UniProt: P01857-1, version 1.
  • the multi-specific anti-CD47 antibody molecules include a humanized light chain or a light chain comprising a variable light chain region as provided herein and a human light chain constant region.
  • the human light chain constant region is a human kappa light chain constant region.
  • bispecific humanized IgGl antibody molecules that bind CD47 and ABCB 1 and comprise two identical light chain variable regions, a first heavy chain variable region, and a second heavy chain variable region, wherein the light chain variable regions each comprise an antigen-binding site for ABCB 1 , the first heavy chain variable region comprises an antigen-binding site for ABCB1, the second heavy chain variable region comprises an antigen-binding site for CD47, and the second VH chain binds CD47 when paired with one of the VL chains.
  • the bispecific antibody molecules that bind CD47 and an ABC transporter, such as ABCB 1 bind to the individual antigens with lower affinity such that the bispecific antibody does not show significant binding for a cell that does not express both antigens (CD47 and the ABC transporter, e.g., ABCB1) at sufficiently high levels, as present on cancer cells.
  • the human IgG (IgGl) constant region additionally includes a KKDD enhancing heterodimer formation through electrostatic steering effects.
  • the human IgGl heavy chain constant region present in the subject antibodies may include additional mutations, e.g., substitutions to modulate Fc function.
  • the LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody dependent cellular cytotoxicity (ADCC). The numbering of the substitutions is based on the EU numbering system.
  • EU index is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • EU index as in Kabat refers to the residue numbering of the human IgGl EU antibody.
  • the multi-specific anti-CD47 antibody molecules can include knobs-into-hole substitutions in their Fc region.
  • a multi -specific anti-CD47 antibody molecule herein is an IgGl subtype, and one of the Fc regions comprises hole amino acid substitutions Y349C, T366S, L368A, and Y407V in combination with substitutions (i) F405V; (ii) F405V, K409D; (iii) K409D; (iv) D399K; (v) F405V, D399K; or (vi) Q347R, wherein numbering is according to the EU numbering scheme.
  • the other Fc region comprises knob amino acid substitutions (i) S354C, T366W; (ii) S354C, T366W, D399K; (iii) S354C, T366W, K409D; or (iv) S354C, T366W, K360E, wherein numbering is according to the EU numbering scheme.
  • one of the Fc regions comprises a set of amino acid substitutions and amino acid residues selected from the group consisting of:
  • the other Fc region comprises a set of amino acid substitutions and amino acid residues selected from the group consisting of:
  • a multi-specific anti-CD47 IgGl antibody molecule wherein one of the Fc regions comprises the set of amino acid substitutions and amino acid residues Y349C, T366S, L368A, Y407V, E356, E357, F405V, and the other Fc region comprises the set of amino acid substitutions and amino acid residues S354C, T366W, E356, E357, wherein numbering is according to the EU numbering scheme.
  • one of the Fc regions comprises the set of amino acid substitutions and amino acid residues Y349C, T366S, L368A, Y407V, E356, E357, K409D
  • the other Fc region comprises the set of amino acid substitutions and amino acid residues S354C, T366W, E356, E357, D399K, wherein numbering is according to the EU numbering scheme.
  • any of the KbRl, KbR3, KbR5, and KbR7 sets of knob sites can be combined with any of the HIR1-HIR7 sets of hole sites.
  • the bispecific antibodies herein comprise HIRl/KbR3, HIRl/KbR5, HIRl/KbR7, HIR2/KbRl, HIR3/KbR4, HIR4/KbR3, HIR6/KbR5, or HIR7/KbR7 combinations.
  • the bispecific antibodies herein comprise the HIR2/KbRl and HIR4/KbR3 combinations, especially the HIR2/KbRl combination.
  • one IgG (IgGl) heavy chain of the bispecific anti-CD47 antibodies herein contain charge pair substitutions at the CH3 domain, thus one of the heavy chains may contain K392D and/or K409D substitutions and the other E356K and/or D399K substitutions to further facilitate assembly.
  • any of the listed sets of amino acid substitutions can be present in either the heavy chain with binding specificity for ABCB 1 or in the heavy chain binding to the TAA.
  • the “KK” and “DD” substitution respectively can be present either in the ABCB 1 -binding heavy chain or in the heavy chain binding the TAA.
  • any of the KbRl, KbR3, KbR5, and KbR7 sets can be combined with any of the HIR-HIR7 sets
  • the HIRl/KbRl, HIR2/KbRl, HIR4/KbR3, HIR3/KBR4, HIR6/KbR5, and HIR7/KbR7 combinations are preferred.
  • Particularly preferred are the HIR2/KbRl (format 1) and HIR4/KbR3 (format 2) combinations.
  • the Format 1 and Format 2 Fc polypeptide sequences with mutations relative to the wild-type human IgGl Fc reference sequence, are shown below.
  • the mutated residues and, in the wild-type sequence, the native residues participating in interactions facilitating pairing are shown in bold.
  • nucleic acids comprising nucleotide sequences encoding a subject antibody.
  • a nucleotide sequence encoding a subject antibody can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g., a cell that is genetically modified to synthesize and/or secrete the encoded antibody).
  • Suitable promoter and enhancer elements are known in the art.
  • suitable promoters include, but are not limited to, lad, lacZ, T3, T7, gpt, lambda P and trc.
  • suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art- known tissue specific promoters.
  • a nucleotide sequence encoding a subject antibody can be present in an expression vector and/or a cloning vector. Where a subject antibody comprises two or more separate polypeptides, nucleotide sequences encoding the two polypeptides can be cloned in the same or separate vectors. Separate polypeptides may be expressed from a single nucleic acid or single vector using various strategies, such as separate promoters, one or more internal ribosomal entry sites (IRES), one or more self-cleaving sequences (e.g., 2A cleavage sequences, e.g., P2A, T2A, E2A, and F2A), combinations thereof, and the like.
  • An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
  • a selectable marker operative in the expression host may be present.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g.
  • viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno- associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther
  • a retroviral vector e.g., Murine Leukemia Virus, s
  • a subject nucleic acid comprises a nucleotide sequence encoding a subject antibody, including multi-specific, e.g., bispecific antibodies.
  • a subject nucleic acid comprises a nucleotide sequence encoding heavy- and/or light-chain CDRs of a CD47 antibody herein. Such CDRs are set forth in Table 2.
  • the anti-CD47 antibodies herein are multi-specific, comprising at least one further specificity, such as binding specificity to an efflux pump polypeptide.
  • the efflux pump polypeptides include members of the ATP-binding cassette (ABC)-superfamily multidrug effectors pumps (ABC proteins) grouped into seven families ranging from ABCA to ABCG, including, without limitation, ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCG2 (BCRP).
  • ABCB1 P-glycoprotein
  • MRP1 MRP1
  • BCRP2 BCRP
  • the encoding nucleic acid further comprises a nucleic acid encoding CDRs of such efflux pump polypeptides, where the CDR-encoding sequences are interspersed with FR-encoding nucleotide sequences.
  • a subject nucleic acid comprises a nucleotide sequence encoding heavy- and/or light-chain CD47 CDRs, where the CDR- encoding sequences are interspersed with FR-encoding nucleotide sequences.
  • the FR-encoding nucleotide sequences are human FR-encoding nucleotide sequences.
  • Nucleic acids may, in some instances, be introduced into a cell, e.g., by contacting the cell with the nucleic acid.
  • Cells with introduced nucleic acids will generally be referred to herein as genetically modified cells.
  • Various methods of nucleic acid delivery may be employed including but not limited to e.g., naked nucleic acid delivery, viral delivery, chemical transfection, biolistics, and the like.
  • aspects of the invention include compositions (e.g., pharmaceutical compositions) that comprise one or more nucleotide sequences (e.g., mRNA sequences) that encode the amino sequence of an antibody described herein.
  • nucleotide sequences e.g., mRNA sequences
  • aspects of the invention include antibody- encoding nucleic acid molecules that encode one or more polypeptide subunits of an antibody, and which can be delivered to a cell and expressed and/or transcribed to facilitate production of the encoded antibody.
  • Such techniques are described, for example, in Van Hoecke, L., Roose, K. How mRNA therapeutics are entering the monoclonal antibody field. J Transl Med 17, 54 (2019).
  • aspects of the invention also include methods of treatment comprising administering antibody-encoding nucleic molecules to a patient to accomplish delivery of the antibody.
  • the present disclosure provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that are genetically modified with a subject nucleic acid.
  • a subject isolated genetically modified cell can produce a subject antibody.
  • a genetically modified cell can deliver an antibody, e.g., to a subject in need thereof.
  • a genetically modified cell may be used in the production, screening, and/or discovery of multi -specific antibodies.
  • Genetically modified cells may also, in some instances, include cells where endogenous gene expression has been reduced, e.g., inhibited, knocked-down, etc., or abolished, e.g., knocked-out.
  • Genetically modified cells may also, in some instances, include cells where expression of a gene has been enhanced, e.g., the expression of an endogenous gene is increased, or the expression of a heterologous gene is increased.
  • Suitable cells include eukaryotic cells, such as a mammalian cell, an insect cell, a yeast cell; and prokaryotic cells, such as a bacterial cell.
  • Introduction of a subject nucleic acid into the host cell can be effected, for example by calcium phosphate precipitation, DEAE dextran mediated transfection, liposome-mediated transfection, electroporation, or other known method.
  • Suitable mammalian cells include primary cells and immortalized cell lines.
  • Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
  • Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.
  • CCL10 PC 12 cells
  • COS cells COS-7 cells
  • RATI cells mouse L cells
  • HEK cells ATCC No. CRL1573
  • HLHepG2 cells HLHepG2 cells, and the like.
  • useful mammalian cells may include cells derived from a mammalian tissue or organ.
  • cells employed are kidney cells, including e.g., kidney cells of an established kidney cell line, such as HEK 293T cells.
  • Suitable yeast cells or fungi or algae cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia memhranaefaciens , Pichia opuntiae, Pichia thermotolerans , Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp. , Saccharomyces cerevisiae, Saccharomyces sp. , Hansenula polymorphci, Kluyveromyces sp.
  • Kluyveromyces lactis Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like.
  • Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148: 1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302.
  • Salmonella strains which can be employed in the present invention include, but are not limited to, Salmonella typhi and .S', typhimurium.
  • Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae .
  • the laboratory strain is one that is non-pathogenic.
  • suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like.
  • the host cell is Escherichia coli.
  • cells of the present disclosure may be immune cells.
  • immune cells generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.
  • HSC hematopoietic stem cells
  • Immune cells includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • T cell includes all types of immune cells expressing CD3 including T- helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg) and gamma-delta T cells.
  • a “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • useful cells of the present disclosure may be immune cells expressing a multi-specific antibody or chimeric receptor incorporating binding components of the multi-specific antibody of the present disclosure and may be useful as immunotherapeutic agents.
  • useful cells expressing an antibody of the present disclosure may include producer T cells.
  • Nonlimiting examples of producer T cells include those described in Tsai & Davila Oncoimmunology. (2016) 5(5): el 122158; the disclosure of which is incorporated herein by reference in its entirety.
  • Producer T cells engineered to include nucleic acid sequence encoding an anti-CD47 antibody of the present disclosure may, in some instances, be employed to deliver the antibody to a subject in need thereof.
  • Cells of the present disclosure also include cells genetically modified to change and/or amend expression of CD47 and optionally an ABC transporter, such as ABCB1, ABCC1, or ABCG2.
  • an ABC transporter such as ABCB1, ABCC1, or ABCG2.
  • Such modified cells are useful for various purposes including assaying the binding of multi-specific antibodies herein, including but not limited to those produced according to the description and methods provided herein.
  • the ABC transporter such as ABCB1 may be knocked out or knocked down in a subject cell line.
  • CD47 may be knocked out or knocked down in a subject cell line.
  • the ABC transporter may be constitutively or inducibly overexpressed in a subject cell line.
  • CD47 may be constitutively or inducibly overexpressed in a subject cell line. In some instances, both the ABC transporter and CD47 may knocked down, knocked out, or constitutively or inducibly overexpressed in a subject cell line. Any convenient and appropriate method for knockdown, knockout and/or overexpression may be employed. Introduced nucleic acid may be stably integrated or present transiently.
  • cells of the present disclosure include a genetically modified human cell line that expresses CD47 and includes an exogenous nucleic acid comprising a sequence encoding an ABC transporter for overexpression of the ABC transporter.
  • CD47 expression may by endogenous or exogenously derived (i.e., introduced) and ABC transporter expression may be stable or transient.
  • cells lines of the present disclosure, that express CD47 may be configured to produce a genetically modified human cell expressing CD47 and stably overexpressing an ABC transporter, such as such as ABCB1, ABCC1, or ABCG2, preferably ABCB1.
  • Cells and cell lines of the present disclosure may be cultured, including e.g., through use of culture methods described herein.
  • a cell, into which nucleic acid have been introduced to genetically modify the cell may be cultured to produce a cell line.
  • Useful cells lines may include but are not limited to e.g., genetically modified cell lines, including human cell lines, expressing CD47 and optionally stably over-expressing ABCB1.
  • Cells of the present disclosure may be employed in various methods of the disclosure, e.g., as test samples, controls, and the like.
  • cells in which an ABC transporter and/or CD47 have been knocked out and/or knocked down may be employed as reference cells, e.g., to which the binding of a multi-specific antibody of the present disclosure may be compared.
  • Other useful reference cells include but are not limited to e.g., non-cancerous cells, as well as normal cells and cells expressing normal levels of various proteins, including normal levels of ABC transporter and/or CD47.
  • compositions including pharmaceutical compositions, comprising an antibody described herein.
  • present disclosure also provides compositions, including pharmaceutical compositions, comprising an antibody-encoding nucleic acid molecule (e.g., an mRNA molecule) described herein.
  • an antibody-encoding nucleic acid molecule e.g., an mRNA molecule
  • a pharmaceutical composition or formulation comprises an effective amount of the subject antibody an/or antibody-encoding nucleic acid molecule in combination with a pharmaceutically acceptable excipient.
  • An “effective amount” means a dosage sufficient to produce a desired result, e.g., reduction in a cancer of a subject, reduction in the growth rate of a cancer in a subject, amelioration of a symptom of cancer, and the like.
  • the desired result is at least a reduction in a symptom of a cancer, reduction in the growth of a cancer, reduction in the size of a cancer, etc., as compared to a control.
  • the effective amount of antibody present in the formulation is determined by considering the desired dose volumes and mode(s) of administration, for example.
  • the pharmaceutical composition may, for example, be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration.
  • formulations of the subject antibodies may be in a solid form, which present advantages, such as improved stability and increased shelf-life as well as simpler storage and transportation.
  • Such solid formulations may be prepared by a variety of drying technologies, including lyophilization and spray dry manufacturing processes. Solid formulations may be reconstituted prior to use.
  • compositions of the subject antibodies might be in a variety of formulations, including lyophilized powders and liquid, typically aqueous, formulations, which may be concentrated solutions that require dilution prior to administration or ready-to-use solutions suitable for subcutaneous administration.
  • the compositions contain the antibody, an excipient to adjust tonicity or osmolality for solutions or a lyoprotectant for lyophilized powders, a buffer, and a surfactant.
  • the ionic tonicity-adjusting excipient may, for example, be sodium chloride
  • non-ionic osmolalityadjusting excipients include, for example, trehalose, sucrose, mannitol, maltose, and sorbitol.
  • Typical lyoprotectants include trehalose and sucrose.
  • a tonicity agent may be included in the antibody formulation to modulate the tonicity of the formulation.
  • exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • isotonic denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.
  • a surfactant may also be added to the antibody formulation to reduce aggregation of the formulated antibody and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene - polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • Suitable polyoxyethylene sorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • suitable polyethylene- polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188TM.
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
  • Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v.
  • a lyoprotectant may also be added in order to protect the labile active ingredient (e.g., a protein) against destabilizing conditions during lyophilization, if any.
  • known lyoprotectants include sugars (including trehalose, glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid).
  • Typical lyoprotectants are trehalose and sucrose. Lyoprotectants can be included in an amount of about 10 mM to 500 nM.
  • compositions of the subject antibodies include aqueous formulations comprising the antibody in a pH-buffered solution.
  • the buffer used in aqueous pharmaceutical formulations herein has a pH in the range from about 4.8 to about 8.0.
  • the pH is in the range from 5.5 to 7.0, from pH 5.5 to 6.5, in the range from pH 5.7 to 6.8, in the range from pH 5.8 to 6.5, in the range from pH 5.9 to 6.5, in the range from pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5.
  • the formulation has a pH of 6.0 or about 6.0.
  • the buffer contains histidine in the concentration of about 15 mM to about 35 mM.
  • the buffer contains histidine in the concentration of about 20 mM to about 30 mM, about 22 mM to about 28 mM, or about 25 mM.
  • the buffer is histidine in an amount of about 20 mM, pH 6.0.
  • the formulation may contain a phosphate buffer, such as sodium phosphate in the concentration of about 20 mM to about 30 mM, about 22 mM to about 28 mM, or about 25 mM.
  • the buffer is sodium phosphate in an amount of about 25 mM, pH 6.2.
  • a subject parenteral formulation is a liquid or lyophilized formulation which may comprise, for example, about 1 mg/mL to about 200 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose or sucrose; and has a pH of 5.5.
  • a subject parenteral formulation comprises a lyophilized formulation comprising: 1) 15 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 2) 75 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5;or 3) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM Sucrose; and has a pH of 5.5; or 4) 75 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 6) 75 mg/mL of a subject antibody; 0.02%
  • a subject parenteral formulation is a liquid formulation comprising: 1) 7.5 mg/mL of a subject antibody; 0.022% Tween 20 w/v; 120 mM L-histidine; and 250 125 mM sucrose; and has a pH of 5.5; or 2) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of 5.5; or 5) 37.5 mg/mL of a subject antibody; 0.02% Twe
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for a subject antibody may depend on the particular antibody employed and the effect to be achieved, and the pharmacodynamics associated with each antibody in the host.
  • a subject antibody can be administered in conjunction with a pharmaceutically acceptable excipient, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • a subject antibody is formulated in a controlled release formulation.
  • Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in which the matrices are in the form of shaped articles, e.g. fdms or microcapsules.
  • sustained-release matrices examples include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3 -hydroxybutyric acid.
  • Possible loss of biological activity and possible changes in immunogenicity of antibodies comprised in sustained-release preparations may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.
  • Controlled release within the scope of this invention can be taken to mean any one of a number of extended-release dosage forms.
  • the following terms may be considered to be substantially equivalent to controlled release, for the purposes of the present invention: continuous release, controlled release, delayed release, depot, gradual release, long-term release, programmed release, prolonged release, proportionate release, protracted release, repository, retard, slow release, spaced release, sustained release, time coat, timed release, delayed action, extended action, layered-time action, long acting, prolonged action, repeated action, slowing acting, sustained action, sustained-action medications, and extended release. Further discussions of these terms may be found in Lesczek Krowczynski, Extended- Release Dosage Forms, 1987 (CRC Press, Inc.).
  • Intranasal formulations are also included within the scope herein. Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • the subject antibodies can be utilized in aerosol formulation to be administered via inhalation.
  • a subject antibody can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • Oral formulations for administering the subject antibodies are also included. Oral administration is challenging, due to the degradation or orally administered antibodies by proteolytic enzymes, such as pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase.
  • proteolytic enzymes such as pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase.
  • Various approaches used to include stability or orally administered antibodies include formulations in liposomes, coating polymers and genetic engineering of resistant forms.
  • a suitable dosage can be determined by an attending physician or other qualified medical practitioner, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently.
  • a subject antibody may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g., between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g., between 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 pg to 10 mg per kilogram of body weight per minute.
  • the antibodies herein can also be administered by fixed dosing, where the fixed dose and dosing schedule are determined and adjusted depending on the target disease.
  • a subject antibody in the subject methods, can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic effect.
  • a subject antibody is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • the main routes of administration for the antibodies herein incudes intravenous (IV), subcutaneous (SC), intramuscular (IM), and pulmonary administration.
  • IV intravenous
  • SC subcutaneous
  • IM intramuscular
  • pulmonary administration e.g., pulmonary administration.
  • the subject antibodies may, for example, be delivered by intravenous infusion, subcutaneous injection, or by intranasal delivery, but other administration routes, such as oral delivery are also possible.
  • the subject antibodies may be administered by intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibodies are administered by intravenous administration.
  • the formulation may be injected using a syringe or via an IV line, for example.
  • the formulation is administered by subcutaneous administration.
  • the subject antibodies may be administered by intravenous infusion typically in about 30 to 90 minutes, every week, biweekly or every three weeks.
  • the antibodies may be administered subcutaneously, as a fixed dose or body weight-adjusted dose over a shorter period of time, such as 2 to 20 minutes, or 2 to 10 minutes, or 2 to 5 minutes, weekly, once every two weeks or once every three weeks.
  • the advantage of subcutaneous injections is that it allows the medical practitioner to perform it in a rather short intervention with the patient.
  • the patient can be trained to perform the subcutaneous injection by himself. Such self-administration is particularly useful during maintenance dosing because no hospital care is needed (reduced medical resource utilization).
  • injections via the subcutaneous route are limited to approximately 2 ml.
  • several unit dose formulations can be injected at multiple sites of the body surface.
  • the subject antibodies can be co-administered with other therapeutic agents, such as, for example, chemotherapeutic agents.
  • co-administering is meant administering two (or more) drugs during the same administration, rather than sequential administration of the two or more drugs. In the case of intravenous administration, this will generally involve combining the two (or more) drugs into the same IV bag prior to co-administration but co-administration from different, separate formulations is also included.
  • the antibody and one or more additionally drugs may also be administered concurrently.
  • a drug that is administered “concurrently” with one or more other drugs is administered during the same treatment cycle, on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs.
  • the concurrently administered drugs are each administered on day-1 of a 3 -week cycle.
  • Methods of the present disclosure include methods of contacting a cell with an antibody of the present disclosure, methods of treating a subject according to a method that involves administering to the subject an antibody of the present disclosure, methods of making elements described in the instant application, including, e.g., antibodies, compositions and formulations, nucleic acids, expression vectors, cells, and the like.
  • methods of the present disclosure include contacting a cancer cell with an antibody of the present disclosure, e.g., to facilitate and/or enhance killing of the cancer cell.
  • an antibody of the present disclosure e.g., to facilitate and/or enhance killing of the cancer cell.
  • multi-specific, e.g., bispecific antibodies killing of the cancer cell may be mediated by an immune response or immune cell acting upon the cancer cell as a result of opsonization of the cancer cell by bispecific targeting when the two targets are co-expressed on the cancer cell.
  • killing of the cancer cell is mediated by an immune response or immune cell acting upon the cancer cell, e.g., as a result of masking or antagonizing of a CD47 epitope present on the surface of the cancer cell by the multi-specific antibody.
  • contacting a cancer cell with an antibody of the present disclosure will generally enhance the killing of the cancer cell, e.g., as compared to the level of killing of the cancer cell in the absence of the antibody.
  • enhanced killing of the cancer cell may be seen as compared to the level of killing observed using the additional active agent alone.
  • the amount of enhancement of cancer cell killing attributable to an antibody of the present invention will vary and may range from at least a 5% increase in cancer cell killing to at least 90% or more, including but not limited to e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, etc. Such increases may be compared to contacting with one or more additional active agents alone.
  • Enhanced killing of a cancer cell may be assessed by a variety of means including but not limited to e.g., observational studies, in vitro cell-based cytotoxicity assays, flow cytometry, cell viability labeling (e.g., using one or more cell viability stains), and the like.
  • the present disclosure provides methods of treating a condition benefiting from the inhibition of the CD47 pathway.
  • methods for the treatment of oncological conditions associated with the CD47 pathway including, without limitation, acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), myelodysplastic syndromes, multiple myeloma (MM), chronic myeloid leukemia (CML), hepatocellular carcinoma (HCC), gastric cancer, non-small cell lung cancer (NSCC), ovarian cancer, breast cancer, head and neck cancer, bladder cancer and brain tumors.
  • AML acute myeloid leukemia
  • NHL non-Hodgkin’s lymphoma
  • MM multiple myeloma
  • CML chronic myeloid leukemia
  • HCC hepatocellular carcinoma
  • gastric cancer non-small cell lung cancer
  • NSCC non-small cell lung cancer
  • ovarian cancer breast cancer, head and neck cancer
  • breast cancer breast cancer
  • head and neck cancer bladder cancer and brain tumor
  • killing of the cancer cell is mediated by inhibition of cellular efflux of the cancer cell, e.g., as a result of ABC transporter, e.g., ABCB1 antagonism on the cancer cell by the multi-specific antibody.
  • the cell contacted with such multi-specific antibody with binding specificity to an ABC transporter, such as ABCB1 may be a multidrug resistant cancer cell.
  • the target condition e.g., malignant cancer is characterized by overexpression of CD47 and another tumor antigen, such as EGFR, HER2, PD-L1, CD 19, CD20, CD38, or CD40.
  • the target condition e.g., malignant cancer is characterized by overexpression of CD47 and another tumor antigen, such as AXL, LIV1, MET, CD73, CD70, LY6E, CD44.
  • Treatment of such cancers may involve multi-specific, e.g., bispecific, antibodies, with binding specificity for one of such tumor antigens.
  • Treatment methods generally involve administering to an individual in need thereof (e.g., an individual having a cancer) an effective amount of a subject anti-CD47 antibody, alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents.
  • Administration of an antibody of the present disclosure may be performed by any convenient and appropriate route of delivery.
  • aspects of the present disclosure include the antibody molecule according to the preceding section of the specification for use in a method of treating cancer in a subject, the method comprising administering the antibody to the subject.
  • the method may comprise administering the antibody in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.
  • the at least one additional active agent is a chemotherapeutic agent, optionally wherein the chemotherapeutic agent is a taxane (e.g., TAXOL®), a vinca alkaloid, or an anthracycline.
  • the chemotherapeutic agents include Paclitaxel, Colchicine, Verapamil, Vinblastine, Topotecan, Doxorubicin, Daunorubicin, Etoposide, and Nilotinib.
  • Also disclosed herein is a chemotherapy agent for use in a method of treating cancer in a subject, the method comprising administering the chemotherapy agent in combination with the antibody described herein to the subject.
  • Administration includes but is not limited to e.g., delivery of the antibody by injection, delivery of the antibody by infusion, delivery of a nucleic acid or expression vector encoding the antibody, delivery of the antibody by administering to the subject a cell that expresses and secretes the multispecific antibody, and the like.
  • Administration of an agent, a nucleic acid encoding an agent, a cell expressing an agent, etc. may include contacting with the agent, contacting with the nucleic acid, contacting with the cell, etc.
  • an effective amount of a subject multi-specific antibody is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to reduce an adverse symptom of cancer by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the severity of the adverse symptom in the absence of treatment with the antibody.
  • an effective amount of a subject multi-specific antibody is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to improve the cancer (i.e., slow the growth of the cancer, stop the growth of the cancer, reverse the growth of the cancer, kill cancer cells (including tumor cells, or the like) in the individual being treated.
  • an effective amount of a subject antibody can reduce a cancer growth rate or reduce a cancer size in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more, compared to in the absence of treatment with the multi-specific antibody.
  • a subject may be treated systemically, including with the subject multispecific antibody, with or without one or more additional reagents.
  • systemic treatment is meant a treatment that is not directed solely to target a specific tumor (such as e.g., a primary tumor or a defined secondary tumor) or a specific cancer containing tissue (such as e.g., the liver in the case of liver cancer, the blood in the case of a blood cancer, etc.).
  • Systemic treatments will generally be directed to the subject’s body as a whole and may include but are not limited to e.g., systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof and the like.
  • a subject may be treated locally, including with the subject multi -specific antibody, with or without one or more additional reagents.
  • local treatment is meant a treatment that is specifically directed to the location of a tumor (such as e.g., a primary tumor or a defined secondary tumor) or specifically directed to a cancer containing tissue (such as e.g., the liver in the case of liver cancer, the blood in the case of a blood cancer, etc.).
  • local treatment may also be administered in such a way as to affect the environment surrounding a tumor, such as tissue surrounding the tumor, such as tissue immediately adjacent to the tumor.
  • Local treatment will generally not affect or not be targeted to tissues distant from the site of cancer including the site of a tumor, such as a primary tumor.
  • Useful local treatments that may be administered in addition to or in combination with a subject multi-specific antibody, e.g., include but are not limited to surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, and the like.
  • a subject treatment method involves administering a subject multispecific antibody and one or more additional therapeutic agents.
  • additional therapeutic agents include, but are not limited to, chemotherapeutic agents, radiation therapy reagents, immunotherapy reagents, other antibody or multi-specific antibody agents, and the like.
  • Additional therapies that may be administered to a subject before, during or after a subject administering a multi -specific antibody of the present disclosure will vary depending on numerous factors including e.g., the type of cancer, the subject’s medical history, general state of health and/or any co-morbidities, and the like.
  • Useful cancer therapies include but are not limited to e.g., radiation therapy, chemotherapy, immunotherapy, and the like.
  • Radiation therapy includes, but is not limited to, x-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.
  • Suitable antibodies for use in cancer treatment include, but are not limited to, naked antibodies, e.g., trastuzumab (Herceptin), bevacizumab (AvastinTM), cetuximab (ErbituxTM), panitumumab (VectibixTM), Ipilimumab (Y ervoyTM), rituximab (Rituxan), alemtuzumab (LemtradaTM), Ofatumumab (ArzerraTM), Oregovomab (OvaRexTM), Lambrolizumab (MK-3475), pertuzumab (PerjetaTM), ranibizumab (LucentisTM) etc., and conjugated antibodies, e.g., gemtuzumab ozogamicin (MylortargTM), Brentuximab vedotin (AdcetrisTM), 90Y-labelled ibritumomab tiuxetan (Z
  • Suitable antibodies for use in cancer treatment also include, but are not limited to, antibodies raised against tumor-associated antigens.
  • antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, Mucins, TAG-72, CAIX, PSMA, Folate-binding protein, Gangliosides (e.g., GD2, GD3, GM2, etc.), Le y , VEGF, VEGFR, Integrin alpha-V-beta-3, Integrin alpha-5 -beta- 1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, Tenascin, etc.
  • Conventional cancer therapies also include targeted therapies for cancer including but not limited to e.g., Ado-trastuzumab emtansine (Kadcyla) targeting HER2 (ERBB2/neu) (approved for use in Breast cancer); Afatinib (Gilotrif) targeting EGFR (HER1/ERBB1), HER2 (ERBB2/neu) (approved for use in Non-small cell lung cancer); Aldesleukin (Proleukin) targeting (approved for use in Renal cell carcinoma, Melanoma); Alectinib (Alecensa) targeting ALK (approved for use in Non-small cell lung cancer); Alemtuzumab (Campath) targeting CD52 (approved for use in B-cell chronic lymphocytic leukemia); Atezolizumab (Tecentriq) targeting PD-L1 (approved for use in Urothelial carcinoma, Non- small cell lung cancer); Avelumab (Bavencio) targeting PD-L1 (approved for use in U
  • Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; ( 4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon- a.; (7) interferon -y; (8) colony-stimulating factors; (9) inhibitors of angiogenesis; (10) poly ADP ribose polymerase (PARP) inhibitors and (11) antagonists of tumor necrosis factor
  • RTK tyrosine kinase
  • PARP poly ADP ribose polymerase
  • Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.
  • Agents that act to reduce cellular proliferation are known in the art and widely used.
  • Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl- CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazen
  • Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5 -fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
  • CYTOSAR-U cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g.
  • anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
  • phenoxizone biscyclopeptides e.g. dactinomycin
  • basic glycopeptides e.g.
  • cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafme.
  • Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
  • Hormone modulators and steroids include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.
  • adrenocorticosteroids e.g. prednisone, dexamethasone, etc.
  • estrogens and pregestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.
  • adrenocortical suppressants e.g.
  • estradiosteroids may inhibit T cell proliferation.
  • chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
  • metal complexes e.g., cisplatin (cis-DDP), carboplatin, etc.
  • ureas e.g. hydroxyurea
  • hydrazines e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • procarbazine e.g. mitoxantrone
  • leucovorin tega
  • mycophenolic acid mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.
  • Taxanes include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel”
  • Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TaxotereTM docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or paclitaxel-albumin). Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives and derivatives bound to proteins e.g. Abraxane described in U.S. Patent No. 7,820,788.
  • taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701.
  • Useful immunotherapies include: anti-PD-l/PD-Ul immunotherapies, and/or other immunotherapy targets, such as e.g., immune check point markers, such as CTUA-4, UAG-3 and TIM- 3, that may be targeted in treatment methods.
  • immunotherapy targets such as e.g., immune check point markers, such as CTUA-4, UAG-3 and TIM- 3, that may be targeted in treatment methods.
  • Anti-PD-l/PD-Ul immunotherapies which include but are not limited to e.g., those therapies that include administering to a subject an effective amount of one or more anti-PD-l/PD-Ul therapeutic antagonists where such antagonists include but are not limited to e.g., OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), TecentriqTM (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS- 202, BMS-8, BMS-37, BMS-242 and the like.
  • OPDIVO® nivolumab
  • KEYTRUDA® pembrolizumab
  • TecentriqTM atezolizumab
  • durvalumab avelumab
  • BMS-936559 MDX-1105
  • CA-170 BMS-
  • CTLA-4 also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 have been approved for treating some cancer types. The co-inhibitory effect of CTLA-4 with other immunotherapies make CTLA-4 a good candidate for use in combination with other immunotherapies to treat certain cancers. TIM-3 may also be targeted for immunotherapy for several cancer types.
  • LAG-3 is in clinical trials for treating cancers.
  • Anti -LAG-3 immunotherapies include those that employ antagonist LAG-3 antibodies that can both activate T effector cells (by downregulating the LAG-3 inhibiting signal into pre-activated LAG-3+ cells) and inhibit induced (i.e. antigen-specific) Treg suppressive activity.
  • Useful LAG-3 antagonistic antibodies include relatlimab (BMS-986016; developed by Bristol-Myers Squibb), IMP701 (developed by Immutep), TSR-033 (anti-LAG-3 mAb; developed by TESARO, Inc.), and the like.
  • Immunotherapies also include T cell -based immunotherapies such as e.g., adoptive cell therapy
  • a subject may be administered a population of CAR T cells engineered to target an antigen expressed by the subject’s cancer.
  • a T cell-based therapy may involve, in some instances, obtaining a cellular sample from a subject, such as a blood sample or a tumor biopsy, and culturing immune cells from the sample ex vivo, with or without genetic modification of the cultured immune cells.
  • immune cells may be obtained from a subject, cultured ex vivo and modified with a CAR specific for an antigen expressed by the cancer to produce a population of CAR T cells. Then, the CAR T cells may be reintroduced into the subject to target the cancer.
  • T cell-based immunotherapies may be configured in various ways, e.g., by targeting various antigens, by collecting/culturing various cell types, etc., depending on a particular cancer to be treated.
  • T cell-based immunotherapies may be administered systemically, e.g., by intravenous injection, or locally, e.g., by infusion (e.g., intraperitoneal infusion, pleural catheter infusion, etc.), direct injection, and the like.
  • aspects of the present disclosure include an anti-CD47 antibody, or an antigen-binding fragment thereof, as described herein, for use in a method of treating cancer in a subject, the method comprising administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with at least one additional immunotherapy agent.
  • the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with an immunotherapy agent that is a PD-1 antagonist.
  • the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with an immunotherapy agent that is a PD-L1 antagonist.
  • the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with OPDIVO® (nivolumab). In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with KEYTRUDA® (pembrolizumab). In one embodiment, the methods involve administering the anti- CD47 antibody or antigen-binding fragment thereof to the subject in combination with TECENTRIQTM (atezolizumab). In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with durvalumab (MEDI4736).
  • the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with avelumab (MSB0010718C). In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with BMS-936559 (MDX-1105). In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with CA-170. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with BMS-202. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with BMS-8.
  • the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with BMS-37. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with BMS-242.
  • the methods involve administering the anti-CD47 antibody or antigenbinding fragment thereof to the subject in combination with an immunotherapy agent that binds to an immune check point marker. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with an immunotherapy agent that binds to CTLA-4. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with an immunotherapy agent that binds to LAG-3. In one embodiment, the methods involve administering the anti-CD47 antibody or antigen-binding fragment thereof to the subject in combination with an immunotherapy agent that binds to TIM-3.
  • a method of treatment described herein may include administering to a subject one or more inhibitors of a multidrug resistance transporter, including but not limited to, e.g., a multidrug resistance transporter other than MDR1.
  • a multidrug resistance transporter other than MDR1.
  • Useful inhibitors of multidrug resistance transporters include, e.g., tyrosine kinase inhibitors, natural products, microRNAs, and small molecule inhibitors.
  • Inhibitors of multidrug resistance transporters include ABC transporter inhibitors. A summary of such MDR modulators or reverters is provided in Choi (2005), Cancer Cell Int, 5:30; the disclosure of which is incorporated by reference herein in its entirety.
  • Individuals suitable for treatment using a method of the present disclosure include an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.); an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who has failed to respond to the treatment; an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who initially responded to the treatment but who subsequently relapsed, i.e., the cancer recurred.
  • the methods of the present disclosure may be employed to target and treat a variety of cancers, including e.g., primary cancer, secondary cancers, re-growing cancers, recurrent cancers, refractory cancers and the like.
  • the methods of the present disclosure may be employed as an initial treatment of a primary cancer identified in a subject.
  • the methods of the present disclosure may be employed as a non-primary (e.g., secondary or later) treatment, e.g., in a subject with a cancer that is refractory to a prior treatment, in a subject with a cancer that is re-growing following a prior treatment, in a subject with a mixed response to a prior treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least a second tumor in the subject), and the like.
  • a non-primary (e.g., secondary or later) treatment e.g., in a subject with a cancer that is refractory to a prior treatment, in a subject with a cancer that is re-growing following a prior treatment, in a subject with a mixed response to a prior treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least a second tumor in the subject), and the like.
  • the methods of the present disclosure may be employed to treat a subject with a drug resistant cancer, such as a multi -drug resistant cancer.
  • Multidrug resistance is the mechanism by which many cancers develop resistance to chemotherapy drugs, resulting in minimal cell death and the expansion of drug-resistant tumors.
  • MDR cancers may involve one or more resistance mechanisms including but not limited to e.g., increased expression of efflux pumps, decreased absorption of drug, inhibition of cell death or apoptosis, modulating drug metabolism, and the like.
  • the methods of the present disclosure may prevent, reverse or circumvent MDR.
  • methods of the present disclosure may include treating a subject having a cancer that is resistant to a first agent with an effective amount of a subject multi-specific antibody described herein in combination with a second agent that is different from the first agent.
  • cancer of a subject may be resistant to a first chemotherapeutic and the subject may be treated by administering an effective amount of a subject multi-specific antibody as described herein in combination with a second chemotherapeutic that is different from the first.
  • first and second chemotherapeutics may be employed depending on e.g., the type of cancer to be treated, the likelihood of developing resistance, etc.
  • Acute Lymphoblastic Leukemia ALL
  • Acute Myeloid Leukemia AML
  • Adrenocortical Carcinoma AIDS-Related Cancers
  • Anal Cancer Appendix Cancer
  • Astrocytomas Atypical Teratoid/Rhabdoid Tumor
  • Basal Cell Carcinoma Basal Cell Carcinoma
  • Bile Duct Cancer Extrahepatic
  • Bladder Cancer Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniophary
  • the methods of treating described herein may, in some instances, be performed in a subject that has previously undergone one or more conventional treatments.
  • the methods described herein may, in some instances, be performed following a conventional cancer therapy including but not limited to e.g., conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc.
  • the methods described herein may be used when a subject has not responded to or is refractory to a conventional therapy.
  • the methods described herein may be used when a subject has responded to a conventional therapy.
  • the method of the present disclosure may be employed to target, treat or clear a subject for minimal residual disease (MRD) remaining after a prior cancer therapy.
  • MRD minimal residual disease
  • Targeting, treating and/or clearance of MRD may be pursued using the instant methods whether the MRD is or has been determined to be refractory to the prior treatment or not.
  • a method of the present disclosure may be employed to target, treat and/or clear a subject of MRD following a determination that the MRD is refractory to a prior treatment or one or more available treatment options other than those employing the herein described multi -specific antibodies.
  • the instant methods may be employed prophylactically for surveillance.
  • a subject in need thereof may be administered a treatment involving one or more of the herein described multi-specific antibodies when the subject does not have detectable disease but is at risk of developing a recurrent cancer, including e.g., a drug resistant cancer.
  • a prophylactic approach may be employed when a subject is at particularly high risk of developing a primary cancer that would be predicted to be drug resistant or expected to become drug resistant.
  • a prophylactic approach may be employed when a subject has been previously treated for a cancer and is at risk of reoccurrence or development of drug resistance.
  • methods of the present disclosure may involve analyzing a cancer for expression of one or more markers or therapeutic targets.
  • methods may involve analyzing a sample of a cancer from a subject to determine whetherthe cancer expresses MDR1 above a predetermined threshold, a TAA (e.g., CD47, PD-L1, or EGFR) above a predetermined threshold, or both.
  • a predetermined threshold e.g., CD47, PD-L1, or EGFR
  • whether a subject is treated with a multi-specific antibody of the present disclosure may depend on the results of the TAA and/or MDR1 testing. For example, in some instances, if a cancer expresses the TAA at or above a predetermined threshold then the subject may be treated with a multi-specific antibody of the present disclosure, and if the cancer expresses the TAA below the predetermined threshold then the subject may not be treated with the multi-specific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multispecific antibody.
  • a cancer expresses MDR1 at or above a predetermined threshold then the subject may be treated with a multi-specific antibody of the present disclosure, and if the cancer expresses MDR1 below the predetermined threshold then the subject may not be treated with the multispecific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multi-specific antibody.
  • the subject may be treated with a multi -specific antibody of the present disclosure, and if the cancer expresses the TAA and MDR1 below the predetermined thresholds then the subject may not be treated with the multi -specific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multispecific antibody.
  • Any convenient assay may be employed for analyzing MDR1 and/or TAA levels, including but not limited to e.g., flow cytometry, nucleic acid-based assays (e.g., amplification, sequencing, etc.), cell cytometry, immunohistochemistry, and the like.
  • Any convenient biological sample may be employed, including but not limited to e.g., cancer biopsy samples.
  • Useful predetermined thresholds for assessing expression of one or more markers and/or targets may be determined by any convenient and appropriate method, including comparison of the measured level of expression to a corresponding control.
  • a useful predetermined threshold for the level of MDR1 and/or TAA assayed in a sample may correspond to a level of MDR1 and/or TAA as measured in a reference cell, such as a healthy/normal cell.
  • the TAA may be CD47, PD-L1, or EGFR.
  • the TAA may be EGFR, HER2, PD-L1, CD19, CD20, CD38, or CD40.
  • the TAA may be AXL, LIV1, MET, CD73, CD70, LY6E, CD44. These TAAs are known to be overexpressed with CD47 in many tumor types, according to, for example, publicly available databases such as the Broad Institute database, which encompasses data from the Cancer Cell Line Encyclopedia (CCLE).
  • methods of the present disclosure also include methods or making and/or identifying anti-CD47 antibodies as described herein.
  • a subject antibody can be produced by any known method, e.g., conventional synthetic methods for protein synthesis; recombinant DNA methods; etc.
  • a subject antibody is a single chain polypeptide
  • it can synthesized using standard chemical peptide synthesis techniques.
  • the synthesis may proceed via liquid-phase or solid-phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing a subject antibody.
  • Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp.
  • the free N-terminal amine of a solidphase attached is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
  • the peptide remains immobilized on the solid-phase and undergoes a filtration process before being cleaved off.
  • Standard recombinant methods can be used for production of a subject antibody.
  • nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions are inserted into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.
  • Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • the expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the antibodies.
  • eukaryotic host cells e.g., COS or CHO cells.
  • nucleic acid sequences can encode each immunoglobulin amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2: 183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single -stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer, and encodes the selected alteration in the target polypeptide DNA.
  • Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.
  • selection markers e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used for cloning a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • yeast Other microbes, such as yeast, are also useful for expression.
  • Saccharomyces e.g., .S'. cerevisiae
  • Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3 -phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • mammalian cells e.g., mammalian cells grown in in vitro cell culture
  • the polypeptides of the present invention e.g., polynucleotides encoding immunoglobulins or fragments thereof.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B-cells or hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148: 1149 (1992).
  • the whole antibodies, their dimers, individual light and heavy chains, or other forms of a subject antibody can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).
  • a subject antibody can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules other than a subject antibody, etc.
  • methods of generating an anti-CD47 antibody of the present disclosure may include producing candidate antibodies and screening for activity.
  • methods may generate a multispecific antibody that specifically binds a cell expressing both CD47 and another target, such as an ABC transporter or a further tumor antigen, through the use of a series of steps.
  • steps of such methods may include: producing a multi-specific antibody or a plurality of antibodies that each include or are expected to include an ABCB1 -binding domain and a CD47-binding domain; contacting a first test cell expressing ABCB1 and CD47 with the multi-specific antibody or plurality of antibodies; contacting a second cell expressing either ABCB1 or CD47 with the multi-specific antibody or plurality of antibodies; comparing the binding of the multispecific antibody, or the antibodies of the plurality, to the first cell with the binding of the multi -specific antibody to the second cell to determine a binding-specificity ratio; and identifying the multi-specific antibody, or one or more of the antibodies of the plurality, as specific for the cell expressing both ABCB1 and CD47 when the ratio is above a predetermined threshold.
  • the threshold may vary and may range from 1.5 : 1 or more, including but not limited to e.g., 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 50: 1, 100: 1, etc.
  • This description is for illustration only, other multi-specific antibodies may be screened for activity in an analogous manner.
  • Various cells may be used in such methods, including but not limited to e.g., the cells described herein.
  • such methods may employ one or more controls, including but not limited to e.g., control cells, control reagents, and the like.
  • Useful control cells include those that have a known expression or known lack of expression of one or more relevant genes or proteins.
  • Useful control reagents may include but are not limited to e.g., control antibodies such as but not limited to e.g., monospecific antibodies to known targets.
  • aspects of the invention include diagnostic assay methods, e.g., in vitro diagnostic immunoassays, which can be used to detect the presence or absence of a protein of interest (e.g., CD47 or an ABC transporter) in a test sample.
  • a protein of interest e.g., CD47 or an ABC transporter
  • the immunoassay format used for the detection of the target protein can be configured in a variety of ways.
  • the immunoassays can include both homogeneous and heterogeneous assays, competitive and non-competitive assays, direct and indirect assays, and “sandwich” assays.
  • Useful formats include, but are not limited to, enzyme immunoassays, for example, enzyme linked immunosorbent assays (EUISA), chemiluminescent immune-assays (CUIA), electrochemiluminescent assays, radioimmunoassay, immunofluorescence, fluorescence anisotropy, immunoprecipitation, equilibrium dialysis, immunodiffusion, immunoblotting, agglutination, luminescent proximity assays, and nephelometry.
  • EUISA enzyme linked immunosorbent assays
  • CLIA chemiluminescent immune-assays
  • electrochemiluminescent assays radioimmunoassay
  • immunofluorescence fluorescence anisotropy
  • immunoprecipitation equilibrium dialysis
  • immunodiffusion immunodiffusion
  • immunoblotting agglutination
  • luminescent proximity assays luminescent proximity assays
  • a biological sample is contacted with an antibody of the present invention.
  • the biological sample can be immobilized on a solid support.
  • the biological sample is contacted with an antibody of the invention that has been immobilized on a solid support.
  • the solid support can be, for example, a plastic surface, a glass surface, a paper or fibrous surface, or the surface of a particle. More specifically, the support can include, e.g., a microplate, a bead, a polyvinylidene difluoride (PVDF) membrane, a nitrocellulose membrane, a nylon membrane, a porous membrane, a non-porous membrane, or any combination thereof.
  • PVDF polyvinylidene difluoride
  • substrates or supports can comprise glass, cellulose-based materials, thermoplastic polymers, such as polyethylene, polypropylene, or polyester, sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane fdms composed of nitrocellulose, nylon, or polysulfone.
  • thermoplastic polymers such as polyethylene, polypropylene, or polyester
  • sintered structures composed of particulate materials e.g., glass or various thermoplastic polymers
  • cast membrane fdms composed of nitrocellulose, nylon, or polysulfone.
  • the substrate may be any surface or support upon which an antibody or a polypeptide can be immobilized, including one or more of a solid support (e.g., glass such as a glass slide or a coated plate, silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal), a semi-solid support (e.g., a polymeric material, a gel, agarose, or other matrix), and/or a porous support (e.g., a fdter, a nylon or nitrocellulose membrane or other membrane).
  • a solid support e.g., glass such as a glass slide or a coated plate, silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal
  • a semi-solid support e.g., a polymeric material, a gel, agarose, or other matrix
  • a porous support e.g., a fdter, a nylon or nitrocellulose membrane or other membrane.
  • synthetic polymers can be used as a substrate, including, e.g., polystyrene, polypropylene, polyglycidylmethacrylate, aminated or carboxylated polystyrenes, polyacrylamides, polyamides, and polyvinylchlorides.
  • the immunoassay format can be a two antibody “sandwich” assay.
  • the biological sample is contacted with an antibody of the invention that has been immobilized on a solid support, for example, a microtiter plate.
  • the sample and the first antibody are incubated under conditions that favor specific binding and the formation of a target protein-antibody complex.
  • unbound constituents of the biological sample are removed.
  • the complex is contacted with a second antibody.
  • the second antibody binds to a different epitope than the epitope bound by the first antibody.
  • the first and second antibodies do not competitively inhibit one another for binding to the target protein.
  • Antibody binding can be measured in a variety of ways.
  • the signal for example, generated by a detectable label, can be analyzed and, if applicable, quantified using an optical scanner or other image acquisition device and software that permits the measurement of the signal, for example, a fluorescent signal, a luminescent signal, a phosphorescent signal, or a radioactive signal, associated with complex formation.
  • Examples of instrumentation for measuring a detectable signal can include, but are not limited to: microplate readers, fluorimeters, spectrophotometers, and gamma counters. Kits
  • kits may include, e.g., any combination of the multi-specific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, or the like, described herein.
  • a subject kit can include one or more of: a subject multi-specific antibody, a nucleic acid encoding the same, or a cell comprising a subject multi-specific nucleic acid.
  • Kits may be configured for various purposes, including, e.g., treatment kits (e.g., where a kit may include a multi-specific antibody and e.g., one or more additional active agents, such as a chemotherapeutic), kits for producing antibodies, kits for screening antibodies, and the like.
  • kits will vary and may, e.g., include: a buffer; a protease inhibitor; etc.
  • a subject kit comprises a subject nucleic acid
  • the nucleic acid may also have restrictions sites, multiple cloning sites, primer sites, etc.
  • the various components of the kit may be present in separate containers or certain compatible components may be pre -combined into a single container, as desired.
  • a subject kit can include instructions for using the components of the kit to practice a subject method.
  • the instructions for practicing a subject method are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., compact disc -read only memory (CD-ROM), digital versatile disk (DVD), diskette, etc.
  • CD-ROM compact disc -read only memory
  • DVD digital versatile disk
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • kits with unit doses of a subject antibody e.g., in injectable doses
  • the container containing the unit doses will be an informational package insert describing the use and attendant benefits of the antibody in treating pathological condition of interest.
  • the article of manufacture may comprise two vials, where the first vial contains the subject antibody and the second vial contains the other drug, such as a chemotherapeutic agent or another antibody.
  • an article of manufacture herein comprises an intravenous (IV) bag containing a stable formulation of a subject antibody suitable for administration to a cancer patient.
  • IV intravenous
  • the formulation in the IV bag is stable for up to 24 hours at 5 °C or 30 °C. Stability of the formulation can be evaluated by one or more assays such as, color, appearance, and clarity (CAC), concentration and turbidity analysis, particulate analysis, size exclusion chromatography (SEC), ion- exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assay.
  • assays such as, color, appearance, and clarity (CAC), concentration and turbidity analysis, particulate analysis, size exclusion chromatography (SEC), ion- exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assay.
  • kits of the invention can include a population of beads (e.g., suitable for an agglutination assay or a lateral flow assay), or a plate (e.g., a plate suitable for an ELISA assay).
  • the kits comprise a device, such as a lateral flow immunoassay device, an analytical rotor, or an electrochemical, optical, or opto-electronic sensor.
  • the population of beads, the plate, and the devices are useful for performing an immunoassay. For example, they can be useful for detecting formation of a first agent-analyte -second agent complex.
  • kits can include various diluents and buffers, labeled conjugates or other agents for the detection of specifically bound antigens or antibodies, and other signal -generating reagents, such as enzyme substrates, cofactors and chromogens.
  • the kits can include one or more reference samples of varying concentrations, for example, a purified recombinant CD47, a purified recombinant ABC transporter, etc.
  • the kits can also include a positive control.
  • kits can include coating reagents, polyclonal or monoclonal capture antibodies specific for an antigen or analyte to be tested, or a cocktail of two or more of the antibodies, purified or semi -purified extracts of these antigens as standards, monoclonal antibody detector antibodies, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup, etc.
  • a kit comprises buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide -antibody complex.
  • kits further comprise instructions for use.
  • the product may also include a legend (e.g., a printed label or insert or other medium describing the product’s use (e.g., an audio- or videotape)).
  • the legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the assay should be performed, indications therefor, and other uses.
  • Example 1 Generation of antibodies that bind specifically to cells expressins human CD47 antisen
  • DNA sequences were determined by double strand sequencing.
  • the full length human CD47 has the following amino acid sequence (Sequence ID: NP_001768):
  • CD47 Homo sapiens CD47 molecule (CD47), transcript variant 1, mRNA NCBI Reference Sequence: NM_001777.3.
  • the full length Macaca mulatta leukocyte surface antigen CD47 (Sequence ID: NP_001253446.1) has the following amino acid sequence:
  • Anti-human CD47 5F9 antibody variable heavy chain sequence is as follows:
  • Anti-human CD47 5F9 antibody variable light chain sequence is as follows:
  • Rhesus LLC-MK2 and human HEK-293 cell lines expressing endogenous CD47 antigen were obtained from the American Type Culture Collection. Although rhesus and cynomolgus are different species of Macaques, the endogenous CD47 sequences are the same for the two species.
  • SA-MES/DX5 was obtained from the European Collection of Authenticated Cell Cultures (ECACC) through the United States distributor (Sigma- Aldrich). Generation of stable cell lines expressing human or cynomolgus CD47
  • Murine C6 cell lines were obtained from the American Type Culture Collection (ATCC No. CCL-107).
  • SA-MES/DX5 was obtained from the European Collection of Authenticated Cell Cultures (ECACC) through the United States distributor (Sigma- Aldrich) (ECACC 95051031).
  • DMEM Dulbecco's Modified Eagle's Medium, Gibco, Grand Island, N.Y., USA
  • FBS 10% (v/v) FBS
  • glutamine glutamine via adhesion culture.
  • Cells were cultivated at 37°C with 5% CO2 at saturated humidity.
  • Transfection of cells was performed by lipid-based transfection using the CRISPR Max reagent (ThermoFisher) according to the manufacturer’s protocol.
  • Hybridoma supernatants were screened for the presence of anti-CD47 antibodies by flow cytometry. The binding to CD47 antigens was then confirmed for supernatants from the isolated subcloned hybridoma clones. cDNA cloning of heavy and light chain variable domains from B cells and hybridoma cells
  • mRNAs from the hybridomas or B cells were purified using DynabeadsTM mRNA DirectTM Micro Purification kit (Thermo #61021). Subsequently, the cDNAs were synthesized and a 3 ’-tailing reaction was performed using 5 ’race technology (Takara # 634858). Finally, the variable domain sequences of the heavy chain and the light chain were applied with UPM and HC reverse primer or LC reverse primer. The resulting PCR products were gel purified and sequenced.
  • Binding specificity of the control antibodies and of the supernatants from hybridoma single cells was tested by ELISA using of recombinant human CD47 protein (Aero biosystems). Briefly, microtiter plates were coated with 100 pL of recombinant human CD47 protein at 3ug/ml in PBS and then blocked with 300 pl of 2% BSA in PBS. Serial dilutions of the control antibodies and the supernatants from single cell hybridoma were added in 1/3 sequential dilutions to each well and incubated for 1 hour at room temperature. The 5F9 known anti-CD47 antibody was used as a positive control.
  • Plates were subsequently washed three times with PBS/Tween and then incubated with HRP- conjugated anti-human or anti rat specific secondary reagents for 1 hour at room temperature. After washing, plates were developed with HRP substrate. The reaction was stopped with 2M H2SO4, and OD was measured at 520 nm.
  • CD47:SIRPa-Bio Inhibitor Screening ELISA Assay (Aero Biosystems) was used. Briefly, microtiter plates were coated with 100 pL of recombinant human CD47 protein at 3pg/mL in PBS and then blocked with 300 pL of 2% BSA in PBS. Serial dilutions of the control antibodies and the supernatants from single cell hybridoma mixed with biotinylated human SIRP alpha were added in 1/3 sequential dilutions to each well and incubated for 1 hour at room temperature. The 5F9 and CC2C6 known anti-CD47 antibodies were used as a positive control.
  • Table 4 lists the following characteristics of the anti-CD47 antibodies: binding to human and cynomolgus CD47 antigens and blocking of the SIRP alpha/human CD47 antigen binding measured by ELISA.
  • the anti-CD47 antibodies bind to human and/or cynomolgus CD47 antigens coated on an ELISA plate. Furthermore, the antibodies can block the binding between the SIRP alpha protein and the human CD47 antigen detected by ELISA.
  • Table 5 lists the following characteristics of the anti-CD47 antibodies: binding to human and cynomolgus CD47 antigens and blocking of the binding between SIRP alpha protein and the human CD47 antigen measured by FACS.
  • some of the anti-CD47 antibodies can bind to both human and/or cynomolgus CD47 antigens expressed on different cell lines.
  • the binding to human CD47 is evaluated both as the ratio between the binding to HEK293 overexpressing hCD47 and HEK293 and as the ratio between the binding to naive MES-SA-DX/5 cell lines and huCD47 Knock out MES-SA- DX/5 cell lines.
  • the binding to cynomolgus CD47 was evaluated as binding to naive Rhesus LLC-MK2 expressing endogenous CD47.

Abstract

L'invention concerne des anticorps anti-CD47, ainsi que des méthodes de production de tels anticorps, des compositions, y compris des compositions pharmaceutiques, contenant de tels anticorps, et leur utilisation pour traiter des troubles qui sont caractérisés par l'expression d'un groupe de différenciation 47 (CD47).
PCT/US2023/062875 2022-02-18 2023-02-17 Anticorps anti-cd47 WO2023159220A1 (fr)

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