WO2023246790A1 - 一种含抗cd47抗体或其抗原结合片段的制剂及其制备方法和应用 - Google Patents

一种含抗cd47抗体或其抗原结合片段的制剂及其制备方法和应用 Download PDF

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WO2023246790A1
WO2023246790A1 PCT/CN2023/101430 CN2023101430W WO2023246790A1 WO 2023246790 A1 WO2023246790 A1 WO 2023246790A1 CN 2023101430 W CN2023101430 W CN 2023101430W WO 2023246790 A1 WO2023246790 A1 WO 2023246790A1
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antibody
seq
antigen
binding fragment
preparation
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PCT/CN2023/101430
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French (fr)
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霍永庭
阮小华
黄丹丹
闫加庆
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广东菲鹏制药股份有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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

Definitions

  • the present disclosure relates to the field of biological preparations, and in particular, to a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof and its preparation method and application.
  • Immune checkpoint inhibitor therapies based on T cell-based CTLA4 antibodies, PD-1 antibodies, PD-L1 antibodies, etc., and cell therapies such as CAR-T and TCR-T are all Immunotherapy has become a hot topic in recent years. These all revolve around how to restore T cell function, in other words, how to improve the ability of the adaptive immune system.
  • the road to targeting immune checkpoints and activating T cell function to improve the ability of the adaptive immune system to conquer cancer is still full of twists and turns.
  • the role of the innate immune system in tumor immunotherapy has not been fully utilized for a long time. In fact, in the entire tumor infiltration area, macrophages account for about 50% of the tumor tissue.
  • the number of macrophages is inversely related to the prognosis of the tumor, which further illustrates the importance of macrophages in tumors. role.
  • the phagocytic effect of macrophages requires the simultaneous action of two signals: one is the activation of the "eat me” signal targeting the cell surface, and the other is the inactivation of the "don't eat me” signal on the same target surface. The lack of any signal is not enough to trigger the phagocytosis effect.
  • CD47 is a type of "don't eat me” signal that inhibits macrophage phagocytosis by binding to signal regulatory protein ⁇ (SIRP ⁇ ) on the surface of macrophages.
  • SIRP ⁇ signal regulatory protein ⁇
  • Tumor cells can also escape macrophage phagocytosis through the expression of CD47 (see, for example, EP2242512 and related documents cited therein).
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • IAP integrin-associated protein
  • SIRP ⁇ monoregulatory protein alpha
  • SIRP ⁇ integrins
  • TSP-1 thrombospondin-1
  • SIRP ⁇ is mainly expressed on bone marrow cells, including macrophages, bone marrow dendritic cells (DCs), granulocytes, mast cells and their precursors (precursors include hematopoietic stem cells).
  • CD47/SIRP ⁇ interaction transmits the "don't eat me” signal and inhibits autophagy.
  • Analysis of the patient's tumors and matched adjacent normal (non-tumor) tissue revealed that the CD47 protein is overexpressed on cancer cells, which effectively helps them evade innate immune surveillance and elimination.
  • Blocking the interaction of CD47-SIRP ⁇ with anti-CD47 antibodies has been shown to effectively induce phagocytosis of tumor cells in vitro and inhibit the growth of various hematological and solid tumors in vivo. Therefore, CD47 is a valid target for cancer therapy, and its appropriate antagonists are needed to prepare human therapeutics.
  • the purpose of this disclosure is to provide a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof and a preparation method and application thereof.
  • the present disclosure provides a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which includes the following components: an anti-CD47 antibody or an antigen-binding fragment thereof, a buffer, and auxiliary materials.
  • the antibody or antigen-binding fragment thereof contains the following CDRs: LCDR1 as shown in SEQ ID NO:1 or an amino acid sequence having at least 95% homology with SEQ ID NO:1, such as SEQ ID NO:2 or SEQ ID NO:2 LCDR2 represented by an amino acid sequence having at least 95% homology to SEQ ID NO:3 or LCDR3 represented by an amino acid sequence having at least 95% homology to SEQ ID NO:3, such as SEQ ID NO :10 or an HCDR1 represented by an amino acid sequence having at least 95% homology to SEQ ID NO:10, as represented by SEQ ID NO:11 or an amino acid sequence having at least 95% homology to SEQ ID NO:11 HCDR2, such as SEQ ID NO:12 or HCDR3 shown in an amino acid
  • the present disclosure provides a method for preparing a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof as described in the previous embodiments, which includes: mixing the components of the preparation.
  • the present disclosure provides the use of the formulation as described in the previous embodiments in the preparation of products for preventing and treating CD47-positive tumors.
  • the present disclosure provides a lyophilized preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which is obtained by freeze-drying the preparation as described in the previous embodiment.
  • the present disclosure provides a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which is obtained after reconstitution of the freeze-dried preparation as described in any of the foregoing embodiments. have to.
  • the present disclosure also provides a formulation as described in any of the preceding embodiments for use as a medicament for preventing and treating CD47-positive tumors.
  • the present disclosure also provides a method of treating CD47-positive tumors, the method comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any of the preceding embodiments.
  • the present disclosure innovatively invented a preparation containing anti-CD47 antibodies or anti-CD47 antibody fragments.
  • the anti-CD47 antibodies or anti-CD47 antibody fragments contained in the preparation did not undergo red blood cell hemagglutination in vitro, and showed extremely weak levels of coagulation with red blood cells. Low or no binding; and the antibody can effectively block the binding of CD47 to SIRP ⁇ , activate and mediate the phagocytic activity of macrophages on tumor cells, demonstrate targeting specificity with CD47-positive tumor cells, and is highly efficient and safe. Good, no drug side effects and other advantages.
  • Figure 1 is the DSC spectrum in Test Example 1.
  • Figure 2 is the DSC spectrum in Test Example 2.
  • Figure 3 is the average fluorescence intensity of CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47 in Experimental Example 5.
  • Figure 4 is the average fluorescence intensity of CD47 antibodies (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47 in Experimental Example 5.
  • Figure 5 is the average fluorescence intensity of CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47 in Experimental Example 6.
  • Figure 6 is the average fluorescence intensity of CD47 antibodies (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47 in Experimental Example 6.
  • Figure 7 shows the results of CD47/SIRP ⁇ binding inhibition of human CD47 by CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42 and hIgG4-isotype control) in Test Example 7.
  • Figure 8 shows the results of CD47/SIRP ⁇ binding inhibition of human CD47 by CD47 antibodies (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55) in Test Example 7.
  • Figure 9 is the measurement result of the ability of the CD47 antibody to promote macrophage phagocytosis of tumor cells in Test Example 8.
  • Figure 10 shows the results of measuring the RBC agglutination ability of the CD47 antibody in Test Example 9.
  • Figure 11 is the measurement result of the CD47 antibody binding ability to human red blood cells in Test Example 10.
  • Figure 12 is the results of the binding analysis between CD47 antibodies and human platelets in Test Example 11.
  • Figure 13 is the analysis result of the effect of CD47 antibody on activating macrophages to phagocytose red blood cells in Test Example 12.
  • Figure 14 shows the anti-tumor results of the CD47 antibody on the human B lymphocyte subcutaneous transplantation tumor model in Test Example 13.
  • Figure 15 shows the anti-tumor results of the CD47 antibody on the human malignant melanoma model in Test Example 14.
  • Embodiments of the present disclosure provide a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which includes the following components: an anti-CD47 antibody or an antigen-binding fragment thereof fragments, buffers and excipients.
  • antibody or antigen-binding fragment thereof refers to a protein that binds a specific antigen, which generally refers to proteins and protein fragments containing complementarity-determining regions (CDR regions).
  • Antibody refers to a full-length antibody.
  • antigen-binding fragment is a substance containing the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope.
  • the antigen-binding fragment specifically recognizes and binds to CD47.
  • the antigen-binding fragment is a fragment that blocks the binding of CD47 to its ligand and activates the function of immune cells.
  • such fragments are selected from Fab (consisting of a complete light chain and Fd), Fv ( Composed of VH and VL), ScFv (single chain antibody, VH and VL are connected by a connecting peptide) or single domain antibody (composed only of VH).
  • the fragments may be prepared by conventional methods, for example, may be produced by recombinant nucleic acid technology, or may be produced by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.
  • CDR complementarity determining region
  • the term “complementarity determining region” or “CDR” refers to the highly variable regions of the heavy and light chains of an immunoglobulin. There are three heavy chain CDRs and three light chain CDRs.
  • CDR and CDRs are used to refer to a region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof to the antigen or epitope it recognizes.
  • the complementarity-determining region of the heavy chain is represented by HCDR
  • the complementarity-determining region of the light chain is represented by LCDR.
  • CDR notation methods in this field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily.
  • the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.
  • Kabat annotation standards are used to mark CDR regions, but CDR regions marked by other methods also fall within the protection scope of the present disclosure.
  • the antibody or antigen-binding fragment thereof contains the following CDRs: LCDR1 as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 95% homology to SEQ ID NO: 1, such as SEQ ID NO :2 or an LCDR2 represented by an amino acid sequence having at least 95% homology with SEQ ID NO:2, as represented by SEQ ID NO:3 or an amino acid sequence having at least 95% homology with SEQ ID NO:3 LCDR3, as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 95% homology to SEQ ID NO: 10 HCDR1, as set forth in SEQ ID NO: 11 or having at least 95% homology to SEQ ID NO: 11 HCDR2 represented by an amino acid sequence, such as SEQ ID NO:12 or HCDR3 represented by an amino acid sequence having at least 95% homology with SEQ ID NO:12, the sequence information is as follows.
  • amino acid sequences that are at least 95% homologous to SEQ ID NO: 1 include, but are not limited to, KSSQSLLNTRTRKNYLA (SEQ ID NO: 21).
  • Amino acid sequences that are at least 95% homologous to SEQ ID NO: 11 include, but are not limited to, MIHPSDSETRLNQKFQG (SEQ ID NO: 22).
  • the antibody or antigen-binding fragment thereof includes: LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, LCDR3 as shown in SEQ ID NO:3, such as HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, and HCDR3 shown in SEQ ID NO:12;
  • the antibody or antigen-binding fragment thereof includes: as shown in SEQ ID NO:1 LCDR1 shown in SEQ ID NO:2, LCDR3 shown in SEQ ID NO:3, HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, and HCDR3 as shown in SEQ ID NO:12;
  • the antibody or antigen-binding fragment thereof includes: LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, as shown in SEQ ID NO:3 LCDR3 as shown, HCDR1 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO
  • the auxiliary materials include: 1% to 20% of sugars and 0.01% to 0.5% of surfactants.
  • the present disclosure provides anti-CD47 antibodies or antigen-binding fragments thereof.
  • the antibodies do not undergo hemagglutination of red blood cells in vitro. What is even more valuable is that they show extremely weak levels of low or no binding to red blood cells; they can effectively block CD47 and SIRP ⁇ . Binds and activates to mediate the phagocytosis activity of macrophages on tumor cells, showing significant targeting specificity with CD47-positive tumor cells. It has high affinity and specificity, does not cause red blood cell agglutination, and interacts with human red blood cells and platelets. Shows extremely weak bonding and good safety.
  • the preparation combination provided by the embodiments of the present disclosure is a combination formula proposed and verified by the inventor through a series of creative efforts, which is conducive to making the above-mentioned anti-CD47 antibody or its antigen-binding fragment stably and effectively exert its effect, and effectively block CD47 and SIRP ⁇
  • the combination enables anti-CD47 antibodies to be better used in clinical applications.
  • the mass volume percentage of surfactant can be 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, 0.22% , 0.24%, 0.26%, 0.28%, 0.3%, 0.32%, 0.34%, 0.36%, 0.38%, 0.4%, 0.42%, 0.44%, 0.46%, 0.48%, 0.5%, any one or any two range between species.
  • the mass volume percentage of surfactant is 0.02%, that is, 0.02% (w/v), which means that every 100 mL volume solution contains 0.02g of surfactant; the rest can be deduced by analogy, the same below.
  • the surfactant is selected from any one of Tween 20 (polysorbate-20), Tween 80 (polysorbate-80), and poloxamer 188.
  • the mass volume percentage of the Tween 80 in the preparation is 0.01% to 0.1%, optionally 0.01% to 0.04%, or The selected range is 0.02% to 0.04%.
  • the mass volume percentage of the poloxamer 188 in the preparation is 0.05% to 0.5%, optionally 0.1% to 0.2%, optionally is 0.1%.
  • the surfactant is 0.02% (w/v) Tween 80.
  • the mass volume percentage (w/v) of sugars can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 Any one or the range between any two of %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.
  • the sugar includes at least one of sucrose and trehalose.
  • the sugar is sucrose.
  • the concentration of sucrose is 3% to 15%, optionally 6% to 10%, optionally 8%. In some embodiments, the mass volume percentage of the sucrose in the preparation is 8%.
  • the carbohydrate is trehalose.
  • the concentration of trehalose is 3% to 15%, optionally 6% to 10%, optionally 8.8%.
  • the sugar is 8% (w/v) sucrose.
  • Buffer refers to a solution that resists changes in pH through the action of its acid-base conjugated components.
  • buffers that control the pH in an appropriate range include, but are not limited to, buffers containing acetate, histidine, citrate, phosphate, and the like.
  • the buffer is a citric acid-sodium citrate buffer configured from citric acid monohydrate and sodium citrate dihydrate, and is composed of L-histidine and L-histidine hydrochloride.
  • Histidine-histidine salt buffer prepared from monohydrate, acetic acid-sodium acetate buffer prepared from glacial acetic acid and sodium acetate trihydrate, or sodium dihydrogen phosphate and disodium hydrogen phosphate.
  • Phosphate-phosphate buffer
  • the buffer is selected from any one of citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, histidine-histidine buffer, and phosphoric acid-phosphate buffer.
  • species pH is 4.5 ⁇ 7.5, the pH can be specifically 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, The range between any one or any two of 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5.
  • the buffer is selected from any one of citric acid-sodium citrate buffer, histidine-histidine buffer and phosphoric acid-phosphate buffer, with a pH of 5.0 to 7.5.
  • the buffer is a histidine-histidine salt buffer with a pH of 5.5 to 6.0.
  • the pH of the histidine-histidine salt buffer is 5.5. Compared with other buffers, histidine-histidine salt buffer can stabilize the conformation of antibody molecules more effectively.
  • the histidine-histidine salt buffer is a histidine-histidine hydrochloride buffer configured from L-histidine and L-histidine hydrochloride monohydrate.
  • the concentration of the buffer is 10-40mM, specifically 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, Any one of 40mM or the range between any two.
  • the concentration of the buffer is 15-25mM.
  • the concentration of the buffer is 20mM.
  • the buffer is a 20 mM histidine-histidine hydrochloride buffer with a pH of 6.0.
  • the formulation further includes methionine.
  • the concentration of methionine is 1 to 20mM, specifically, it can be any one or any of 1mM, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM. The range between the two.
  • the concentration of methionine is 4.5-5.5mM.
  • the concentration of methionine is 5mM.
  • the concentration of the anti-CD47 antibody or antigen-binding fragment thereof is 20 to 60 mg/mL, specifically 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, or 45 mg/mL. , 50mg/mL, 55mg/mL, 60mg/mL any one or the range between any two.
  • the concentration of the anti-CD47 antibody or antigen-binding fragment thereof is 45-55 mg/mL.
  • the concentration of the anti-CD47 antibody or antigen-binding fragment thereof is 50 mg/mL.
  • the anti-CD47 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region.
  • the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 4-9 or has at least 95% homology with any one of SEQ ID NO: 4-9.
  • the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 13 to 18 or has at least 95% homology with any one of SEQ ID NO: 13 to 18. sequence.
  • sequence. The corresponding sequences of the antibody and its heavy chain variable region and light chain variable region are as follows:
  • SEQ ID NO:4 The sequence shown in SEQ ID NO:4 is as follows:
  • SEQ ID NO:5 The sequence shown in SEQ ID NO:5 is as follows:
  • SEQ ID NO:6 The sequence shown in SEQ ID NO:6 is as follows:
  • sequence shown in SEQ ID NO:7 is as follows:
  • sequence shown in SEQ ID NO:8 is as follows:
  • sequence shown in SEQ ID NO:9 is as follows:
  • sequence shown in SEQ ID NO:13 is as follows:
  • sequence shown in SEQ ID NO:14 is as follows:
  • sequence shown in SEQ ID NO:15 is as follows:
  • sequence shown in SEQ ID NO:16 is as follows:
  • sequence shown in SEQ ID NO:17 is as follows:
  • sequence shown in SEQ ID NO:18 is as follows:
  • the anti-CD47 antibody or antigen-binding fragment thereof further includes a constant region.
  • the constant region includes a heavy chain constant region and a light chain constant region
  • the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD
  • the light chain constant region is a kappa or lambda chain.
  • the species source of the constant region is selected from mouse, rabbit, sheep, monkey, or human.
  • the anti-CD47 antibody is a 7A11H14 antibody
  • the heavy chain of 7A11H14 is as shown in SEQ ID NO: 19
  • the light chain is as shown in SEQ ID NO: 20:
  • the antibody is any one or more of a CDR-grafted antibody, a multimeric antibody, or a bispecific antibody.
  • the antigen-binding fragment is any one or more of F(ab') 2 , Fab, scFv and Fv.
  • the CD47 is human CD47, murine CD47, or monkey CD47.
  • the embodiments of the present disclosure also provide a method for preparing a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments, which includes: The components of the formulation are mixed.
  • the preparation method further includes: using a buffer in the preparation to fight against CD47 The solution of the antibody or its antigen-binding fragment is replaced; after replacement, it is mixed with the excipients.
  • inventions of the present disclosure also provide the use of the preparation as described in any of the foregoing embodiments in the preparation of products for preventing and treating CD47-positive tumors.
  • Embodiments of the present disclosure also provide a formulation as described in any of the preceding embodiments for use as a medicament for preventing and treating CD47-positive tumors.
  • Embodiments of the present disclosure also provide a method of treating CD47-positive tumors, the method comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any of the preceding embodiments.
  • prevention and treatment in this article includes prevention and/or treatment, and treatment can be understood as improving the condition or curing.
  • CD47-positive tumors include tumors that express CD47, specifically including but not limited to solid tumors and hematological tumors, such as hematological malignancies, non-Hodgkin lymphoma, multiple myeloma, B-cell lymphoma, liver Cell carcinoma, ovarian cancer, colorectal cancer, lung cancer, breast cancer, melanoma and kidney tumors, etc.
  • the embodiments of the present disclosure also provide a lyophilized preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which is obtained by freeze-drying the preparation as described in any of the foregoing embodiments.
  • embodiments of the present disclosure also provide a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof, which is obtained by reconstituting the freeze-dried preparation as described in any of the foregoing embodiments.
  • Examples 1 to 4 and Experimental Examples 1 to 3 exemplarily demonstrate the technical effect when the anti-CD47 antibody in the preparation is 7A11H14.
  • 7A11H14 comes from the application number 202011544262.6, and the invention name is "A CD47-targeting antibody” As disclosed in the Chinese application for "Antibodies and Applications thereof" (submitted on December 23, 2020) (the sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the sequence of the light chain variable region is shown in SEQ ID NO: 8 As shown, the constant region is the Fc region of human IgG4 containing S228P variation), which includes a heavy chain as shown in SEQ ID NO: 19, and a light chain as shown in SEQ ID NO: 20.
  • the anti-CD47 antibody or antigen-binding fragment thereof in the preparation can also be disclosed in the Chinese application with application number 202011544262.6 and the invention title "Antibody targeting CD47 and its application”
  • Other anti-CD47 antibodies or antigen-binding fragments can achieve the same or similar technical effects.
  • a preparation containing anti-CD47 antibody or antigen-binding fragment thereof which includes: 50mg/mL anti-CD47 antibody, 20mM histidine-histidine salt buffer system (pH6.0), 8% (w/v) sucrose and 0.02% (w/v) Tween 80.
  • the defined concentration of each component is the concentration of each component in the preparation.
  • w/v refers to the mass volume percentage.
  • 8% (w/v) sucrose means that each 100 mL volume solution contains 8 g. of sucrose, and so on for the others.
  • a preparation containing anti-CD47 antibody or antigen-binding fragment thereof which includes: 50mg/mL anti-CD47 antibody, 20mM histidine-histidine salt buffer system (pH6.0), 8.8% (w/v) sucrose and 0.02% (w/v) Tween 80.
  • a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof which includes: 50 mg/mL anti-CD47 antibody, 20 mM histidine-histidine salt buffer system (pH 6.0), 8.8% (w/v) sucrose, 0.04% (w/v) Tween 80, and 5mM methionine.
  • a preparation containing an anti-CD47 antibody or an antigen-binding fragment thereof which includes: 50 mg/mL anti-CD47 antibody, 20 mM histidine-histidine salt buffer system (pH 6.0), 8.8% (w/v) sucrose, and 0.1% (w/v) poloxamer 188.
  • Test Example 1 Effect of different buffers and pH on the stability of anti-CD47 antibodies
  • each prepared pH buffer to perform ultrafiltration replacement on the small test samples. After the replacement is completed, the concentration of each sample is diluted to 50 mg/ml, filtered, aliquoted, put into vials, plugged and capped, and placed out (samples are placed in a 40°C stability box and incubated for 4 weeks), and then scanned by differential calorimetry (DSC). , molecular sieve chromatography (SEC) and capillary isoelectric focusing electrophoresis (CIEF) to detect the stability of the antibody at 40°C for 4 weeks.
  • DSC differential calorimetry
  • SEC molecular sieve chromatography
  • CIEF capillary isoelectric focusing electrophoresis
  • the citric acid-sodium citrate buffer system is a citric acid-sodium citrate buffer composed of citric acid monohydrate and sodium citrate dihydrate;
  • the histidine-histidine salt buffer system is composed of L - Histidine-histidine hydrochloride buffer is composed of histidine and L-histidine hydrochloride monohydrate, and
  • acetic acid-sodium acetate buffer system is composed of glacial acetic acid and sodium acetate trihydrate.
  • Acetic acid-sodium acetate buffer, phosphoric acid-phosphate buffer system is a phosphoric acid-phosphate buffer composed of sodium dihydrogen phosphate and disodium hydrogen phosphate;
  • DSC Differential calorimetry scanning
  • MicroCal TM VP DSC was used to detect the sample transition melting point temperature (Tm) and onset unfolding temperature (Tm onset). The experimental parameters were set so that the scanning temperature increased from 10°C to 95°C at a scanning rate of 200°C/h. MicroCal TM VP DSC automatic analysis software was used for data analysis.
  • the results are shown in Figure 1 and Table 2.
  • the experimental results show that in the same buffer system, the Tmonset value increases with the increase of pH.
  • the Tm onset value is the highest in the phosphoric acid-phosphate buffer system (63.2°C ⁇ 63.6°C), and the Tmonset values in the histidine-histidine buffer system and citric acid-sodium citrate buffer system are 56.0°C ⁇ 61.4 respectively. °C and 54.9°C ⁇ 61.2°C.
  • the Tmonset value is the lowest (51.4°C ⁇ 60.1°C).
  • the data show that the antibody conformation stability is better in the histidine-histidine buffer system, citric acid-sodium citrate buffer system and phosphoric acid-phosphate buffer system.
  • the anti-CD47 antibody has good stability in the histidine-histidine salt buffer system.
  • the CD47 antibody was measured using ProteinSimple's capillary isoelectric focusing electrophoresis analyzer and fluorine-coated electrophoresis capillary.
  • the master mix is 0.5 ⁇ L isoelectric point marker (pI7.05), 0.5 ⁇ L isoelectric marker (pI 9.50), 4.0 ⁇ L ampholyte 3-10, 35 ⁇ L 1% methylcellulose, 37.5 ⁇ L 8M urea solution and 2.5 ⁇ L Ultra-pure water. Dilute the sample to 1.0 mg/mL with ultrapure water. Add 20 ⁇ L of diluent and 80 ⁇ L of premix to a centrifuge tube, mix and centrifuge. The sample injection temperature is 10°C, the first stage of focusing is 1500V for one minute, and the second stage is 3000V for eight minutes. The area percentage of the main peak, acidic peak and alkali peak is reported.
  • results are shown in Table 4.
  • the results show that the main peak of iCIEF in the citric acid-sodium citrate buffer system decreased by 14% to 22.7%, and the main peak of iCIEF in the acetic acid-sodium acetate buffer system decreased by 10.9% to 13.3%.
  • Histidine-histidine salt The iCIEF main peak in the buffer system decreased by 11.2% to 16.5%, and the SEC-HPLC main peak purity in the phosphoric acid-phosphate buffer system decreased by 26.2% to 30.0%.
  • Appearance inspection includes color, clarity and presence of visible particles.
  • MicroCal TM VP DSC was used to detect the sample transition melting point temperature (Tm) and onset unfolding temperature (Tm onset). The experimental parameters were set so that the scanning temperature increased from 10°C to 95°C at a scanning rate of 200°C/h. MicroCal TM VP DSC automatic analysis software was used for data analysis.
  • HACH insoluble particle analyzer in a biological safety cabinet to detect the size and number of insoluble particles in the sample. Clean the system with ultrapure water before use and test for insoluble particles in the ultrapure water. After the environment passes the test (the number of 10 ⁇ m particles is less than 25 particles per 25 ml), the sample is tested. Each sample was continuously injected 4 times, each injection was 1 mL. The first injection test result was discarded. After the results were reported, the particles with particle sizes ⁇ 2 ⁇ m, ⁇ 5 ⁇ m, ⁇ 10 ⁇ m and ⁇ 25 ⁇ m per ml were detected in the 3 consecutive test results. Quantity average.
  • Table 10-1 shows the test results after the samples were incubated in a 40°C stability box for 4 weeks.
  • Table 10-2 shows the test results after 5 rounds of freeze-thaw cycles (-70°C to room temperature)
  • Table 10-3 shows the test results after 3 days of shaking (300rpm, 25°C)
  • SEC-HPLC analysis shows that the stability of CD47 antibody protein in formulations F01, F04 and F06 is poor.
  • the CD47 antibody was measured using ProteinSimple's capillary isoelectric focusing electrophoresis analyzer and fluorine-coated electrophoresis capillary.
  • the master mix is 0.5 ⁇ L isoelectric point marker (pI7.05), 0.5 ⁇ L isoelectric marker (pI 9.50), 4.0 ⁇ L ampholyte 3-10, 35 ⁇ L 1% methylcellulose, 37.5 ⁇ L 8M urea solution and 2.5 ⁇ L Ultra-pure water. Dilute the sample to 1.0 mg/mL with ultrapure water. Add 20 ⁇ L of diluent and 80 ⁇ L of premix to a centrifuge tube, mix and centrifuge. The sample injection temperature is 10°C, the first stage of focusing is 1500V for one minute, and the second stage is 3000V for eight minutes. The area percentage of the main peak, acidic peak and alkali peak is reported.
  • the humanized variable domains were combined with secretion signals and human kappa and human FcIgG4S228P constant domains, cloned into a mammalian expression system, and transfected into 293 cells to generate humanized mAbs. Humanized variants are expressed as full-length IgG molecules, secreted into culture medium and purified using Protein A.
  • the humanized antibody and positive control antibody Hu5F9-G4 were transiently expressed and purified in Expi293 cells (the Hu5F9-G4 sequence has been disclosed in US Patent US2015/0183874A1).
  • pCDNA3.4 For transient expression of the antibody in Expi293 cells, use the vector pCDNA3.4. First clone the heavy and light chains of the antibody into separate pCDNA3.4 vectors. Use PEI (purchased from Polysciences) chemical transfection reagent, follow the chemical transfection The method is to transfer the pCDNA3.4 vector carrying the heavy chain and light chain of the antibody molecule into Expi293 cells, and transiently transfect the cultured Expi293 cells according to the protocol provided by the manufacturer.
  • PEI purchased from Polysciences
  • Expi293 ThermoFisher Scientific; A14635 cells were passaged, inoculated with Dynamis medium (gibco; A2617502) at a density of 2E6 in a 1L shake flask (conning; 431147), and placed in a cell culture shaker (Adolf Kuhner; ISF4- XC) incubated at 37°C; 8% CO2; 120rpm;
  • Biofilm interference technology was used to determine the equilibrium dissociation constant (KD) of the disclosed antibody binding to human CD47 (hCD47).
  • ForteBio affinity determination is performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs; 2013.5(2): p.270-8), specifically: the sensor is analyzed Equilibrate offline for 30 minutes in the buffer, and then detect online for 60 seconds to establish a baseline. Load the purified antibody obtained in Experiment 3 online to the AHC sensor (ForteBio) for ForteBio affinity measurement, and then expose the sensor with the loaded antibody. After exposure to 100 nM CD47 antigen for 5 minutes, the sensor was transferred to assay buffer and dissociated for 5 minutes for dissociation rate measurement. Kinetic analysis was performed using a 1:1 binding model.
  • the CD47 antibody affinity measurement results are shown in Table 12. The results show that the CD47 antibody has high affinity.
  • Binding of CD47 antibodies of the disclosure to human CD47 is measured in a flow cytometry-based assay. The specific steps are:
  • the cancer cell line CCRF-CEM (Cell Bank of the Chinese Academy of Sciences, Shanghai) expressing human CD47 was used and belongs to human acute lymphoblastic leukemia T lymphocytes.
  • CCRF-CEM cells (0.1 ⁇ 10 6 cells) were mixed with experimental antibodies (CD47 antibody and Hu5F9-G4 antibody of the present disclosure) of different concentrations (the highest concentration is 30ug/mL, three times diluted, a total of 10 concentrations) in a solution containing 3% bovine serum albumin (BSA) in PBS and incubate on ice for 30 minutes.
  • BSA bovine serum albumin
  • the EC50 results of CD47 antibody binding to human CD47 are shown in Table 13.
  • the average fluorescence intensity of CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47 is shown in the figure.
  • CD47 antibody The average fluorescence intensity of (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) combined with human CD47 is shown in Figure 4.
  • the results show that the CD47 antibody of the present disclosure binds to positive
  • the control antibody has comparable specific binding ability to human CD47 at the cellular level.
  • CHO-cynoCD47 cells By transfecting the pCDNA3.4 vector carrying full-length monkey CD47, a stable CHO cell line overexpressing monkey CD47 (CHO-cynoCD47 cells) was generated.
  • CHO-cynoCD47 cells (0.1 ⁇ 10 6 cells) were incubated with different concentrations (the highest Experimental antibodies (CD47 antibody and Hu5F9-G4 antibody of the present disclosure) with a concentration of 10ug/mL, three times diluted, a total of 10 concentrations, were incubated on ice for 30 minutes in PBS containing 3% bovine serum albumin (BSA). .
  • BSA bovine serum albumin
  • the EC50 results of CD47 antibody binding to monkey CD47 are shown in Table 14.
  • the average fluorescence intensity of CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47 As shown in Figure 5, the average fluorescence intensity of CD47 antibodies (7A11H14, 7A11H15, 7A11H33, 7A11H34, 7A11H35, 7A11H55, positive control antibody Hu5F9-G4 and hIgG4-isotype control) combined with monkey CD47 is shown in Figure 6.
  • the results show that, In comparison, the antibodies of the present disclosure and the positive control antibodies have comparable specific binding abilities to monkeys at the cellular level.
  • Antibody dilution Use FCM buffer (1XPBS+3% BSA) to dilute the disclosed CD47 antibody and control antibody Hu5F9-G4 to 90ug/mL, then 3-fold gradient dilution to 10 concentrations, and dilute the subtype control hIgG4 to 30ug/mL , 1.1ug/mL, 0.04ug/mL, and the ligand hSIRP ⁇ -mFC (AcroBiosystems) was diluted to 10ug/mL.
  • CCRF-CEM Chinese Academy of Sciences Cell Bank
  • CD47/SIRP ⁇ binding inhibition results of CD47 antibodies (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, Hu5F9-G4 and hIgG4-isotype control) on human CD47 are shown in Figure 7.
  • the CD47 antibodies (7A11H52, 7A11H14, 7A11H15, 7A11H33, 7A11 H34 , 7A11H35, 7A11H55) on the CD47/SIRP ⁇ binding inhibition results of human CD47 are shown in Figure 8.
  • the results show that the CD47 antibody of the present disclosure can significantly inhibit and block the binding of CD47 and SIRP ⁇ at the cellular level. Compared with the positive control antibody, this Disclosed CD47 antibodies showed considerable blocking ability.
  • Test Example 8 Detection of the ability of CD47 antibodies to promote macrophage phagocytosis of tumor cells
  • CD47 antibodies of the disclosure to promote macrophage phagocytosis of tumor cells was determined in a flow cytometry-based assay. The specific steps are:
  • PBMC peripheral blood mononuclear cells
  • CD14-positive monocytes were isolated from the PBMC using hCD14 magnetic beads (Miltenyi/130-050-201), and configured with rhGM-CSF (R&D; 7954-GM-010) complete culture medium, the final concentration of rhGM-CSF is 50ng/mL, the concentration of CD14-positive monocytes is 5E5/mL, add 20mL/dish to the cell culture dish; transfer to 5% CO 2 In a 37°C cell culture incubator, replace the medium with fresh medium (containing 50ng/mL GM-CSF) in half every 3 days and continue culturing for 4 days. On the 8th day, aspirate the macrophage supernatant into a 15mL centrifuge tube, add pre-cooled DPBS at the same time, and collect the cells directly with a cell scraper;
  • Allophycocyanin (APC)-labeled CD14 antibody purchased from Biolegend; B259538 was added and placed on ice (protected from light) in PBS containing 0.1% BSA. Incubate for 30 minutes. Cells were washed at least twice and analyzed by flow cytometry. The phagocytosed cell population is a cell population that is CD14 positive in living cells and is also positive for the fluorescent dye CFSE (carboxyfluorescein diacetate, succinimidyl ester, carboxyfluorescein diacetate, succinimidyl ester). Hu5F9-G4 antibody was used as a positive control antibody.
  • CFSE fluorescent dye
  • the measurement results of the CD47 antibody's ability to promote macrophages to phagocytose tumor cells are shown in Figure 9.
  • the results show that the CD47 antibody of the present disclosure has the ability to promote macrophages to phagocytose tumor cells, and the ability to promote macrophages to phagocytose tumor cells is consistent with the positive control.
  • Antibody Hu5F9-G4 has comparable potency.
  • RBC hemagglutination assay was performed to characterize the RBC agglutination ability of CD47 antibodies.
  • CD47 antibodies were screened for RBC agglutination by observing their ability to prevent human RBC sedimentation. The specific methods are:
  • Human red blood cells were diluted to 2% in PBS and mixed with instilled CD47 antibodies (the concentrations were 200ug/mL, 100ug/mL, 50ug/mL, 25ug/mL, 12.5ug/mL, 6.25ug/mL, and 1.5625ug/mL). , 0.78125ug/mL, 0.390625ug/mL, 0.195313ug/mL, 0.097656ug/mL) and incubate at 37°C for 2 hours in a round-bottom 96-well plate.
  • the RBC agglutination ability measurement results of the CD47 antibody are shown in Figure 10.
  • the results show that when the CD47 antibody concentration reaches 200ug/mL, it does not cause cell aggregation, indicating that the CD47 antibody of the present disclosure has the effect of significantly reducing red blood cell hemagglutination.
  • the CD47 antibody is clinically useful It can significantly reduce side effects and has good safety in treating cancer.
  • CD47 monoclonal antibodies have the property of binding to human red blood cells.
  • CD47 antibody inhibitors there are potential risks of interference with red blood cells and tumor off-targeting. If antibodies with low binding activity to red blood cells can be screened, the risk of off-targeting can be reduced and their safety improved. Specific steps are as follows:
  • Antibody dilution Use FACS buffer to dilute the CD47 antibody to an initial concentration of 20 ⁇ g/mL, a volume of 180uL, and a 3-fold gradient dilution (60+120).
  • the CD47 monoclonal antibody has the activity characteristic of binding to platelets and has many side effects caused by platelet reduction. Antibodies with low platelet binding can reduce the risk of off-target and improve their safety. The specific method is as follows:
  • Antibody dilution Use FACS buffer to dilute the antibody to 20 ⁇ g/ml, with a volume of 240ul;
  • PE fluorescent secondary antibody 1:500 dilution PE goat anti-human IgG Fc; biolegend; 409304
  • add 100ul/well to the corresponding 96-well plate
  • add 1ul APC anti human CD61 (biolegend) to each well.
  • 336411 resuspend the cells and incubate at 2°C-8°C for 30min;
  • Hu5F9-G4 antibody was used as a positive control antibody.
  • Test Example 12 Analysis of the effect of CD47 antibody on activating macrophages to phagocytose red blood cells
  • the detection method is the same as in Test Example 8, except that red blood cells are replaced with tumor cells as target cells, and whether the CD47 antibody of the present disclosure has an activating effect on macrophages phagocytosis of red blood cells is detected.
  • Hu5F9-G4 antibody was used as a positive control antibody.
  • mice purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.
  • Hu5F9-G4 antibody and TJC-4 antibody were used as positive control antibodies
  • hIgG4 was used as isotype control antibody.
  • the anti-tumor results of the CD47 antibody against the human B lymphocyte subcutaneous transplanted tumor model are shown in Figure 14.
  • the results show that the CD47 antibody of the present disclosure is significantly better than the positive control antibody in the anti-human B lymphocyte subcutaneous transplanted tumor effect.
  • the anti-tumor results of the CD47 antibody against human malignant melanoma model are shown in Figure 15.
  • the results show that compared with the positive control antibody, the CD47 antibody of the present disclosure has more advantages in the anti-human malignant melanoma effect.

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Abstract

本公开提供了一种含抗CD47抗体或其抗原结合片段的制剂及其制备方法和应用，本发明的抗CD47抗体能有效阻断CD47与SIRPα结合，但其与红细胞表现出极弱水平结合，展现出与CD47阳性肿瘤细胞的靶向特异性，本发明的制剂具有较高稳定性。

Description

一种含抗CD47抗体或其抗原结合片段的制剂及其制备方法和应用
相关申请的交叉引用
本公开要求于2022年06月21日向中国国家知识产权局提交的、专利申请号为CN202210709122.2，发明名称为“一种含抗CD47抗体或其抗原结合片段的制剂及其制备方法和应用”的专利申请的优先权和权益，并且通过援引将其全文并入本公开。
技术领域
本公开涉及生物制剂领域，具体而言，涉及一种含抗CD47抗体或其抗原结合片段的制剂及其制备方法和应用。
背景技术
癌症免疫疗法是近年来生物科学领域的重头大戏，基于T细胞的CTLA4抗体、PD-1抗体、PD-L1抗体等的免疫检查点抑制剂疗法和CAR-T、TCR-T等细胞疗法皆是近年来大热的免疫疗法。这些都是围绕如何恢复T细胞功能来进行，换言之，主要围绕如何提高获得性免疫系统能力。但是，以免疫检查点(checkpoint)为靶点，激活T细胞功能，以提高获得性免疫系统的能力，进而攻克癌症的道路仍充满曲折。固有免疫系统在肿瘤免疫治疗中的作用长期没有得到发挥。事实上，在整个肿瘤浸润区域，巨噬细胞在肿瘤组织约占50％，更重要的是巨噬细胞的数量同肿瘤的预后呈现反向联系，这进一步说明巨噬细胞在肿瘤中有很重要的作用。巨噬细胞发挥吞噬效应需要两个信号同时起作用：一个是靶向细胞表面的“吃我”信号的激活，另一个是相同目标表面“别吃我”信号的失活。任何一个信号的缺少都不足以引发吞噬效应的发生。越来越多的证据表明，CD47是一类“别吃我”信号，它通过与巨噬细胞表面的信号调节蛋白α(Signal regulatory proteinα，SIRPα)相互结合抑制巨噬细胞的吞噬作用。肿瘤细胞也可以通过CD47的表达逃避巨噬细胞吞噬作用(例如参见EP2242512及其中引用的相关文献)。
CD47也称为整联蛋白相关蛋白(IAP)，是具有氨基末端的免疫球蛋白结构域和羧基末端的多重跨膜区的50kDa膜蛋白。它与多种配体相互作用，包括但不限于单调节蛋白α(SIRPα)，SIRPγ，整联蛋白和血小板反应蛋白-1(TSP-1)。SIRPα主要在骨髓细胞上表达，包括巨噬细胞，骨髓树突细胞(DC)，粒细胞，肥大细胞及其前体(前体包括造血干细胞)。CD47/SIRPα相互作用传递“不要吃我”信号，抑制自体吞噬作用。对患者肿瘤和匹配的邻近正常(非肿瘤)组织的分析显示，CD47蛋白在癌细胞上过表达，这有效地帮助它们逃避先天免疫监视和消除。阻断CD47-SIRPα与抗CD47抗体的相互作用已经显示出有效诱导体外肿瘤细胞的吞噬作用并抑制体内各种血液和实体肿瘤的生长。因此，CD47是癌症治疗的有效靶标，并且需要其适当的拮抗剂来制备人类治疗剂。
发明内容
本公开的目的在于提供一种含抗CD47抗体或其抗原结合片段的制剂及其制备方法和应用。
本公开是这样实现的：
本公开提供了一种含抗CD47抗体或其抗原结合片段的制剂，其包括以下组分：抗CD47抗体或其抗原结合片段、缓冲液和辅料。所述抗体或其抗原结合片段含有以下CDRs：如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ ID NO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3，如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，如SEQ ID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3；按组分在所述制剂中的质量体积百分数计，所述辅料包括：1％～20％的糖类和0.01％～0.5％的表面活性剂。
本公开提供了如前述实施例所述的含抗CD47抗体或其抗原结合片段的制剂的制备方法，其包括：将所述制剂的组分混合。
本公开提供了如前述实施例所述的制剂在制备用于防治CD47阳性肿瘤的产品中的应用。
本公开提供了一种含抗CD47抗体或其抗原结合片段的冻干制剂，其由如前述实施例所述的制剂冷冻干燥后获得。
本公开提供了一种含抗CD47抗体或其抗原结合片段的制剂，其由如前述任意实施例所述的冻干制剂复溶后获 得。
本公开还提供了用作药物的如前述任意实施例所述的制剂，所述药物用于防治CD47阳性肿瘤。
本公开还提供了一种治疗CD47阳性肿瘤的方法，所述方法包括向有需要的受试者施用治疗有效量的如前述任意实施例所述的制剂。
本公开具有以下有益效果：
本公开创新性发明了一种含有抗CD47抗体或抗CD47抗体片段的制剂，该制剂中包含的抗CD47抗体或抗CD47抗体片段在体外未发生红细胞血凝集，其与红细胞表现出极弱水平的低结合或不结合；且该抗体能有效阻断CD47与SIRPα结合，激活介导巨噬细胞对肿瘤细胞的吞噬活性，展现出与CD47阳性肿瘤细胞的靶向特异性，具有高效、安全性好、无药物副作用等优点。
此外，制剂中其他组分及其配比都是发明人经一系列创造性劳动后提出并验证的，适用于搭配上述抗CD47抗体或抗CD47抗体片段使用，能够有效维持抗CD47抗体或抗CD47抗体片段的功能，延长其使用期限，具有较高稳定性，为抗CD47抗体的相关研究和应用提供了途径。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为试验例1中的DSC图谱。
图2为试验例2中的DSC图谱。
图3是试验例5中CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度。
图4是试验例5中CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度。
图5是试验例6中CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、7A11H52、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度。
图6是试验例6中CD47抗体(7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度。
图7是试验例7中CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42和hIgG4-同型对照)对人CD47的CD47/SIRPα结合抑制结果。
图8是试验例7中CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55)对人CD47的CD47/SIRPα结合抑制结果。
图9是试验例8中CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力测定结果。
图10是试验例9中CD47抗体的RBC凝集能力测定结果。
图11是试验例10中CD47抗体与人类红细胞结合能力的测定结果。
图12是试验例11中CD47抗体与人类血小板结合分析结果。
图13是试验例12中CD47抗体对激活巨噬细胞吞噬红细胞的作用分析结果。
图14是试验例13中CD47抗体对人B淋巴细胞皮下移植瘤模型的抗肿瘤结果。
图15是试验例14中CD47抗体对人恶性黑色素瘤模型的抗肿瘤结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
本公开实施例提供了一种含抗CD47抗体或其抗原结合片段的制剂，其包括以下组分：抗CD47抗体或其抗原结 合片段、缓冲液和辅料。
在本公开中，“抗体或其抗原结合片段”此技术术语是结合特定抗原的蛋白，其泛指包含互补决定区(CDR区)的蛋白及蛋白片段。“抗体”指全长抗体。
术语“抗原结合片段”是包含抗体CDR的的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至靶抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。在一些实施方式中，抗原结合片段具有特异性识别并结合CD47的作用。在一些实施方式中，抗原结合片段是具有阻断CD47与其配体结合，激活免疫细胞功能的片段，在一个方面中，此类片段选自Fab(由完整的轻链和Fd构成)，Fv(由VH和VL构成)，ScFv(单链抗体，VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。所述片段可通过常规方法制备获得，例如可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
术语“互补性决定区”或“CDR”是指免疫球蛋白的重链和轻链的高度可变区。有三种重链CDR和三种轻链CDR。术语“CDR”和“CDRs”用于指包含一种或多种或者甚至全部的对抗体或其抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。
在本公开中，重链的互补决定区用HCDR表示，轻链的互补决定区用LCDR表示。本领域常用的CDR标示方法包括：Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。在本公开具体实施例中，采用Kabat注释标准标示CDR区，但其他方法标示的CDR区也属于本公开的保护范围。在一些实施例中，所述抗体或其抗原结合片段含有以下CDRs：如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ ID NO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3，如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，如SEQ ID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3，序列信息如下。
在一些实施例中，与SEQ ID NO:1具有至少95％同源性的氨基酸序列包括但不限于KSSQSLLNTRTRKNYLA(SEQ ID NO:21)。与SEQ ID NO:11具有至少95％同源性的氨基酸序列包括但不限于MIHPSDSETRLNQKFQG(SEQ ID NO:22)。
在一些实施例中，所述抗体或其抗原结合片段包括：如SEQ ID NO:1所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:11所示的HCDR2，和如SEQ ID NO:12所示的HCDR3；所述抗体或其抗原结合片段包括：如SEQ ID NO:1所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:11所示的HCDR2，和如SEQ ID NO:12所示的HCDR3；或者所述抗体或其抗原结合片段包括：如SEQ ID NO:1所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:22所示的HCDR2，和如SEQ ID NO:12所示的HCDR3。
在一些实施例中，按组分在所述制剂中的质量体积百分数(w/v)计，所述辅料包括：1％～20％的糖类和0.01％～0.5％的表面活性剂。
本公开提供了抗CD47抗体或其抗原结合片段，该抗体在体外未发生红细胞血凝集，更难能可贵的是与红细胞表现出极弱水平的低结合或不结合；能够有效地阻断CD47与SIRPα结合、激活介导巨噬细胞对肿瘤细胞的吞噬活性，展现出了显著的与CD47阳性肿瘤细胞的靶向特异性，亲和力高、特异性强，不会引起红细胞凝集，且与人类红细胞、血小板表现出极弱的结合，安全性好。
本公开实施例提供的制剂组合是发明人经一系列创造性劳动，提出并验证的组合配方，有利于使得上述抗CD47抗体或其抗原结合片段稳定而有效地发挥其作用，有效阻断CD47与SIRPα结合，使得抗CD47抗体能更好的应用于临床。
在一些实施例中，表面活性剂的质量体积百分数可以为0.01％、0.02％、0.04％、0.06％、0.08％、0.1％、0.12％、0.14％、0.16％、0.18％、0.2％、0.22％、0.24％、0.26％、0.28％、0.3％、0.32％、0.34％、0.36％、0.38％、0.4％、0.42％、0.44％、0.46％、0.48％、0.5％中的任意一种或任意两种之间的范围。例如，表面活性剂的质量体积百分数0.02％，也即0.02％(w/v),表示每100mL体积溶液含0.02g的表面活性剂；其它依此类推，下同。
在一些实施例中，所述表面活性剂选自吐温20(聚山梨酯-20)、吐温80(聚山梨酯-80)和泊洛沙姆188中的任意一种。
在一些实施例中，当所述表面活性剂为吐温80时，所述吐温80在所述制剂中的质量体积百分数为0.01％～0.1％，可选地为0.01％～0.04％，可选地为0.02％～0.04％。
当所述表面活性剂为泊洛沙姆188时，所述泊洛沙姆188在所述制剂中的质量体积百分数为0.05％～0.5％，可选地为0.1％～0.2％，可选地为0.1％。
在一些实施例中，所述表面活性剂为0.02％(w/v)的吐温80。
在一些实施例中，糖类的质量体积百分数(w/v)可以为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％中的任意一种或任意两种之间的范围。
在一些实施例中，所述糖类包括蔗糖和海藻糖中的至少一种。
在一些实施例中，所述糖类为蔗糖。
在一些实施例中，所述蔗糖的浓度为3％～15％，可选地为6％～10％，可选地为8％。在一些实施例中，所述蔗糖在所述制剂中的质量体积百分数为8％。
在一些实施例中，所述糖类为海藻糖。
在一些实施例中，所述海藻糖的浓度为3％～15％，可选地为6％～10％，可选地为8.8％。
在一些实施例中，所述糖类为8％(w/v)的蔗糖。
“缓冲液”指通过其酸-碱共轭组分的作用而耐受pH变化的溶液。将pH控制在适当范围中的缓冲液的例子包括但不限于：包含醋酸盐、组氨酸、柠檬酸盐或磷酸盐等的缓冲液。
在一些实施例中，缓冲液为由柠檬酸一水合物与柠檬酸钠二水合物配置而成的柠檬酸-柠檬酸钠缓冲液，由L-组氨酸与L-组氨酸盐酸盐一水合物配置而成的组氨酸-组氨酸盐缓冲液，由冰醋酸与三水醋酸钠配置而成的醋酸-醋酸钠缓冲液，或者由磷酸二氢钠与磷酸氢二钠配置而成的磷酸-磷酸盐缓冲液；
在一些实施例中，所述缓冲液选自：柠檬酸-柠檬酸钠缓冲液、醋酸-醋酸钠缓冲液、组氨酸-组氨酸盐缓冲液和磷酸-磷酸盐缓冲液中的任意一种，pH为4.5～7.5，pH具体可以为4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、和7.5中的任意一种或任意两种之间的范围。
在一些实施例中，所述缓冲液选自柠檬酸-柠檬酸钠缓冲液、组氨酸-组氨酸盐缓冲液和磷酸-磷酸盐缓冲液中的任意一种，pH为5.0～7.5。
可选地，所述缓冲液为组氨酸-组氨酸盐缓冲液，pH为5.5～6.0。
可选地，所述组氨酸-组氨酸盐缓冲液的pH为5.5。相对于其他缓冲液而言，组氨酸-组氨酸盐缓冲液能够更有效地使得抗体分子维持构象稳定。
在一些实施例中，组氨酸-组氨酸盐缓冲液由L-组氨酸与L-组氨酸盐酸盐一水合物配置而成的组氨酸-盐酸组氨酸缓冲液。
在一些实施例中，所述缓冲液的浓度为10～40mM，具体可以为10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM、32mM、34mM、36mM、38mM、40mM中的任意一种或任意两种之间的范围。
可选地，所述缓冲液的浓度为15～25mM。
可选地，所述缓冲液的浓度为20mM。
在一些实施例中，所述缓冲液为pH为6.0的20mM的组氨酸-盐酸组氨酸缓冲液。
在一些实施例中，所述制剂还包括甲硫氨酸。
在一些实施例中，所述甲硫氨酸的浓度为1～20mM，具体可以为1mM、2mM、4mM、6mM、8mM、10mM、12mM、14mM、16mM、18mM、20mM中的任意一种或任意两种之间的范围。
可选地，所述甲硫氨酸的浓度为4.5～5.5mM。
可选地，所述甲硫氨酸的浓度为5mM。
在一些实施例中，所述抗CD47抗体或其抗原结合片段的浓度为20～60mg/mL具体可以为20mg/mL、25mg/mL、30mg/mL、35mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL中的任意一种或任意两种之间的范围。
可选地，所述抗CD47抗体或其抗原结合片段的浓度为45～55mg/mL。
可选地，所述抗CD47抗体或其抗原结合片段的浓度为50mg/mL。
在一些实施例中，所述抗CD47抗体或其抗原结合片段包括有重链可变区和轻链可变区。
在一些实施例中，所述重链可变区的氨基酸序列如SEQ ID NO:4～9中任一项所示或与SEQ ID NO:4～9任一项所示具有至少95％同源性的序列；所述轻链可变区的氨基酸序列如SEQ ID NO:13～18中任一项所示或与SEQ ID NO:13～18任一项所示具有至少95％同源性的序列。抗体及其重链可变区和轻链可变区对应的序列如下：
SEQ ID NO:4所示序列如下：
SEQ ID NO:5所示序列如下：
SEQ ID NO:6所示序列如下：
SEQ ID NO:7所示序列如下：
SEQ ID NO:8所示序列如下：
SEQ ID NO:9所示序列如下：
SEQ ID NO:13所示序列如下：
SEQ ID NO:14所示序列如下：
SEQ ID NO:15所示序列如下：
SEQ ID NO:16所示序列如下:
SEQ ID NO:17所示序列如下:
SEQ ID NO:18所示序列如下:
在一些实施例中，所述抗CD47抗体或其抗原结合片段还包括恒定区。
在一些实施例中，所述恒定区包括重链恒定区和轻链恒定区，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；轻链恒定区为κ或λ链。
在一些实施例中，所述恒定区的种属来源选自鼠、兔、羊、猴、或人。
在一些实施例中，所述抗CD47抗体为7A11H14抗体，7A11H14的重链如SEQ ID NO:19所示，轻链如SEQ ID NO:20所示：
7A11H14的重链氨基酸序列(SEQ ID NO:19)：
7A11H14的轻链氨基酸序列(SEQ ID NO:20)：
在一些实施例中，所述抗体为CDR移植抗体、多聚体抗体或双特异性抗体中的任一种或几种。
在一些实施例中，所述抗原结合片段为F(ab’)2、Fab、scFv和Fv中的任一种或几种。
在一些实施方式中，所述CD47为人CD47、鼠CD47或猴CD47。
本公开提供的含抗CD47抗体或其抗原结合片段的制剂适用于申请号为“202011544262.6”、发明名称为“一种靶向CD47的抗体及其应用”的中国申请(2020年12月23递交)中公开的多个抗CD47抗体或其抗原结合片段。也适用于以中国专利申请202011544262.6为优先权的PCT专利申请(申请号：PCT/CN2021/140404，申请日：2021-12-22)，这些专利申请的全文通过引用并入本公开。
本公开实施例还提供了如前述任意实施例所述的含抗CD47抗体或其抗原结合片段的制剂的制备方法，其包括： 将所述制剂的组分混合。
在一些实施例中，当所述制剂中的抗CD47抗体或其抗原结合片段为抗CD47抗体或其抗原结合片段的溶液时，所述制备方法还包括：采用所述制剂中的缓冲液对抗CD47抗体或其抗原结合片段的溶液进行置换；置换后与辅料混合。
本公开实施例还提供了如前述任意实施例所述的制剂在制备用于防治CD47阳性肿瘤的产品中的应用。
本公开实施例还提供了用作药物的如前述任意实施例所述的制剂，所述药物用于防治CD47阳性肿瘤。
本公开实施例还提供了一种治疗CD47阳性肿瘤的方法，所述方法包括向有需要的受试者施用治疗有效量的如前述任意实施例所述的制剂。
本文中的“防治”包括预防和/或治疗，治疗可以理解为改善病情或治愈。
本文中的“CD47阳性肿瘤”包括表达CD47的肿瘤，具体包括但不限于实体肿瘤和血液肿瘤，例如，血液系统恶性肿瘤、非霍奇金淋巴瘤、多发性骨髓瘤、B细胞淋巴瘤、肝细胞癌、卵巢癌、结直肠癌、肺癌、乳腺癌、黑色素瘤和肾脏肿瘤等。
本公开实施例还提供了一种含抗CD47抗体或其抗原结合片段的冻干制剂，所述冻干制剂由如前述任意实施例所述的制剂冷冻干燥后获得。
此外，本公开实施例还提供了一种含抗CD47抗体或其抗原结合片段的制剂，其由如前述任意实施例所述的冻干制剂复溶后获得。
实验材料：实施例1～4以及试验例1～3中示例性地展示了制剂中的抗CD47抗体为7A11H14时的技术效果，7A11H14来源于申请号为202011544262.6，发明名称为“一种靶向CD47的抗体及其应用”的中国申请(2020年12月23递交)中公开的(重链可变区的序列如SEQ ID NO:14所示，轻链可变区的序列如SEQ ID NO:8所示，恒定区为含有S228P变异的人类IgG4的Fc区)，其包括如SEQ ID NO:19所示的重链，和如SEQ ID NO:20所示的轻链。需要说明的是，在其他实施例中，制剂中的抗CD47抗体或其抗原结合片段也可以采用申请号为202011544262.6，发明名称为“一种靶向CD47的抗体及其应用”的中国申请中公开的其他抗CD47抗体或抗原结合片段，均可达到相同或相似的技术效果。
实施例1
一种含抗CD47抗体或其抗原结合片段的制剂，其包括：50mg/mL抗CD47抗体，20mM组氨酸-组氨酸盐缓冲体系(pH6.0)，8％(w/v)蔗糖以及0.02％(w/v)吐温80。
可以理解的是，各组分限定的浓度为各组分在制剂中的浓度，本公开中“w/v”是质量体积百分数，例如8％(w/v)蔗糖表示每100mL体积溶液含8g的蔗糖，其它依此类推。
实施例2
一种含抗CD47抗体或其抗原结合片段的制剂，其包括：50mg/mL抗CD47抗体，20mM组氨酸-组氨酸盐缓冲体系(pH6.0)，8.8％(w/v)蔗糖以及0.02％(w/v)吐温80。
实施例3
一种含抗CD47抗体或其抗原结合片段的制剂，其包括：50mg/mL抗CD47抗体，20mM组氨酸-组氨酸盐缓冲体系(pH6.0)，8.8％(w/v)蔗糖，0.04％(w/v)吐温80，以及5mM甲硫氨酸。
实施例4
一种含抗CD47抗体或其抗原结合片段的制剂，其包括：50mg/mL抗CD47抗体，20mM组氨酸-组氨酸盐缓冲体系(pH6.0)，8.8％(w/v)蔗糖，以及0.1％(w/v)泊洛沙姆188。
试验例1不同缓冲液和pH对抗CD47抗体稳定性的影响
将抗CD47抗体分别以50mg/mL的浓度超滤置换至以下表1配制20mM柠檬酸缓冲液(20mM Citrate)、20mM醋酸缓冲液(20mM acetate)、20mM组氨酸缓冲液(20mM histidine)和20mM磷酸缓冲液(20mM PB)中，分别将溶液按照表1的pH调节为4.5、5.0、5.5、6.0、6.5、7.0和7.5。
用配制完成的各个pH缓冲液分别对小试样品进行超滤置换。置换完成后，将各样品的浓度稀释至50mg/ml过滤分装西林瓶中加塞轧盖后放样(将样品置于40℃稳定性箱中孵育4周)，通过差式量热扫描(DSC)、分子筛色谱(SEC)和毛细管等电聚焦电泳(CIEF)检测抗体在40℃放置4周的稳定性。具体试验方案见表1。
表1各实验组的缓冲液和pH信息
备注：柠檬酸-柠檬酸钠缓冲体系为由柠檬酸一水合物与柠檬酸钠二水合物配置而成的柠檬酸-柠檬酸钠缓冲液；组氨酸-组氨酸盐缓冲体系为由L-组氨酸与L-组氨酸盐酸盐一水合物配置而成的组氨酸-盐酸组氨酸缓冲液，醋酸-醋酸钠缓冲体系为由冰醋酸与三水醋酸钠配置而成的醋酸-醋酸钠缓冲液，磷酸-磷酸盐缓冲液体系为由磷酸二氢钠与磷酸氢二钠配置而成的磷酸-磷酸盐缓冲液；
(1)差式量热扫描(DSC)
采用差式量热扫描仪MicroCalTM VP DSC进行样品转变熔点温度(Tm)和起始解折叠温度(Tm onset)检测。设置实验参数使得扫描温度以200℃/h的扫描速率从10℃上升到95℃。数据分析采用MicroCalTM VP DSC自动分析软件。
表2.DSC结果
结果见图1和表2，实验结果显示：在相同缓冲体系中，Tm onset值随着pH的升高而增加。在磷酸-磷酸盐缓冲液体系中Tm onset值最高(63.2℃～63.6℃)，在组氨酸-组氨酸盐缓冲体系和柠檬酸-柠檬酸钠缓冲体系Tm onset值分别为56.0℃～61.4℃和54.9℃～61.2℃，在醋酸-醋酸钠缓冲体系中Tm onset值最低(51.4℃～60.1℃)。
数据表明，在组氨酸-组氨酸盐缓冲体系、柠檬酸-柠檬酸钠缓冲体系和磷酸-磷酸盐缓冲液体系中抗体构象稳定性较好。
(2)分子筛色谱(SEC-HPLC)
在安捷伦高效液相色谱系统上使用SEC色谱柱(300×7.8mm,5μm)运行的。样品温度设置为5℃，柱温设置为25℃。流动相组成为50mM PB,300mM NaCl,pH 6.8±0.1，流速设置为1.0mL/min。使用流动相将样品稀释至10 mg/mL浓度进样(100μg样品)至系统并运行20min，检测波长设置为280nm。使用安捷伦CDS软件进行数据分析。
表3.SEC-HPLC检测结果
结果见表3，结果显示：在柠檬酸-柠檬酸钠缓冲体系中SEC-HPLC主峰纯度下降1.9％～6.7％；醋酸-醋酸钠缓冲体系中SEC-HPLC主峰纯度下降2.1～2.9％；组氨酸-组氨酸盐缓冲体系中SEC-HPLC主峰纯度下降1.7％～1.9％；磷酸-磷酸盐缓冲液体系中SEC-HPLC主峰纯度下降3.7％～6.6％。
综上所示，抗CD47抗体在组氨酸-组氨酸盐缓冲体系中具有较好的稳定性。
(3)毛细管等电聚焦电泳(iCIEF)
采用ProteinSimple公司的毛细管等电聚焦电泳分析仪和氟涂层电泳毛细管对CD47抗体进行的测定。预混液为0.5μL等电点标记物(pI7.05)，0.5μL等电标记物(pI 9.50)，4.0μL两性电解质3-10，35μL 1％甲基纤维素，37.5μL8M尿素溶液和2.5μL超纯水。用超纯水将样品稀释到1.0mg/mL。取20μL稀释液和80μL预混液加到离心管中，混合并离心。样品进样温度为10℃，聚焦第一阶段为1500V一分钟，第二阶段为3000V八分钟，报告主峰、酸蜂和碱峰面积百分比。
表4.iCIEF检测结果

结果见表4，结果显示：柠檬酸-柠檬酸钠缓冲体系中iCIEF主峰下降14％～22.7％，醋酸-醋酸钠缓冲体系中iCIEF主峰下降10.9％～13.3％，组氨酸-组氨酸盐缓冲体系中iCIEF主峰下降11.2％～16.5％，磷酸-磷酸盐缓冲液体系中SEC-HPLC主峰纯度下降26.2％～30.0％。
其中，组氨酸-组氨酸盐缓冲体系中，pH5.5、pH6.0的条件下，主峰纯度下降幅度小(分别下降11.2％、11.4％)。
试验例2不同辅料对抗CD47抗体稳定性的影响
将CD47抗体超滤置换至添加不同辅料的缓冲液中，在40℃条件放置4周、或经振摇(300rpm，25℃)3天、或冻融循环(-70℃至室温)5轮，然后分别用外观、DSC、SEC-HPLC、iCIEF和不溶性微粒测试(HIAC)分析溶液中CD47抗体蛋白的分布情况，并分别以未经处理的初始样品和未添加糖类的样品作为对照。详细的制剂配方信息如表5所示。
表5.详细的制剂配方信息

(1)外观
外观检测包括颜色、澄明度和有无可见颗粒。
表6.外观检测结果

详细结果详见表6，结果显示：在T0点，制剂配方F01-F31外观均为无色、微乳光、无可见颗粒。经振摇(300rpm，25℃)3天、或冻融循环(-70℃至室温)5轮、或40℃孵育2周后，制剂配方F01-F31均无可见颗粒的产生。经40℃孵育4周后，制剂配方F01-F08、F10-F20、F22-F31外观仍为无色、微乳光、无可见颗粒，但制剂配方F21出现小于等于5颗的可见颗粒，制剂配方F09出现5颗以上的可见颗粒。
(2)差式量热扫描(DSC)
采用差式量热扫描仪MicroCalTM VP DSC进行样品转变熔点温度(Tm)和起始解折叠温度(Tm onset)检测。设置实验参数使得扫描温度以200℃/h的扫描速率从10℃上升到95℃。数据分析采用MicroCalTM VP DSC自动分析软件。
表7.DSC分析结果
结果见表7和图2，实验结果显示：在相同缓冲体系中，添加不同种类的辅料，制剂配方F01-F09的Tmonset值没有明显差别，范围在57.9℃～60.3℃。
数据表明CD47抗体蛋白在不同的制剂配方中具有相近的热稳定性。
(3)不溶性微粒测试(HIAC)
在生物安全柜中用HACH不溶性微粒分析仪检测样品中不溶性微粒的尺寸和颗粒数。使用前先用超纯水清洗系统，测试超纯水中的不溶性微粒。环境通过测试后(10μm微粒数小于25粒每25毫升)测试样品。每个样品连续进样4次，每次进样1mL，舍弃第1次进样检测结果，结果报告后3次连续检测结果中每毫升粒径≥2μm，≥5μm，≥10μm和≥25μm的粒子数量平均值。
表8.样品经振荡处理后的HIAC分析结果
表9.经40℃孵育4周处理后的HIAC分析结果
详细结果参见表8和表9，结果显示：经振摇(300rpm，25℃)3天后，制剂配方F05不溶性微粒数在几种水平上(≥5μm；≥10μm)高于其他制剂配方；经40℃孵育4周后，所有制剂配方微粒数无明显增长。
(4)分子筛色谱(SEC-HPLC)
在安捷伦高效液相色谱系统上使用SEC色谱柱(300×7.8mm,5μm)运行的。样品温度设置为5℃，柱温设置为25℃。流动相组成为50mM PB,300mM NaCl,pH 6.8±0.1，流速设置为1.0mL/min。使用流动相将样品稀释至10mg/mL浓度进样至系统并运行20min，检测波长设置为280nm。使用安捷伦CDS软件进行数据分析。
表10-1. 40℃孵育4周SEC-HPLC检测结果

备注：表10-1为将样品置于40℃稳定性箱中孵育4周后检测结果
表10-2.冻融5轮SEC-HPLC检测结果
备注：表10-2为冻融循环(-70℃至室温)5轮后检测结果
表10-3.振摇3天SEC-HPLC检测结果

备注：表10-3为振摇(300rpm，25℃)3天后检测结果
详细结果详见表10-1、10-2、10-3，结果显示：经冻融循环(-70℃至室温)5轮后，所有制剂配方SEC-HPLC结果均无明显变化，表明CD47抗体蛋白在所有候选制剂配方中均表现出良好的冻融稳定性。经振摇(300rpm，25℃)3天后，制剂配方F01和F04中SEC-HPLC主峰纯度分别下降3.0％和0.8％，其他配方中主峰纯度下降0.0％～0.4％。经40℃孵育4周后，制剂配方F06中SEC-HPLC主峰纯度下降3.0％，其他制剂配方中主峰纯度下降1.3％～2.4％。
综上所述，SEC-HPLC分析表明，制剂配方F01、F04和F06中CD47抗体蛋白的稳定性较差。
(5)毛细管等电聚焦电泳(iCIEF)
采用ProteinSimple公司的毛细管等电聚焦电泳分析仪和氟涂层电泳毛细管对CD47抗体进行的测定。预混液为0.5μL等电点标记物(pI7.05)，0.5μL等电标记物(pI 9.50)，4.0μL两性电解质3-10，35μL 1％甲基纤维素，37.5μL8M尿素溶液和2.5μL超纯水。用超纯水将样品稀释到1.0mg/mL。取20μL稀释液和80μL预混液加到离心管中，混合并离心。样品进样温度为10℃，聚焦第一阶段为1500V一分钟，第二阶段为3000V八分钟，报告主峰、酸蜂和碱峰面积百分比。
表11.iCIEF检测结果
结果见表11，结果显示：制剂配方F06的稳定性较差。
试验例3抗CD47抗体的生产
将人源化可变结构域与分泌信号和人类κ以及人类FcIgG4S228P恒定结构域组合，将其克隆到哺乳动物表达系统中，并且转染到293细胞中以生成人源化mAb。将人源化变体表达为全长IgG分子、分泌到培养基中并使用蛋白质A纯化。
在Expi293细胞中瞬时表达并纯化人源化抗体和阳性对照抗体Hu5F9-G4(Hu5F9-G4序列已在美国专利US2015/0183874A1中公开)。
对于Expi293细胞中抗体的瞬时表达，使用载体pCDNA3.4，首先将抗体的重链和轻链克隆到单独的pCDNA3.4载体中，使用PEI(购自Polysciences)化学转染试剂、按照化学转染的方法将带有抗体分子重链和轻链的pCDNA3.4载体转入Expi293细胞中，按照生产商提供的方案瞬时转染培养的Expi293细胞。
瞬时转染前一天Expi293(ThermoFisher Scientific；A14635)细胞传代，用Dynamis培养基(gibco；A2617502)按2E6的密度接种1L摇瓶(conning；431147)，放入细胞培养摇床(Adolf Kuhner；ISF4-XC)中37℃；8％CO2；120rpm培养；
转染当天，Expi293细胞用细胞计数仪(Countstar；IC1000)计数，用新鲜Dynamis培养稀释调整细胞密度为2.9E6；准备转染；PEI：DNA＝3:1；混匀5min，将二者轻柔混匀20次，静置15～30min。将DNA-PEI混合物加入Expi293细胞中，混匀，放入细胞培养摇床(Adolf Kuhner；ISF4-XC)中37℃；8％CO2；120rpm培养；转染4h之后补加双抗(gibco；15140122)和抗凝剂(gibco；0010057)；
收获上清纯化：转染连续培养7天，之后收样，先低速1000rpm；10min；4℃离心(湘仪H2050R)，再高速12000rpm；30min；4℃；收集细胞培养上清，0.22um过滤。将培养上清应用于Protein A Sepharose柱(GE Healthcare)。柱用PBS洗涤，然后用洗脱缓冲液(0.1M柠檬酸钠缓冲液，pH 3.0)洗脱蛋白质。收集的组分用1M Tris pH9.0中和。最后，将纯化的样品与PBS进行透析。
试验例4 CD47抗体亲和力测定
1、实验方法
采用生物膜干涉技术(ForteBio)测定本公开抗体结合人CD47(hCD47)的平衡解离常数(KD)。ForteBio亲和力测定按照现有的方法(Estep，P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs；2013.5(2)：p.270-8)进行，具体为：传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线，在线加载试验例3获得的经纯化的抗体至AHC传感器(ForteBio)上进行ForteBio亲和测量，再将具有加载的抗体的传感器暴露于100nM的CD47抗原中作用5分钟，之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1：1结合模型进行动力学的分析。
2、实验结果
CD47抗体亲和力测定结果如表12所示，结果显示CD47抗体具有高亲和力。
表12 CD47抗体亲和力测定结果
试验例5抗CD47抗体亲和力测定
1、实验方法
在基于流式细胞术的测定法中测量本公开CD47抗体与人CD47的结合。具体步骤为：
采用表达人CD47的癌细胞系CCRF-CEM(上海中国科学院细胞库)属于人急性淋巴细胞白血病T淋巴细胞。将CCRF-CEM细胞(0.1×106个细胞)与不同浓度(最高浓度为30ug/mL，三倍稀释，共10个浓度)的实验抗体(本公开CD47抗体以及Hu5F9-G4抗体)，在含3％牛血清白蛋白(BSA)的PBS中，冰上孵育30分钟。然后将细胞洗涤至少两次，用FCM buffer(1XPBS+3％BSA)配制PE Goat anti human IgG Fc(1:500x稀释)荧光二抗，按100uL/孔加入对于的96孔板中，4度冰箱孵育30min。取出96孔板，250g离心5min，小心去上清后，加入FCM buffer 200uL/孔，再次250g离心5min，小心去上清，将细胞洗涤至少两次用1xPBS 100uL/孔重悬，并通过流式细胞术进行分析，并根据其MFI用GraphPad拟合浓度依赖的曲线。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与人CD47结合的EC50结果如表13所示，CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度如图3所示，CD47抗体 (7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度如图4所示，结果显示，本公开CD47抗体与阳性对照抗体对细胞水平的人CD47具有相当的特异性结合能力。
表13 CD47抗体与人CD47的结合能力测定结果
试验例6抗CD47抗体与猴CD47的结合
1、实验方法
通过转染携带全长猴CD47的pCDNA3.4载体，产生过表达猴CD47的CHO细胞稳定细胞株(CHO-cynoCD47细胞)，将CHO-cynoCD47细胞(0.1×106个细胞)与不同浓度(最高浓度为10ug/mL，三倍稀释，共10个浓度)的实验抗体(本公开CD47抗体以及Hu5F9-G4抗体)，在含3％牛血清白蛋白(BSA)的PBS中，冰上孵育30分钟。然后将细胞洗涤至少两次，用FCM buffer(1XPBS+3％BSA)配制PE Goat anti human IgG Fc(1:500x稀释)荧光二抗，按100uL/孔加入对于的96孔板中，4度冰箱孵育30min。取出96孔板，250g离心5min，小心去上清后，加入FCM buffer200uL/孔，再次250g离心5min，小心去上清，将细胞洗涤至少两次用1x PBS 100uL/孔重悬，并通过流式细胞术进行分析，并根据其MFI用GraphPad拟合浓度依赖的曲线。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与猴CD47结合的EC50结果如表14所示，CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、7A11H52、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度如图5所示，CD47抗体(7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度如图6所示，结果显示，相比，本公开抗体与阳性对照抗体对细胞水平形式猴具有相当的特异性结合能力。
表14 CD47抗体与猴CD47结合的EC50结果

试验例7抗CD47抗体对人CD47配体SIRPα与CD47相互作用的阻断
1、实验方法
通过流式细胞术测定本公开CD47抗体阻断人CD47与SIRPα的结合能力。具体步骤为：
抗体稀释：用FCM buffer(1XPBS+3％BSA)将本公开CD47抗体和对照抗体Hu5F9-G4稀释成90ug/mL，然后3倍梯度稀释成10个浓度，将亚型对照hIgG4稀释成30ug/mL、1.1ug/mL、0.04ug/mL，配体hSIRPα-mFC(AcroBiosystems)稀释至10ug/mL。
将CCRF-CEM(上海中国科学院细胞库)细胞按0.1×106个细胞/孔加入到96孔V型板，并且在CD47抗体量增加的条件下监控hSIRPα-mFC结合。使用PE Goat anti mouse IgG Fc二抗(Biolegend)确定结合的SIRPα。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、Hu5F9-G4和hIgG4-同型对照)对人CD47的CD47/SIRPα结合抑制结果如图7所示，CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55)对人CD47的CD47/SIRPα结合抑制结果如图8所示，结果显示，本公开CD47抗体能够在细胞水平显著抑制阻断CD47与SIRPα的结合，与阳性对照抗体相比，本公开CD47抗体表现出相当的阻断能力。
试验例8 CD47抗体促进巨噬细胞吞噬肿瘤细胞能力的检测
1、实验方法
在基于流式细胞术的测定法中测定本公开CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力。具体步骤为：
取捐赠者的新鲜血液，分离得到外周血单核细胞(PBMC)，并通过hCD14磁珠(Miltenyi/130-050-201)从PBMC中分离CD14阳性单核细胞，配置含rhGM-CSF(R&D；7954-GM-010)的完全培养基，rhGM-CSF终浓度为50ng/mL，CD14阳性单核细胞的浓度为5E5/mL，按20mL/皿加入到细胞培养皿中；转移至5％CO2 37℃细胞培养箱中，每3天对半更换新鲜培养基；(含50ng/mLGM-CSF)继续培养4天。在第8天将巨噬细胞上清吸到15mL离心管中，同时加入预冷的DPBS，直接用细胞刮刀收集细胞；
选择人CD47高表达的肿瘤细胞系Jurkat(上海中国科学院细胞库)作为靶细胞类型，将靶肿瘤细胞按照CellTraceTM CFSE试剂盒的说明，进行荧光标记。将标记好的肿瘤细胞与上述已经完成分化的巨噬细胞按照1：1的比例共培养，同时加入终浓度10ug/mL、1ug/mL、0.1ug/mL抗体在37℃孵育2小时。然后将细胞洗涤至少两次，将细胞小心吹下，加入别藻青蛋白(allophycocyanin，APC)标记的CD14抗体(购自Biolegend；B259538)，在含0.1％BSA的PBS中冰上(避光)孵育30分钟。将细胞洗涤至少两次并通过流式细胞术进行分析。被吞噬的细胞群体为活细胞中CD14阳性并且荧光染料CFSE(carboxyfluorescein diacetate，succinimidyl ester，羧基荧光素双乙酸盐，琥珀酰亚胺酯)也为阳性的细胞群体。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力测定结果如图9所示，结果显示，本公开CD47抗体具有促进巨噬细胞吞噬肿瘤细胞的能力，且促进巨噬细胞吞噬肿瘤细胞的能力与阳性对照抗体Hu5F9-G4的能力相当。
试验例9 CD47抗体对人类红细胞(RBC)血凝集分析
1、实验方法
进行红细胞血凝集分析来表征CD47抗体的RBC凝集能力。通过观察抗体避免人RBC发生沉降的能力就RBC凝集对CD47抗体进行筛选。具体方法为：
人类红细胞在PBS中稀释到2％，与滴入的CD47抗体(浓度依次为200ug/mL、100ug/mL、50ug/mL、25ug/mL、12.5ug/mL、6.25ug/mL、1.5625ug/mL、0.78125ug/mL、0.390625ug/mL、0.195313ug/mL、0.097656ug/mL)在圆底96孔板内在37℃孵育2小时。未沉淀的红细胞的存在是证明红细胞血凝聚的证据，与未凝聚的红细胞沉淀形成清晰的红点相比，未沉淀的红细胞呈雾状。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体的RBC凝集能力测定结果如图10所示，结果显示，当CD47抗体浓度达到200ug/mL时均未引起细胞凝聚，表明本公开CD47抗体具有显著降低红细胞血凝聚的作用，CD47抗体在临床治疗癌症中能够显著降低其副作用、安全性好。
试验例10 CD47抗体与人类红细胞(RBC)结合分析
1、实验方法
CD47单抗存在与人类红细胞结合的特性，对CD47抗体抑制剂来说，存在药效受红细胞干扰及肿瘤脱靶的潜在风险。若能筛选到与红细胞结合活性低的抗体则能够降低脱靶的风险，提高其安全性。具体步骤如下：
(1)抗体稀释：用FACS buffer将CD47抗体稀释成初始浓度20μg/mL，体积180uL，3倍梯度稀释(60+120)，
11个浓度；
(2)细胞计数并铺板：将RBC细胞离心250g 5min后弃去上清，用FACS buffer调整细胞密度为2E+06，按100uL/管均分到96孔V型板中；
(3)将上述稀释好的抗体加入细胞中，100uL/孔，2℃～8℃孵育0.5h；
(4)取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200uL/孔，再次250g离心5min，小心去上清；
(5)用FACS buffer配制PE goat anti-human IgG Fc(biolegend)荧光二抗(1:500稀释)，按100uL/孔加入对应的96孔板中，重悬细胞，2℃～8℃孵育30min；
(6)取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200uL/孔，再次250g离心5min，小心去上清；
(7)用1xPBS 100uL/孔重悬，FACS检测。使用流式细胞仪(Beckman，cytoflex)分析数据，GraphPad Prism作图。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与人类红细胞结合能力的测定结果如图11所示，结果显示，与阳性对照抗体相比，本公开CD47抗体与人类红细胞的结合活性显著降低，其作为药物时的安全性提高。
试验例11 CD47抗体与血小板结合分析
1、实验方法
同CD47单抗结合人类红细胞一样，CD47单抗存在与血小板结合的活性特征，存在血小板降低带来的诸多副作用。与血小板结合低的抗体能够降低脱靶的风险，提高其安全性。具体方法如下：
抗体稀释：用FACS buffer将抗体稀释成20μg/ml，体积240ul；
细胞计数并铺板：将全血细胞稀释20倍，按100uL/管均分到96孔V型板中；
将上述稀释好的抗体加入到细胞中，100uL/孔，2℃-8℃孵育0.5h；
取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200ul/孔，再次250g离心5min，小心去上清；
用FACS buffer配制PE荧光二抗1:500稀释(PE goat anti-human IgG Fc；biolegend；409304)，按100ul/孔加入对于的96孔板中，同时每孔中加入1ul APC anti human CD61(biolegend；336411),重悬细胞，2℃-8℃孵育30min；
取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200ul/孔，再次250g离心5min，小心去上清；
用1xPBS 200uL/孔重悬，FACS检测。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与血小板结合分析结果如图12所示，结果显示本公开的CD47抗体与血小板的结合活性显著低于阳性对照抗体，显示出了更优的安全性。
试验例12 CD47抗体对激活巨噬细胞吞噬红细胞的作用分析
1、实验方法
检测方法同试验例8，将红细胞替换为肿瘤细胞作为靶细胞，检测本公开的CD47抗体是否对巨噬细胞吞噬红细胞存在激活作用。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体对激活巨噬细胞吞噬红细胞的作用分析结果如图13所示，结果显示本公开的CD47抗体极低介导巨 噬细胞对红细胞的吞噬，且介导作用明显低于阳性对照抗体，显示出了更优的安全性。
试验例13抗肿瘤活性分析
1、实验方法
选用70只NOD SCID雌性小鼠(购于浙江维通利华实验动物技术有限公司)构建人B淋巴细胞皮下移植瘤模型(Raji)和人恶性黑色素瘤模型(A375)，评估本公开的CD47抗体的抗肿瘤活性。以Hu5F9-G4抗体和TJC-4抗体作为阳性对照抗体，hIgG4为同型对照抗体。
2、实验结果
CD47抗体对人B淋巴细胞皮下移植瘤模型的抗肿瘤结果如图14所示，结果显示本公开的CD47抗体在抗人B淋巴细胞皮下移植瘤效果上显著好于阳性对照抗体。
CD47抗体对人恶性黑色素瘤模型的抗肿瘤结果如图15所示，结果显示与阳性对照抗体相比，本公开的CD47抗体在抗人恶性黑色素瘤效果上更加有优势。
以上所述仅为本公开可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (15)

  1. 一种含抗CD47抗体或其抗原结合片段的制剂，其包括以下组分：抗CD47抗体或其抗原结合片段、缓冲液和辅料；
    所述抗体或其抗原结合片段含有以下CDRs：如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ ID NO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3，如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，和如SEQ ID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3；
    按组分在所述制剂中的质量体积百分数计，所述辅料包括：1％～20％的糖类和0.01％～0.5％的表面活性剂；
    可选地，所述抗体或其抗原结合片段包括：如SEQ ID NO:21所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:11所示的HCDR2，和如SEQ ID NO:12所示的HCDR3；或者
    所述抗体或其抗原结合片段包括：如SEQ ID NO:1所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:11所示的HCDR2，和如SEQ ID NO:12所示的HCDR3；或者
    所述抗体或其抗原结合片段包括：如SEQ ID NO:1所示的LCDR1，如SEQ ID NO:2所示的LCDR2，如SEQ ID NO:3所示的LCDR3，如SEQ ID NO:10所示的HCDR1，如SEQ ID NO:22所示的HCDR2，和如SEQ ID NO:12所示的HCDR3。
  2. 根据权利要求1所述的含抗CD47抗体或其抗原结合片段的制剂，所述表面活性剂选自吐温20、吐温80和泊洛沙姆188中的任意一种；
    优选地，所述表面活性剂为吐温80，所述吐温80在所述制剂中的质量体积百分数为0.01％～0.1％，优选为0.01％～0.04％，更优选为0.02％；或者
    优选地，所述表面活性剂为泊洛沙姆188，所述泊洛沙姆188在所述制剂中的质量体积百分数为0.05％～0.5％，优选为0.1％～0.2％，更优选为0.1％。
  3. 根据权利要求1或2所述的含抗CD47抗体或其抗原结合片段的制剂，所述糖类包括蔗糖和海藻糖中的至少一种；
    优选地，所述糖类为蔗糖；所述蔗糖的浓度优选为3％～15％(w/v)，优选为6％～10％(w/v)，更优选为8％(w/v)；或者
    优选地，所述糖类为海藻糖；所述海藻糖的浓度优选为3％～15％(w/v)，优选为6％～10％(w/v)，更优选为8％(w/v)。
  4. 根据权利要求1至3任一项所述的含抗CD47抗体或其抗原结合片段的制剂，所述制剂还包括甲硫氨酸；
    优选地，所述甲硫氨酸的浓度为1～20mM，优选为4.5～5.5mM，更优选为5mM。
  5. 根据权利要求1至4任一项所述的含抗CD47抗体或其抗原结合片段的制剂，所述缓冲液选自：柠檬酸-柠檬酸钠缓冲液、醋酸-醋酸钠缓冲液、组氨酸-组氨酸盐缓冲液和磷酸-磷酸盐缓冲液中的任意一种，pH为4.5～7.5；
    优选地，所述缓冲液选自柠檬酸-柠檬酸钠缓冲液、组氨酸-组氨酸盐缓冲液和磷酸-磷酸盐缓冲液中的任意一种，pH为5.0～7.5；
    优选地，所述缓冲液为组氨酸-组氨酸盐缓冲液，pH优选为5.5～6.0，pH更优选为6.0；
    优选地，所述缓冲液的浓度为10～40mM；优选15～25mM，更优选为20mM。
  6. 根据权利要求1～5任一项所述的含抗CD47抗体或其抗原结合片段的制剂，所述抗CD47抗体或其抗原结合片段的浓度为20～60mg/mL；
    优选地，所述抗CD47抗体或其抗原结合片段的浓度为45～55mg/mL；
    更优选地，所述抗CD47抗体或其抗原结合片段的浓度为50mg/mL。
  7. 根据权利要求1～6任一项所述的含抗CD47抗体或其抗原结合片段的制剂，所述抗CD47抗体或其抗原结合片段包括有重链可变区和轻链可变区；
    优选地，所述重链可变区的序列如SEQ ID NO:4～9中任一项所示或与SEQ ID NO:4～9任一项所示具有至少95％同源性的序列；所述轻链可变区的序列如SEQ ID NO:13～18中任一项所示或与SEQ ID NO:13～18任一项所示具有至 少95％同源性的序列；
    优选地，所述重链可变区包括如SEQ ID NO:8所示的序列，和所述轻链可变区包括如SEQ ID NO:14所示的序列。
  8. 根据权利要求1～7任一项所述的含抗CD47抗体或其抗原结合片段的制剂，所述抗CD47抗体或其抗原结合片段还包括恒定区；
    优选地，所述恒定区包括重链恒定区和轻链恒定区，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；轻链恒定区为κ或λ链；
    优选地，所述恒定区的种属来源选自鼠、兔、羊、猴、或人；
    优选地，所述抗体为CDR移植抗体、多聚体抗体或双特异性抗体中的任一种或几种；
    优选地，所述抗原结合片段为F(ab’)2、Fab、scFv和Fv中的任一种或几种；
    更选地，所述抗CD47抗体包括如SEQ ID NO:19所示的重链，和如SEQ ID NO:20所示的轻链。
  9. 根据权利要求8所述的含抗CD47抗体或其抗原结合片段的制剂，其包括：
    (a)50mg/mL的抗CD47抗体，所述抗CD47抗体包括如SEQ ID NO:19所示的重链，和如SEQ ID NO:20所示的轻链；
    (b)pH为6.0的20mM的组氨酸-盐酸组氨酸缓冲液；
    (c)8％(w/v)的蔗糖；和
    (d)0.02％(w/v)的吐温80。
  10. 如权利要求1～9任一项所述的含抗CD47抗体或其抗原结合片段的制剂的制备方法，其包括：将所述制剂的组分混合；
    优选地，当所述制剂中的抗CD47抗体或其抗原结合片段为抗CD47抗体或其抗原结合片段的溶液时，所述制备方法还包括：采用所述制剂中的缓冲液对抗CD47抗体或其抗原结合片段的溶液进行置换；置换后与辅料混合。
  11. 如权利要求1～9任一项所述的制剂在制备用于防治CD47阳性肿瘤的产品中的应用。
  12. 用作药物的如权利要求1～9任一项所述的制剂，所述药物用于防治CD47阳性肿瘤。
  13. 一种治疗CD47阳性肿瘤的方法，所述方法包括向有需要的受试者施用治疗有效量的权利要求1～9任一项所述的制剂。
  14. 一种含抗CD47抗体或其抗原结合片段的冻干制剂，其由如权利要求1～9任一项所述的制剂冷冻干燥后获得。
  15. 一种含抗CD47抗体或其抗原结合片段的制剂，其由如权利要求14所述的冻干制剂复溶后获得。
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