WO2022095970A1 - 双特异抗体及其应用 - Google Patents

双特异抗体及其应用 Download PDF

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WO2022095970A1
WO2022095970A1 PCT/CN2021/129045 CN2021129045W WO2022095970A1 WO 2022095970 A1 WO2022095970 A1 WO 2022095970A1 CN 2021129045 W CN2021129045 W CN 2021129045W WO 2022095970 A1 WO2022095970 A1 WO 2022095970A1
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amino acid
acid sequence
seq
antibody
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French (fr)
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陈奕藩
梁秋莲
裴树军
俞金泉
李胜峰
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Bio Thera Solutions Ltd
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Bio Thera Solutions Ltd
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Priority to US18/035,591 priority Critical patent/US20240067728A1/en
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Priority to EP21888662.0A priority patent/EP4242232A4/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2827—Immunoglobulins [IG], 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 against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to bispecific antibodies and applications thereof.
  • Cancer immunotherapy is a revolutionary breakthrough in cancer treatment in recent years, and it is a research boom in the field of bioscience. With the approval of more new antibody drugs, the prospect of cancer treatment has been transformed from a supplementary element of traditional therapy to a central and standard one. Cancer treatment options. On the road of targeting immune checkpoints (Checkpoints) to activate T cell function, thereby improving the ability of the acquired immune system to overcome cancer, the development of antibody therapies around the PD-L1/PD-1 pathway has become a leading role in the field. hot spot.
  • Checkpoints immune checkpoints
  • programmed death-ligand 1 When the body is inflamed or infected, programmed death-ligand 1 (PD-L1) can be induced to be expressed on hematopoietic, endothelial or epithelial cells, and by binding to T cells and B cells and programmed death 1 (PD-1) on monocytes to suppress the immune response.
  • the cytoplasmic region of PD-1 contains two tyrosine-based signaling domains, ITIM (Immunoreceptor Tyrosine Inhibitory Motif) and ITSM (Immunoreceptor Tyrosine Switching Motif).
  • PD-L1 is overexpressed in many cancers including a wide variety of solid tumors as well as hematological tumors such as bladder, breast, colon, lung, melanoma, ovarian, gastric, thyroid, primary Mediastinal large B-cell lymphoma and classic Hodgkin lymphoma.
  • PD-L1 which is overexpressed on tumor cells, binds to PD-1 on T cells, activates the ITIM domain of PD-1, causes T cell dysfunction and exhaustion, and prevents cytotoxic T cells from effectively targeting tumor cells, Sending a "don't find me" signal to the immune system enables tumor cells to acquire adaptive immune tolerance, thereby promoting tumor invasion and resulting in poor prognosis.
  • the innate immune system is the first nonspecific line of defense against infection and malignant cell transformation.
  • monocytes, macrophages and dendritic cells function as antigen presenting cells (APCs) through phagocytosis.
  • APCs antigen presenting cells
  • the ability of APCs to engulf tumor cells by phagocytosis is an indispensable bridge between innate and adaptive immunity.
  • Targeting checkpoints that regulate phagocytosis (Phagocytosis checkpoints, such as CD47-SIRP ⁇ ), provides a new avenue for developing cancer immunotherapies.
  • CD47 integrated protein, IAP, CD47
  • IAP interleukin associated protein
  • SIRP ⁇ Signal regulatory protein ⁇
  • CD47 is highly expressed in almost all tumor cells, such as thyroid cancer, ovarian cancer, prostate cancer, cervical cancer, bladder cancer, head and neck cancer, gastric cancer, acute myeloid leukemia, B cell and T cell acute leukemia , non-Hodgkin's lymphoma, etc., and CD47 overexpression is associated with poor clinical prognosis.
  • tumor cells such as thyroid cancer, ovarian cancer, prostate cancer, cervical cancer, bladder cancer, head and neck cancer, gastric cancer, acute myeloid leukemia, B cell and T cell acute leukemia , non-Hodgkin's lymphoma, etc.
  • BsAbs Bispecific monoclonal antibodies
  • bispecific antibodies have also ushered in a research and development boom. Many bispecific antibodies are in the clinical development stage, and 3 bispecific antibodies have been approved for marketing.
  • the European Union approved the first therapeutic bispecific antibody, Catumaxomab (targeting CD3 and EpCAM), with the structure Triomab, for the treatment of cancerous ascites.
  • Catumaxomab targeting CD3 and EpCAM
  • Triomab Triomab
  • bispecific antibodies have the advantages of high specificity, strong targeting, and low dosage. They are not only of great significance in the clinical treatment of tumors, but also have made breakthroughs in the treatment of inflammatory diseases and have a very broad application. prospect.
  • PD-L1 and CD47 proteins are simultaneously overexpressed on tumor cells. Through the PD-L1/PD1 and CD47/SIRP ⁇ pathways, they escape the monitoring of the innate immune system and acquire adaptive immune tolerance. Therefore, blocking the PD-L1/PD1 and CD47/SIRP ⁇ pathways at the same time and activating the innate and adaptive immune systems may exert a better tumor suppressing effect.
  • the present invention provides multivalent and multispecific antibodies or antigen-binding fragments and uses thereof.
  • the present invention provides a novel anti-PD-L1 antibody with better binding to PD-L1 and good druggability.
  • the present invention provides a novel anti-CD47 antibody that does not bind to human erythrocytes, does not cause human erythrocyte agglutination, has good target specificity, and has less side effects.
  • the present invention adopts the "rod-into-holes" symmetrical IgG-like bispecific antibody structure with a common light chain, realizes the correct assembly of different heavy chains and light chains, and increases the yield of bispecific antibodies, It is easy to stably express in cultured cells in vitro and does not require complex production processes.
  • the bispecific antibody structure used in the present invention can maintain the binding affinity of each antigen-binding site to corresponding different epitopes, and will not interfere with each other when binding to different epitopes, and has good druggability. Further, the bispecific antibody of the present invention has stable physical and biological properties, which allows the antibody to have better manufacturability and developability.
  • the present invention provides bispecific antibodies or antigen-binding fragments.
  • the antibodies or antigen-binding fragments provided herein can bind two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a variable region a that specifically binds to PD-L1; wherein the variable region a comprises ( One or more amino acid sequences in a)-(f):
  • VHa CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-8, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 4-8 amino acid sequence of conservative amino acid substitutions;
  • VHa CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9-18, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 9-14 amino acid sequence with conservative amino acid substitutions;
  • VHa CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 19 or 20;
  • VLa CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 36-40, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 36-40 amino acid sequence with conservative amino acid substitutions;
  • VLa CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 41-44, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 41-44 amino acid sequence with conservative amino acid substitutions;
  • VLa CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 45-48, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48 amino acid sequence with conservative amino acid substitutions.
  • variable region a comprises a heavy chain variable region a (VHa), the heavy chain variable region a comprises VHa CDR1, VHa CDR2, VHa CDR3; wherein VHa CDR1 comprises as SEQ ID NO: The amino acid sequence shown in any one of 4-8; VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-18; VHa CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19 or 20 .
  • VHa heavy chain variable region a
  • VHa CDR1 comprises as SEQ ID NO: The amino acid sequence shown in any one of 4-8;
  • VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-18;
  • VHa CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19 or 20 .
  • variable region a comprises a light chain variable region a (VLa) comprising VLa CDR1, VLa CDR2 and VLa CDR3; wherein VLa CDR1 comprises as in SEQ ID NO: The amino acid sequence shown in any one of 36-40; VLa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; VLa CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48 amino acid sequence shown.
  • VLa light chain variable region a
  • variable region a comprises VHa and VLa; the VHa comprises VHa CDRl, VHa CDR2, VHa CDR3; the VLa comprises VLa CDRl, VLa CDR2 and VLa CDR3.
  • VHa CDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 4-8;
  • VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 9-18;
  • VHa CDR3 comprises as SEQ ID NO: 9-18 The amino acid sequence shown in NO: 19 or 20;
  • VLa CDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 36-40;
  • VLa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44 Amino acid sequence;
  • VLa CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 45-48.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the The VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-14, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 9-14
  • the VHa CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 19.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the Described VHa CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, or compared with the amino acid sequence shown in SEQ ID NO: 14 with one or more conservative amino acid substitutions;
  • Described VHa CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 19.
  • the VLa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 36;
  • the Described VLa CDR2 comprises the amino acid sequence as shown in SEQ ID NO:41, or compared with the amino acid sequence shown in SEQ ID NO:41 with one or more conservative amino acid substitutions;
  • Described VLa CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 45, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 45.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the Described VHa CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, or compared with the amino acid sequence shown in SEQ ID NO: 14 with one or more conservative amino acid substitutions;
  • Described VHa CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 19;
  • the VLa CDR1 comprises the amino acid shown in SEQ ID NO: 36 sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 36;
  • the VLa CDR2 comprises the amino acid sequence shown in SEQ ID NO: 41, or compared with the amino acid sequence shown in SEQ ID NO: 36;
  • the VHa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49-63, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 49-63 A homologous amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 49-63.
  • the VHa comprises the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 54, or the amino acid sequence set forth in SEQ ID NO: 54 :
  • the amino acid sequence shown in 54 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the VLa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75-80, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 75-80 A homologous amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • the VLa comprises the amino acid sequence set forth in SEQ ID NO:75, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO:75, or the amino acid sequence set forth in SEQ ID NO:75 :
  • the amino acid sequence shown in 75 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • variable region a comprises VHa and VLa.
  • the VHa comprises the amino acid sequence shown in SEQ ID NO:54, or an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO:54, or the amino acid sequence shown in SEQ ID NO:54 An amino acid sequence with one or more conservative amino acid substitutions compared to the sequence;
  • the VLa comprises the amino acid sequence shown in SEQ ID NO: 75, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO: 75 The amino acid sequence of , or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 75.
  • the present invention provides a bispecific antibody or antigen-binding fragment further comprising a light chain constant region a (CLa), and a heavy chain constant region a (CHa).
  • the CHa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86, or the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86 An amino acid sequence with at least 90% homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 81, 83-86.
  • the CLa comprises the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 82, or the amino acid sequence set forth in SEQ ID NO: 82 :
  • the amino acid sequence shown in 82 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a heavy chain a, a heavy chain x, and a light chain a and a light chain x as described herein ;
  • the heavy chain a and the light chain a are paired to form a PD-L1 antigen binding site, and the heavy chain x and the light chain x are paired to form other antigen binding sites.
  • the heavy chain a comprises VHa CDR1, VHa CDR2, VHa CDR3 described herein.
  • the heavy chain a comprises a VHa as described herein.
  • the heavy chain a comprises VHa and CHa as described herein.
  • the light chain a comprises VLa CDR1, VLa CDR2, and VLa CDR3 described herein. In some embodiments, the light chain a comprises VLa as described herein. In some embodiments, the light chain a comprises VLa and CLa as described herein. In some embodiments, the heavy chain x is the same as the heavy chain b described herein. In some embodiments, the light chain x is the same as the light chain a.
  • the other antigen is CD47, LAG3, TGF ⁇ , CTLA-4, 4-1BB, PD1, TIGIT, KIR, c-Met, VISTA, or BCMA. In some embodiments, the other antigen is CD47.
  • the CDR of the heavy chain x is selected from the heavy chains in Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio) or IBI188 (Innovative Bio), etc. CDRs.
  • the variable region of the heavy chain x is selected from Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio) or IBI188 (Innovative Bio), etc. heavy chain variable region.
  • the CDR of the light chain x is selected from the light chains in Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio) or IBI188 (Innovative Bio), etc. CDRs.
  • the variable region of the light chain x is selected from Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio) or IBI188 (Innovative Bio), etc. light chain variable region.
  • the light chain x is the same as the light chain a.
  • the heavy chain a comprises the amino acid sequence set forth in SEQ ID NO: 92 or 94, or an amino acid sequence with at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 92 or 94 , or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 92 or 94.
  • the affinity index KD of the bispecific antibody or antigen-binding fragment provided by the present invention to PD-L1 is ⁇ 10 nM. In some embodiments, the affinity index KD of the bispecific antibody or antigen-binding fragment provided by the present invention to PD-L1 is ⁇ 1 nM. In some embodiments, the affinity index KD of the bispecific antibody or antigen-binding fragment provided by the present invention to PD-L1 is ⁇ 0.5 nM.
  • the present invention provides a bispecific antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprising a variable region b that specifically binds to CD47; wherein the variable region b comprises (g)-( One or more amino acid sequences in l):
  • VHb CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21-23, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 21-23 amino acid sequence with conservative amino acid substitutions;
  • VHb CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24-28, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 24-28 amino acid sequence with conservative amino acid substitutions;
  • VHb CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 29-35, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 29-35 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 36-40, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 36-40 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 41-44, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 41-44 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 45-48, or having one or more amino acid sequences compared to the amino acid sequence shown in any one of SEQ ID NOs: 45-48 amino acid sequence with conservative amino acid substitutions.
  • variable region b comprises a heavy chain variable region b (VHb) comprising VHb CDRl, VHb CDR2, VHb CDR3; wherein VHb CDRl comprises as in SEQ ID NO:
  • VHb CDRl comprises as in SEQ ID NO:
  • VHb CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-28;
  • VHb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-35 amino acid sequence shown.
  • variable region b comprises a light chain variable region b (VLb) comprising VLb CDR1, VLb CDR2 and VLb CDR3; wherein VLb CDR1 comprises as in SEQ ID NO: The amino acid sequence shown in any one of 36-40; VLb CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; VLb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48 amino acid sequence shown.
  • VLb CDR1 comprises as in SEQ ID NO: The amino acid sequence shown in any one of 36-40;
  • VLb CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44;
  • VLb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48 amino acid sequence shown.
  • variable region b comprises VHb and VLb; the VHb comprises VHb CDRl, VHb CDR2, VHb CDR3; and the VLb comprises VLb CDRl, VLb CDR2, and VLb CDR3.
  • VHb CDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 21-23;
  • VHb CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-28;
  • VHb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-28 The amino acid sequence set forth in any one of NO: 29-35;
  • VLb CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-40;
  • VLb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-44 The amino acid sequence shown in item;
  • the VLb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48.
  • the VHb CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 21;
  • the Described VHb CDR2 comprises the amino acid sequence as shown in SEQ ID NO:24, or compared with the amino acid sequence shown in SEQ ID NO:24 with one or more conservative amino acid substitutions;
  • Described VHb CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 29, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 29.
  • the VLb CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 36;
  • the Described VLb CDR2 comprises the amino acid sequence as shown in SEQ ID NO:41, or compared with the amino acid sequence shown in SEQ ID NO:41 with one or more conservative amino acid substitutions;
  • Described VLb CDR3 comprises as SEQ ID The amino acid sequence shown in NO: 45, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 45.
  • the VHb CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 21;
  • the Described VHb CDR2 comprises the amino acid sequence as shown in SEQ ID NO:24, or compared with the amino acid sequence shown in SEQ ID NO:24 with one or more conservative amino acid substitutions;
  • Described VHb CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 29, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 29;
  • the VLb CDR1 comprises the amino acid shown in SEQ ID NO: 36 sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 36;
  • the VLb CDR2 comprises the amino acid sequence shown in SEQ ID NO: 41, or compared with the amino acid sequence shown in SEQ ID NO: 36
  • the VHb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-74, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 64-74 A homologous amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 64-74.
  • the VHb comprises the amino acid sequence set forth in SEQ ID NO: 64, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 64, or the amino acid sequence set forth in SEQ ID NO: 64 :
  • the amino acid sequence shown in 64 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the VLb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75-80, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 75-80 A homologous amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • the VLb comprises the amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 75, or the amino acid sequence set forth in SEQ ID NO: 75 :
  • the amino acid sequence shown in 75 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • variable region b comprises VHb and VLb.
  • the VHb comprises the amino acid sequence shown in SEQ ID NO:64, or an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO:64, or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence with one or more conservative amino acid substitutions compared to the sequence;
  • the VLb comprises the amino acid sequence shown in SEQ ID NO: 75, or has at least 90% homology with the amino acid sequence shown in SEQ ID NO: 75
  • the present invention provides a bispecific antibody or antigen-binding fragment further comprising a light chain constant region b (CLb) and a heavy chain constant region b (CHb).
  • the CHb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86, or the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86 An amino acid sequence with at least 90% homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 81, 83-86.
  • the CLb comprises the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 82, or the amino acid sequence set forth in SEQ ID NO: 82 :
  • the amino acid sequence shown in 82 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a heavy chain b described herein, a heavy chain y, and a light chain b and a light chain described herein Light chain y; pairing of the heavy chain b and the light chain b to form the CD47 antigen binding site, and the pairing of the heavy chain y and the light chain y to form other antigen binding sites.
  • the heavy chain b comprises VHb CDRl, VHb CDR2, VHb CDR3 described herein.
  • the heavy chain b comprises a VHb described herein.
  • the heavy chain b comprises the VHb and CHb described herein.
  • the light chain b comprises VLb CDRl, VLb CDR2, and VLb CDR3 described herein. In some embodiments, the light chain b comprises a VLb described herein. In some embodiments, the light chain b comprises VLb and CLb described herein. In some embodiments, the light chain y is the same as the light chain b.
  • the other antigen is PD-L1, PD-1, BCMA, MSLN, CD19, CD20, or VEGF. In some embodiments, the other antigen is PD-L1.
  • the CDRs of the heavy chain y are selected from the heavy chain CDRs of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the variable region of the heavy chain y is selected from the heavy chain variable regions of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the CDRs of the light chain y are selected from the light chain CDRs of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • variable region of the light chain is selected from the light chain variable regions of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the heavy chain y is the same as the heavy chain a described herein. In some embodiments, the light chain y is the same as the light chain b.
  • the heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 93 or 95, or an amino acid sequence with at least 90% homology to the amino acid sequence shown in SEQ ID NO: 93 or 95 , or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 93 or 95.
  • the bispecific antibody or antigen-binding fragment provided by the present invention has an affinity index KD ⁇ 30nM for CD47. In some embodiments, the bispecific antibody or antigen-binding fragment provided by the present invention has an affinity index KD ⁇ 10 nM for CD47. In some embodiments, the bispecific antibody or antigen-binding fragment provided by the present invention has an affinity index KD ⁇ 5nM for CD47.
  • the bispecific antibodies or antigen-binding fragments provided herein do not cause hemagglutination.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a variable region a that specifically binds to PD-L1 and a variable region b that specifically binds to CD47.
  • variable region a comprises one or more amino acid sequences in (a)-(f):
  • VHa CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-8, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 4-8 amino acid sequence with conservative amino acid substitutions;
  • VHa CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9-18, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 9-18 amino acid sequence with conservative amino acid substitutions;
  • VHa CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 19 or 20;
  • VLa CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 36-40, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 36-40 amino acid sequence with conservative amino acid substitutions;
  • VLa CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 41-44, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 41-44 amino acid sequence with conservative amino acid substitutions;
  • VLa CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 45-48, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48 amino acid sequence with conservative amino acid substitutions.
  • variable region b comprises one or more amino acid sequences of (g)-(1):
  • VHb CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21-23, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 21-23 amino acid sequence with conservative amino acid substitutions;
  • VHb CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24-28, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 24-28 amino acid sequence with conservative amino acid substitutions;
  • VHb CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 29-35, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 29-35 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 36-40, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 36-40 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 41-44, or having one or more amino acid sequences compared to the amino acid sequence set forth in any one of SEQ ID NOs: 41-44 amino acid sequence with conservative amino acid substitutions;
  • VLb CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 45-48, or having one or more amino acid sequences compared to the amino acid sequence shown in any one of SEQ ID NOs: 45-48 amino acid sequence with conservative amino acid substitutions.
  • variable region a comprises a heavy chain variable region a (VHa), the heavy chain variable region a comprises VHa CDR1, VHa CDR2, VHa CDR3; wherein VHa CDR1 comprises as SEQ ID NO:
  • VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-18;
  • VHa CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19 or 20
  • the variable region b comprises a heavy chain variable region b (VHb), and the heavy chain variable region b comprises VHb CDR1, VHb CDR2, VHb CDR3; wherein VHb CDR1 comprises as in any of SEQ ID NOs: 21-23
  • the VHb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 24-28;
  • the VHb CDR3 comprises the amino acid sequence set forth in any one of
  • variable region a comprises a light chain variable region a (VLa) comprising VLa CDR1, VLa CDR2 and VLa CDR3; wherein VLa CDR1 comprises as in SEQ ID NO: The amino acid sequence shown in any one of 36-40; VLa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; VLa CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48 The amino acid sequence shown; the variable region b comprises a light chain variable region b (VLb), the light chain variable region b comprises VLb CDR1, VLb CDR2 and VLb CDR3; wherein VLb CDR1 comprises as SEQ ID NO:36 - the amino acid sequence set forth in any one of 40; VLb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-44; VLb CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 45-48 amino
  • variable region a comprises VHa and VLa; the variable region b comprises VHb and VLb.
  • the VHa comprises VHa CDR1, VHa CDR2, VHa CDR3; the VLa comprises VLa CDR1, VLa CDR2 and VLa CDR3.
  • VHa CDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 4-8;
  • VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 9-18;
  • VHa CDR3 comprises as SEQ ID NO: 9-18 The amino acid sequence shown in NO: 19 or 20.
  • VHb comprises VHb CDR1, VHb CDR2, VHb CDR3; the VLb comprises VLb CDR1, VLb CDR2 and VLb CDR3.
  • VHb CDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 21-23;
  • VHb CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-28;
  • VHb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-28
  • VLb CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-40;
  • VLb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-44
  • the VLb CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48.
  • VLa CDR1 and VLb CDR1 comprise amino acid sequences independently selected from those shown in any one of SEQ ID NOs: 36-40;
  • VLa CDR2 and VLb CDR2 comprise independently selected from amino acid sequences shown in any one of SEQ ID NOs: 41-44 The amino acid sequences of ;
  • VLa CDR3 and VLb CDR3 comprise independently selected from the amino acid sequences shown in any one of SEQ ID NOs: 45-48.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the The VHa CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-14, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 9-14
  • the amino acid sequence of the VHa CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 19;
  • the VHb The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 21;
  • the VHb CDR2 comprises the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 24, or the amino acid sequence
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the Described VHa CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, or compared with the amino acid sequence shown in SEQ ID NO: 14 with one or more conservative amino acid substitutions;
  • Described VHa CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 19;
  • the VHb CDR1 comprises the amino acid shown in SEQ ID NO: 21 Sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 21;
  • the VHb CDR2 comprises the amino acid sequence shown in SEQ ID NO: 24, or compared with the amino acid sequence shown in SEQ ID NO: 21;
  • the VLa CDRl or VLb CDRl comprises the amino acid sequence set forth in SEQ ID NO:36, or amino acids having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:36 Sequence;
  • the VLa CDR2 or VLb CDR2 comprises the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 41;
  • the The VLa CDR3 or VLb CDR3 comprises the amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:45.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 4;
  • the Described VHa CDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, or compared with the amino acid sequence shown in SEQ ID NO: 14 with one or more conservative amino acid substitutions;
  • Described VHa CDR3 comprises as shown in SEQ ID The amino acid sequence shown in NO: 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 19;
  • the VHb CDR1 comprises the amino acid shown in SEQ ID NO: 21 Sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 21;
  • the VHb CDR2 comprises the amino acid sequence shown in SEQ ID NO: 24, or compared with the amino acid sequence shown in SEQ ID NO: 21;
  • the VLa CDR1 or VLb CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 36;
  • the VLa CDR2 Or the VLb CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 41, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 41;
  • the VLa CDR3 or VLb CDR3 comprises The amino acid sequence shown in SEQ ID NO:45, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:45.
  • the VLa CDRl and the VLb CDRl comprise the same amino acid sequence.
  • the VLa CDR2 and the VLb CDR2 comprise the same amino acid sequence.
  • the VLa CDR3 and the VLb CDR3 comprise the same amino acid sequence.
  • the VLa CDR1 and the VLb CDR1 comprise the same amino acid sequence
  • the VLa CDR2 and the VLb CDR2 comprise the same amino acid sequence
  • the VLa CDR3 and the VLb CDR3 comprise the same amino acid sequence.
  • variable region a comprises a heavy chain variable region VHa and a light chain variable region VLa; wherein the VHa comprises the amino acid sequence shown in any one of SEQ ID NOs: 49-63 , or an amino acid sequence having at least 90% homology with the amino acid sequence shown in any one of SEQ ID NOs: 49-63; and/or the VLa comprises as shown in any one of SEQ ID NO: 75-80 The amino acid sequence shown, or an amino acid sequence with at least 90% homology to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • variable region a comprises a heavy chain variable region VHa and a light chain variable region VLa; wherein the VHa comprises the amino acid sequence shown in SEQ ID NO: 54, or the same as SEQ ID NO
  • the amino acid sequence shown in: 54 has at least 90% homology; and/or the VLa comprises the amino acid sequence shown in SEQ ID NO: 75, or at least the amino acid sequence shown in SEQ ID NO: 75 Amino acid sequences with 90% homology.
  • variable region b comprises a heavy chain variable region VHb and a light chain variable region VLb; wherein the VHb comprises the amino acid sequence shown in any one of SEQ ID NOs: 64-74 , or an amino acid sequence having at least 90% homology with the amino acid sequence shown in any one of SEQ ID NOs: 64-74; and/or the VLb comprises the amino acid sequence shown in any one of SEQ ID NOs: 75-80 The amino acid sequence shown, or an amino acid sequence with at least 90% homology to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • variable region b comprises a heavy chain variable region VHb and a light chain variable region VLb; wherein the VHb comprises the amino acid sequence shown in SEQ ID NO: 64, or the same as SEQ ID NO
  • the amino acid sequence shown in: 64 has at least 90% homology; and/or the VLb comprises the amino acid sequence shown in SEQ ID NO: 75, or at least the amino acid sequence shown in SEQ ID NO: 75 Amino acid sequences with 90% homology.
  • the VHa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49-63, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 49-63 A homologous amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 49-63;
  • the VHb comprises as SEQ ID NO: 64- The amino acid sequence shown in any one of 74, or the amino acid sequence with at least 90% homology to the amino acid sequence shown in any one of SEQ ID NOs: 64-74, or the amino acid sequence shown in SEQ ID NO: 64-74 The amino acid sequence shown in any one is compared to an amino acid sequence with one or more conservative amino acid substitutions.
  • the VHa comprises the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 54, or the amino acid sequence set forth in SEQ ID NO: 54 Compared with the amino acid sequence shown in: 54, the amino acid sequence with one or more conservative amino acid substitutions; the VHb comprises the amino acid sequence shown in SEQ ID NO: 64, or at least the amino acid sequence shown in SEQ ID NO: 64 An amino acid sequence with 90% homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 64.
  • the VLa and VLb comprise amino acid sequences independently selected from the amino acid sequences set forth in any of SEQ ID NOs: 75-80, or the amino acids set forth in any of SEQ ID NOs: 75-80 An amino acid sequence with at least 90% homology in the sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • the VLa and VLb comprise an amino acid sequence independently selected from the amino acid sequence set forth in SEQ ID NO:75, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO:75, Or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 75.
  • variable region a comprises VHa and VLa; the variable region b comprises VHb and VLb.
  • the VHa comprises the amino acid sequence shown in SEQ ID NO:54, or an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO:54, or the amino acid sequence shown in SEQ ID NO:54 Sequences are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the VHb comprises the amino acid sequence shown in SEQ ID NO:64, or an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO:64, or the amino acid sequence shown in SEQ ID NO:64 Sequences are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the VLa or VLb comprises the amino acid sequence shown in SEQ ID NO:75, or the amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO:75, or the amino acid sequence shown in SEQ ID NO:75
  • the amino acid sequence is compared to an amino acid sequence with one or more conservative amino acid substitutions.
  • the VLa and the VLb comprise the same amino acid sequence.
  • the antibodies or antigen-binding fragments described herein further comprise a light chain constant region a (CLa), as well as a heavy chain constant region a (CHa), a light chain constant region b (CLb), and a heavy chain constant region b (CHb).
  • CLa light chain constant region a
  • CHa heavy chain constant region a
  • CHb light chain constant region b
  • CHb heavy chain constant region b
  • the CHa is of the IgGl subtype. In some embodiments, the CHb is of the IgGl subtype.
  • the CHa and/or CHb comprise an IgGl subtype of one or more of the following amino acid mutations: Y349C, S354C, T366W, T366S, L368A, and Y407V, wherein amino acid positions are Eu numbering.
  • the CHa and/or CHb comprise the following amino acid mutated IgGl subtype: N297A, wherein the amino acid positions are Eu numbering.
  • one of the heavy chain constant regions in CHa and CHb comprises one or more of the following amino acid mutations: N297A, Y349C, T366S, L368A, and Y407V.
  • the other heavy chain constant region in the CHa and CHb comprises one or more of the following amino acid mutations: N297A, S354C, and T366W.
  • the CHa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86, or the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86 An amino acid sequence with at least 90% homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 81, 83-86.
  • the CHb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86, or the amino acid sequence set forth in any one of SEQ ID NOs: 81, 83-86 An amino acid sequence with at least 90% homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 81, 83-86.
  • the CHa comprises the amino acid sequence set forth in SEQ ID NO: 81, 83 or 85, or is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 81, 83 or 85 An amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 81, 83 or 85.
  • the CHb comprises the amino acid sequence set forth in SEQ ID NO: 81, 84 or 86, or is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 81, 84 or 86 An amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 81, 84 or 86.
  • the CHa comprises the amino acid sequence set forth in SEQ ID NO:83 and the CHb comprises the amino acid sequence set forth in SEQ ID NO:84. In some embodiments, the CHa comprises the amino acid sequence set forth in SEQ ID NO:85 and the CHb comprises the amino acid sequence set forth in SEQ ID NO:86. In some embodiments, the CHa comprises the amino acid sequence set forth in SEQ ID NO:84 and the CHb comprises the amino acid sequence set forth in SEQ ID NO:83. In some embodiments, the CHa comprises the amino acid sequence set forth in SEQ ID NO:86 and the CHb comprises the amino acid sequence set forth in SEQ ID NO:85.
  • the CLa comprises the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 82, or the amino acid sequence set forth in SEQ ID NO: 82 : The amino acid sequence shown in 82 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the CLb comprises the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 82, or the amino acid sequence set forth in SEQ ID NO: 82 : The amino acid sequence shown in 82 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain a comprises the amino acid sequence set forth in SEQ ID NO: 92 or 94, or an amino acid sequence with at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 92 or 94 , or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 92 or 94;
  • the heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 93 or 95, or An amino acid sequence with at least 90% homology to the amino acid sequence shown in SEQ ID NO: 93 or 95, or an amino acid with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 93 or 95 sequence.
  • the light chain a and light chain b both comprise the amino acid sequence shown in SEQ ID NO:96, or an amino acid with at least 90% homology to the amino acid sequence shown in SEQ ID NO:96 sequence.
  • the affinity index KD of the bispecific antibody or antigen-binding fragment provided by the present invention to PD-L1 is ⁇ 10 nM. In some embodiments, the affinity index KD of the bispecific antibody or antigen-binding fragment provided by the present invention to PD-L1 is ⁇ 1 nM. In some embodiments, the bispecific antibody or antigen-binding fragment provided by the present invention has an affinity index KD ⁇ 30 nM for CD47. In some embodiments, the bispecific antibody or antigen-binding fragment provided by the present invention has an affinity index KD ⁇ 10 nM for CD47. In some embodiments, the bispecific antibody or antigen-binding fragment provided by the invention has an affinity index KD ⁇ 5 nM for CD47.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a variable region a that specifically binds to PD-L1, a variable region b that specifically binds to CD47, and a light chain A constant region, and a heavy chain constant region;
  • the heavy chain constant region is of the IgG1 subtype, or an IgG1 subtype comprising one or more of the following amino acid mutations: Y349C, S354C, T366W, T366S, L368A, and Y407V, where the amino acid positions Number Eu.
  • the heavy chain constant region further comprises the following amino acid mutated IgGl subtype, wherein the amino acid position is Eu numbering: N297A.
  • the heavy chain constant region comprises a first heavy chain constant region and a second heavy chain constant region, the first heavy chain constant region comprising one or more of the following amino acid mutations:
  • the second heavy chain constant region comprises one or more of the following amino acid mutations:
  • N297A, Y349C, T366S, L368A and Y407V N297A, Y349C, T366S, L368A and Y407V.
  • the first heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 81, 83 or 85, or is at least 90 different from the amino acid sequence set forth in SEQ ID NO: 81, 83 or 85
  • the amino acid sequence of % homology; and/or the second heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 81, 84 or 86, or the amino acid sequence shown in SEQ ID NO: 81, 84 or 86 Amino acid sequences with at least 90% homology.
  • the VHa in the variable region a is linked to the first heavy chain constant region, and the VHb in the variable region b is linked to the second heavy chain constant region; or, the The VHa in variable region a is linked to the second heavy chain constant region, and the VHb in variable region b is linked to the first heavy chain constant region.
  • the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:82, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:82.
  • the antibody or antigen-binding fragment comprises variable region a described herein, and variable region b described herein.
  • the present invention provides a bispecific antibody or antigen-binding fragment comprising a heavy chain a, a heavy chain b and two identical light chains.
  • variable region (VL) of the light chain comprises VL CDR1, VL CDR2 and VL CDR3; wherein VL CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-40 VL CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; VL CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 45-48.
  • the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 36;
  • the Described VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:41, or compared with the amino acid sequence shown in SEQ ID NO:41 with one or more conservative amino acid substitutions;
  • Described VL CDR3 comprises as SEQ ID The amino acid sequence shown in NO: 45, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 45.
  • the VL comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75-80, or is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 75-80 A homologous amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 75-80.
  • the VL comprises the amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 75, or the amino acid sequence set forth in SEQ ID NO: 75 :
  • the amino acid sequence shown in 75 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the light chain comprises the amino acid sequence set forth in SEQ ID NO: 96, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 96, or an amino acid sequence with SEQ ID NO: 96
  • the amino acid sequence shown in NO: 96 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain a and one of the light chains are paired to form a PD-L1 antigen binding site
  • the heavy chain b and the other of the light chains are paired to form CD47, LAG3, TGF ⁇ , CTLA-4 , 4-1BB, PD-1, TIGIT, KIR, c-Met, VISTA or BCMA antigen binding sites.
  • the heavy chain a and one of the light chains pair to form a PD-L1, PD-1, BCMA, MSLN, CD19, CD20 or VEGF antigen binding site, the heavy chain b and the other The light chains pair to form the CD47 antigen binding site.
  • the heavy chain a and one of the light chains are paired to form a PD-L1 antigen binding site, and the heavy chain b and another of the light chains are paired to form a CD47 antigen binding site.
  • the heavy chain a comprises the amino acid sequence set forth in SEQ ID NO: 92 or 94, or an amino acid sequence with at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 92 or 94
  • the heavy chain b comprises the amino acid sequence shown in SEQ ID NO: 93 or 95, or an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO: 93 or 95
  • the light chain It comprises the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence with at least 90% homology to the amino acid sequence shown in SEQ ID NO: 96.
  • the antibody or antigen-binding fragment has an affinity index KD ⁇ 1 nM for PD-L1.
  • the antibody or antigen-binding fragment has an affinity index KD ⁇ 10 nM for CD47.
  • the antibody or antigen-binding fragment has an affinity index KD ⁇ 30 nM for CD47.
  • the present invention provides a polynucleotide encoding the above-mentioned antibody or antigen-binding fragment.
  • the polynucleotide is an isolated polynucleotide.
  • the present invention provides an expression vector comprising the above-mentioned polynucleotide.
  • the expression vector is an isolated expression vector.
  • the present invention provides a host cell comprising the above-mentioned polynucleotide or the above-mentioned expression vector.
  • the host cell is an isolated host cell.
  • the cells are CHO cells, HEK cells (eg, HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells, or murine L cells.
  • the present invention provides methods of making the antibodies or antigen-binding fragments described herein, comprising culturing the above-described host cells in a culture medium to produce the antibodies or antigen-binding fragments. In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment from the host cell or the culture medium.
  • the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment, the above-mentioned polynucleotide, or the above-mentioned expression vector, or the above-mentioned cell, and a pharmaceutically acceptable accepted vector.
  • the present invention provides the application of the above-mentioned antibody or antigen-binding fragment, the above-mentioned polynucleotide, or the above-mentioned expression vector, the above-mentioned cell or the above-mentioned composition in the preparation of a medicine for treating diseases.
  • the disease is an autoimmune disease, acute and chronic inflammatory disease, infectious disease (eg, chronic infectious disease or sepsis), cancer.
  • the disease is a disorder associated with PD-L1 and CD47.
  • the disease is a condition associated with PD-L1 and CD47 including, but not limited to, various hematological diseases and solid tumors, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia ( ALL), Non-Hodgkin Lymphoma (NHL), Multiple Myeloma (MM), Lymphoma, Breast Cancer, Stomach Cancer, Lung Cancer, Esophageal Cancer, Colon Cancer, Ovarian Cancer, Cervical Cancer, Kidney Cancer, Pancreatic Cancer, Bladder Cancer cancer, glioma, melanoma and other solid tumors.
  • the cancer is gastrointestinal cancer, such as colon cancer.
  • the present invention provides a method for treating a disease, comprising administering to a patient in need of treatment an effective dose of the above-mentioned bispecific antibody or antigen-binding fragment, the above-mentioned polynucleotide, or the above-mentioned expression vector, the above-mentioned cells or the above-mentioned composition.
  • the disease is an autoimmune disease, acute and chronic inflammatory disease, infectious disease (eg, chronic infectious disease or sepsis), cancer.
  • the disease is a disorder associated with PD-L1 and CD47.
  • the disease is a condition associated with PD-L1 and CD47 including, but not limited to, various hematological diseases and solid tumors, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia ( ALL), Non-Hodgkin Lymphoma (NHL), Multiple Myeloma (MM), Lymphoma, Breast Cancer, Stomach Cancer, Lung Cancer, Esophageal Cancer, Colon Cancer, Ovarian Cancer, Cervical Cancer, Kidney Cancer, Pancreatic Cancer, Bladder Cancer cancer, glioma, melanoma and other solid tumors.
  • the cancer is gastrointestinal cancer, such as colon cancer.
  • the present invention relates to a kit or article of manufacture comprising the above-described bispecific antibody or antigen-binding fragment, the above-described polynucleotide, or the above-described expression vector.
  • the present invention provides bispecific antibodies or antigen-binding fragments and uses thereof.
  • the bispecific antibodies or antigen-binding fragments of the present invention can bind to two or more antigens, or two or more epitopes of the same antigen.
  • the antibodies or antigen-binding fragments of the present invention can be used for the treatment or prevention of various diseases, such as autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases, cancer, and also for the diagnosis and prognosis of related diseases.
  • FIG 1 illustrates the structure of the bispecific antibody of the present invention.
  • Fig. 2 is a group diagram
  • Fig. 2A and Fig. 2B respectively show the purity of two anti-PD-L1/CD47 bispecific antibodies BsAb-46 and BsAb-71 detected by SEC.
  • Figure 3 Binding ability of anti-PD-L1 scFv to PD-L1-His-Biotin antigen protein, where the clone number indicates the positive yeast clone expressing the corresponding anti-PD-L1 scFv.
  • Figure 4 shows the anti-PD-L1 antibody composed of R2-4 as light chain and different anti-PD-L1 VH based on MOA method, and IgG1 antibody (IgG-Isotype) as a negative control to PD-1/ Effects of PD-L1 signaling pathway.
  • Figure 5 shows the effects of different light chain anti-PD-L1 antibodies and IgG1 antibody (IgG-Isotype) as a negative control on PD-1/PD-L1 signal transduction pathway detected by MOA method.
  • Figure 6 shows the effect of anti-CD47 antibody of different light chains, positive control antibody Hu5F9-G4 and IgG1 control antibody (IgG-Isotype) on hemagglutination; wherein Hu5F9 represents Hu5F9-G4.
  • the appearance shows that the red blood cell mass flowing into a line does not cause hemagglutination, the slight aggregation is a point, and the agglutination is obvious, the whole hole is blurred.
  • Figure 7 shows the ability of anti-CD47 antibody and Hu5F9-G4 as a positive control antibody to promote macrophage phagocytosis of tumor cells; wherein Hu5F9 represents Hu5F9-G4.
  • FIG. 8 is a group diagram showing the binding ability of the anti-PD-L1/CD47 bispecific antibody to PDL1 and CD47, respectively.
  • Figure 8A binds to PD-L1-His first, and then binds to CD47-His;
  • Figure 8B binds to CD47-His first, and then binds to PD-L1-His;
  • Figure 8C shows the anti-PD-L1/CD47 bispecific antibody BsAb-71-N297A
  • the upper part of the picture shows the first binding of PD-L1-His, and then the binding of CD47-His; the lower part of the picture shows the first binding of CD47-His and then the binding of PD-L1-His; His, hCD47-His stands for CD47-His.
  • FIG. 9 is a panel diagram showing an exemplary anti-PD-L1/CD47 bispecific antibody, an anti-PD-L1 antibody as a parent, and an anti-CD47 antibody 47-R2- as a negative control detected by FACS. 4 Binding to CHO cells overexpressing PD-L1.
  • Figures 9B and 9D show exemplary anti-PD-L1/CD47 bispecific antibodies, anti-CD47 antibody 47-R2-4 as parent, and anti-PD-L1 antibody L1-R2-4 as negative control detected by FACS -71 binding to CHO cells overexpressing CD47.
  • the horizontal axis represents the antibody concentration
  • the vertical axis represents the mean fluorescence intensity (MFI).
  • FIG. 10 is a group diagram showing the anti-PD-L1/CD47 bispecific antibody of the present invention, the anti-PD-L1 antibody L1-R2-4-71 as the parent and the IgG1 antibody as the negative control ( IgG-Isotype) on PD-1/PD-L1 signal transduction pathway.
  • FIG. 10A shows the results of anti-PD-L1/CD47 bispecific antibodies BsAb-36, BsAb-46, BsAb-47, and BsAb-71
  • FIG. 10B shows the results of anti-PD-L1/CD47 bispecific antibody BsAb-71-N297A.
  • FIG 11 shows the effect of exemplary anti-PD-L1/CD47 bispecific antibody, anti-CD47 positive antibody Hu5F9-G4, anti-CD47 parental antibody 47-R2-4 and IgG1 control antibody (IgG-Isotype) on hemagglutination;
  • Hu5F9 represents Hu5F9-G4.
  • Figure 12 shows the ability of an exemplary anti-PD-L1/CD47 bispecific antibody, the parental antibody against CD47 47-R2-4 and an IgG1 control antibody (IgG-Isotype) to promote phagocytosis of tumor cells by macrophages.
  • FIG. 13 is a panel diagram showing exemplary anti-PD-L1/CD47 bispecific antibody, anti-CD47 positive antibody Hu5F9-G4, parental anti-CD47 antibody 47-R2-4, and negative anti-CD47 antibody detected by FACS Binding of control anti-PD-L1 antibody L1-R2-4-71 to human erythrocyte cells.
  • Figure 13B shows exemplary anti-PD-L1/CD47 bispecific antibody, positive anti-CD47 antibody Hu5F9-G4, parental anti-CD47 antibody 47-R2-4, and anti-PD-L1 as negative control detected by FACS Binding of antibody L1-R2-4-71 to human T-lymphoblastic leukemia Jurkat cells.
  • the horizontal axis represents the antibody concentration
  • the vertical axis represents the mean fluorescence intensity (MFI).
  • Figure 14 Effect of anti-PD-L1/CD47 bispecific antibody on IL2 cytokine release from PBMC under exogenous stimulation.
  • Figure 15 shows anti-PD-L1 antibody L1-R2-4-71, anti-CD47 antibody 47-R2-4, anti-PD-L1 antibody L1-R2-4-71 and anti-CD47 antibody compared to IgG1 control antibody Tumor inhibitory activity of 47-R2-4 combination and anti-PD-L1/CD47 bispecific antibody BsAb-71-N297A in MC38-hCD47(Tg)/C57BL/6-hSIRP ⁇ mouse model.
  • an entity refers to one or more of such entities, eg "an antibody” should be understood to mean one or more antibodies, thus the term “an” (or “an” ), “one or more” and “at least one” are used interchangeably herein.
  • references herein to "about” or “approximately” refer to the recited value and ranges of ⁇ 10%, ⁇ 5%, ⁇ 1%, or ⁇ 0.1% thereof.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide”, and refers to a molecule consisting of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any single chain or chains of two or more amino acids, and does not refer to a particular length of the product.
  • the definition of “polypeptide” includes a peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” may Used in place of, or used interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or non-native Amino acid modifications that occur.
  • a polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a given nucleic acid sequence, and it may be produced by any means including chemical synthesis.
  • Amino acid refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid directly or in a precursor form.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides, so-called codons or base triplets. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons called “degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine Amino acid (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I) ), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • Constant amino acid substitution refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein.
  • amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
  • isolated refers to other components in the cell's natural environment, such as DNA or RNA, respectively of one or more of the isolated molecules.
  • isolated refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not, and would not exist in, their natural state.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are typically prepared by at least one purification step. In some embodiments, the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any of these values The range between any two values of , including the endpoint, or any value therein.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, non-limiting examples may be combined to produce polynucleotides that do not normally exist or peptide.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (including endpoints) between any two values in a numerical value or any value therein.
  • At least 90% identity is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, About 98% identity, about 99% identity, or a range (including endpoints) between any two of these values, or any value therein.
  • a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (eg, 90%, 95%, 98% or 99%) "identity or sequence identity" to another sequence refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned.
  • the alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignments are preferably performed using default parameters.
  • Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides having the same or similar biological activity.
  • a polynucleotide is a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when polynucleotides For RNA, thymine is replaced by uracil (U).
  • a "polynucleotide sequence” can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, eg for functional genomics and homology searches.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (eg probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation to a labeling component.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • encoding when applied to a polynucleotide refers to a polynucleotide referred to as “encoding” a polypeptide, transcribed and/or in its native state or when manipulated by methods well known to those skilled in the art Or translation can yield the polypeptide and/or fragments thereof.
  • Antibody refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide in the molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand binding portions thereof (CH), a light chain constant region (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein.
  • the CDR regions include the CDR regions of the light chain (VL CDR1-3) and the CDR regions of the heavy chain (VH CDR1-3).
  • the antibodies or antigen-binding fragments of the present invention are bispecific antibodies, including antibody fragments that specifically bind to antigen a and antigen b.
  • the first polypeptide chain comprises the structure VHa-CHa
  • the second polypeptide chain comprises the structure VLa-CLa
  • the third polypeptide chain comprises the structure VHb-CHb
  • the fourth polypeptide chain comprises the structure VLb-CLb.
  • the second polypeptide chain has the same amino acid sequence as the fourth polypeptide chain.
  • antibody fragment refers to a portion of an antibody, and the composition of the antibody fragment of the invention may be similar to that of F(ab') 2 , F(ab) 2 , Fab', Fab in monospecific antibody fragments , Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody.
  • antibody fragment includes aptamers, Spiegelmers and diabodies.
  • antigen-binding fragment also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
  • Single-chain variable fragment refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region has been removed and the linker introduced, the protein retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described, for example, in US Pat. No. 5,892,019.
  • antibody includes a wide variety of biochemically distinguishable polypeptides. Those of skill in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, etc., are well characterized and the functional specificities conferred are known.
  • the immunoglobulin molecule is of the IgG class.
  • the four chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.
  • Antibodies, antigen-binding fragments or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding Fragments (eg, Fab-like, Fab'-like, and F(ab') 2 ), single-chain-like Fvs (scFv).
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can bind to a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequence extends from the N-terminus of the Y-shaped fork-end to the C-terminus at the bottom of each chain.
  • the variable region of immunoglobulin kappa light chain is V ⁇ ; the variable region of immunoglobulin lambda light chain is V ⁇ .
  • Both light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used according to function.
  • the variable regions of the light (VL) and heavy chain (VH) portions determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. By convention, the numbering of constant regions increases as they become further from the antigen binding site or amino terminus of the antibody.
  • the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains comprise the carboxy-terminus of the heavy and light chains, respectively.
  • each antigen-binding domain In naturally occurring antibodies, the six “complementarity determining regions" or “CDRs” present in each antigen-binding domain are the short, A non-contiguous amino acid sequence that specifically binds an antigen. The remaining other amino acids in the antigen binding domain, referred to as the "framework” region, show less inter-molecular variability.
  • the framework regions mostly adopt a ⁇ -sheet conformation, and the CDRs form loop structures to which they are attached, or in some cases form part of a ⁇ -sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions.
  • An antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • an antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • amino acids comprising CDRs and framework regions can be identified by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services , Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • the boundaries of the CDRs of the variable regions of the same antibody may vary according to different assignment systems.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise the CDR sequences of the present invention, but whose variable region sequences comprise the CDR sequences of the present invention, but which are The claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention.
  • CDRs as defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof.
  • Kabat et al. also define a numbering system applicable to variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independent of experimental data other than the sequence itself.
  • Kabat Numbering means the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983).
  • Antibodies may also use the EU or Chothia numbering system.
  • the antibodies disclosed herein can be derived from any animal, including birds and mammals.
  • the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • the heavy chain constant region includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.
  • the heavy chain constant region may comprise a hinge region derived in part from an IgGl molecule and in part from an IgG3 molecule.
  • a part of the heavy chain may comprise a chimeric hinge region derived in part from an IgGl molecule and in part from an IgG4 molecule.
  • a “light chain constant region” includes a portion of the amino acid sequence from an antibody light chain.
  • the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
  • a “light chain-heavy chain pair” refers to a collection of light and heavy chains that can dimerize through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • the "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • the "CH1 domain” includes the first constant region of an immunoglobulin heavy chain.
  • the CH2 domains are not tightly paired with other domains, but rather two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule.
  • the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • the "hinge region” includes a portion of the heavy chain region connecting the CH1 domain and the CH2 domain.
  • the hinge region comprises about 25 residues and is resilient, allowing the two N-terminal antigen binding regions to move independently.
  • the hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by a disulfide bond.
  • Chimeric antibody refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial or modified) are derived from a second species.
  • the variable regions are derived from a non-human source (eg, mouse or primate), and the constant regions are derived from a human source.
  • Specifically binds generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment with a specific antigen through complementary binding of its antigen-binding domain to an epitope.
  • Specificity can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody “A” has a greater relative affinity for the same antigen than antibody “B”, antibody “A” can be considered to be more specific for that antigen than antibody "B”.
  • Specific binding can be described by the equilibrium dissociation constant (KD), a smaller KD means tighter binding.
  • Antibodies that "specifically bind" antigen a include antibodies with an equilibrium dissociation constant KD less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM, or less than or equal to about 0.5 nM with antigen a .
  • EC50 or concentration for 50% of maximal effect ( EC50 ) refers to the concentration that elicits 50% of the maximal effect.
  • a “bispecific” antibody refers to an antibody having two antigen-binding sites, which may be different epitopes of the same antigen or different epitopes of different antigens.
  • common light chain means that the light chain can be assembled with different heavy chains at the same time to form a complete antibody with corresponding functions; the light chain can be used in the expression of bispecific antibodies or in the expression of two antibodies. in the mixture.
  • effector functions refers to those biological activities attributable to the Fc region of an immunoglobulin.
  • immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, Fc Receptor binding, immune complex mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (eg B cell receptors) and B cell activation.
  • Treatment means both therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent, slow, ameliorate, or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable As a result, remission of symptoms, reduction of disease severity, stabilization of disease state (ie, no worsening), delay or slowdown of disease progression, improvement, alleviation, alleviation or disappearance of disease state (whether in part or in whole), prolongation and Expected duration of survival when not receiving treatment, etc.
  • Patients in need of treatment include patients already suffering from a condition or disorder, a patient susceptible to a condition or disorder, or a patient in need of prevention of such a condition or disorder, which may or may be expected from administration of the antibodies or pharmaceutical compositions disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prevention, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cattle, and the like.
  • bispecific antibodies Compared with monoclonal antibodies, bispecific antibodies have significant advantages such as greater specificity, targeting tumor cells and reducing off-target toxicity. Compared with monoclonal antibodies, bispecific antibodies add a specific antigen-binding site, which shows the following advantages in treatment: (1) The two antigen-binding sites can respectively bind tumor cells and immune cells, and bind T immune cells to T cells. Aggregate around tumor cells to enhance tumor killing; (2) It can block two different mediator pathways at the same time to play unique or overlapping functions, and mediate multiple immune signaling pathways to enhance cell killing toxicity; (3) The binding of two different cell surface antigens may potentially increase the binding specificity and reduce off-target side effects. Therefore, bispecific antibodies show broad application prospects in tumor immunotherapy and inflammation therapy.
  • the bispecific antibodies or antigen-binding fragments of the invention can specifically bind to PD-L1. In some embodiments, the bispecific antibodies or antigen-binding fragments of the invention can specifically bind mammalian PD-L1. In some embodiments, the PD-L1 is human PD-L1. In some embodiments, the antibody molecule binds to one or more extracellular domains of PD-L1.
  • antibody binding affinity is determined using surface plasmon resonance techniques (eg, Biacore affinity measurements).
  • the bispecific antibodies or antigen-binding fragments of the invention have one or more of the following properties:
  • the bispecific antibody or antigen-binding fragment of the invention binds PD-L1 (eg, human PD-L1) with high affinity, eg, binds to PD-L1 with an equilibrium dissociation constant (KD) of less than about 12 nM, such as less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 4 nM, 3 nM, 2 nM or 1 nM. In some embodiments, the KD is less than or equal to about 0.9 nM, or 0.5 nM.
  • KD equilibrium dissociation constant
  • the bispecific antibody or antigen-binding fragment of the invention binds to cells expressing human PD-L1, in some embodiments, eg, with an EC50 of less than or equal to about 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5nM. In some embodiments, the binding is determined by flow cytometry (eg, FACS). In some embodiments, the cells expressing human PD-L1 are CHO cells expressing human PD-L1.
  • the bispecific antibody or antigen-binding fragment of the invention blocks the related activity of PD-L1/PD-1, eg, has an EC50 of less than or equal to about 6 ⁇ g/ml, 5 ⁇ g/ml, 4 ⁇ g/ml , 3 ⁇ g/ml, 2 ⁇ g/ml, or 1 ⁇ g/ml.
  • the related activity of PD-L1 is the binding of PD-L1 to PD-1.
  • the antibody or fragment thereof of the invention is at less than or equal to about less than or equal to about 6 ⁇ g/ml, 5 ⁇ g/ml, 4 ⁇ g/ml, 3 ⁇ g/ml, 2 ⁇ g/ml, or 1 ⁇ g/ml in an MOA assay , or inhibit the binding of PD-L1 to PD-1 with an EC50 of approximately 0.1-3 ⁇ g/ml, 0.1-0.4 ⁇ g/ml or 0.1-0.3 ⁇ g/ml.
  • the cells are CHO cells that overexpress human PD-L1.
  • the bispecific antibody or antigen-binding fragment of the present invention is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the bispecific antibodies or antigen-binding fragments of the invention inhibit one or more activities of PD-L1, eg, resulting in one or more of the following: increased tumor-infiltrating lymphocytes, T cell receptor-mediated proliferation Increased, or decreased immune evasion of cancer cells.
  • the bispecific antibody or antigen-binding fragment is capable of inhibiting the proliferation of tumors that are immune escaped tumors.
  • the tumor is a gastrointestinal tumor (eg, cancer), eg, colon cancer.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a heavy chain variable region a (VHa) that specifically binds PD-L1, and a light chain variable region a (VLa).
  • VHa contains 3 CDRs (VHa CDR1, VHa CDR2, VHa CDR3)
  • VLa contains 3 CDRs (VLa CDR1, VLa CDR2, VLa CDR3).
  • the CDRs of the VHa are selected from the heavy chain CDRs in Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the VHa is selected from the heavy chain variable regions of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the CDRs of the VLa are selected from the light chain CDRs of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • the VLa is selected from the light chain variable regions of Atezolizumab, Avelumab, Durvalumab, Envafolimab, or Cosibelimab.
  • VHa CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-8; VHa CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-18; VHa CDR3 Contains the amino acid sequence shown in SEQ ID NO: 19 or 20.
  • the VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4; the VHa CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-14; the VHa CDR3 comprises the amino acid sequence set forth in SEQ ID NO: The amino acid sequence shown in 19.
  • VHa CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4;
  • VHa CDR2 comprises the amino acid sequence set forth in SEQ ID NO:14;
  • VHa CDR3 comprises the amino acid sequence set forth in SEQ ID NO:19 .
  • VLa CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-40; VLa CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-44; VLa CDR3 Contains the amino acid sequence shown in any one of SEQ ID NOs: 45-48.
  • VLa CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36; VLa CDR2 comprises the amino acid sequence set forth in SEQ ID NO:41; VLa CDR3 comprises the amino acid sequence set forth in SEQ ID NO:45 .
  • VHa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49-63; in some embodiments, VLa comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75-80 sequence.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a variable region a consisting of a VHa having an amino acid sequence set forth in SEQ ID NO:50 and a VLa having an amino acid sequence set forth in SEQ ID NO:75.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a variable region a consisting of a VHa having an amino acid sequence set forth in SEQ ID NO:51 and a VLa having an amino acid sequence set forth in SEQ ID NO:75.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a variable region a consisting of a VHa having an amino acid sequence set forth in SEQ ID NO:52 and a VLa having an amino acid sequence set forth in SEQ ID NO:75.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a variable region a consisting of VHa having the amino acid sequence set forth in SEQ ID NO:54 and VLa having the amino acid sequence set forth in SEQ ID NO:75.
  • the bispecific antibodies or antigen-binding fragments of the invention can specifically bind to CD47. In some embodiments, the bispecific antibodies or antigen-binding fragments of the invention can specifically bind mammalian CD47. In some embodiments, the CD47 is human CD47. In some embodiments, the antibody molecule binds to one or more extracellular domains of CD47.
  • the bispecific antibodies or antigen-binding fragments of the invention have one or more of the following properties:
  • the bispecific antibody or antigen-binding fragment of the invention binds CD47 (eg, human CD47) with high affinity, eg, with an equilibrium dissociation constant (KD) of less than about 700 nM, such as less than or equal to About 30 nM, less than or equal to about 10 nM, less than or equal to about 5 nM.
  • KD equilibrium dissociation constant
  • the bispecific antibody or antigen-binding fragment of the invention does not cause significant cell agglutination, eg, the bispecific antibody or antigen-binding fragment of the invention does not cause significant red blood cell hemagglutination.
  • the level of agglutination in the presence of the bispecific antibody or antigen-binding fragment of the invention is reduced by at least 5%, at least 10%, at least 10%, if compared to the level of agglutination in the presence of the anti-CD47 positive control antibody Hu5F9-G4 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%, then the bispecific antibody or antigen-binding fragment of the present invention does not cause significant the level of agglutination.
  • the bispecific antibodies or antigen-binding fragments of the invention do not cause significant cellular agglutination.
  • the bispecific antibody or antigen-binding fragment of the present invention does not bind to human erythrocytes.
  • the level of human erythrocyte binding in the presence of the bispecific antibody or antigen-binding fragment of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%, then the bispecific antibody or antigen of the present invention binds Fragments did not bind appreciably to human erythrocytes.
  • the bispecific antibodies or antigen-binding fragments of the invention do not bind significantly to human erythrocytes at antibody concentrations between 200 pM and 100 nM.
  • the bispecific antibody or antigen-binding fragment of the invention is a blocking antibody that blocks the binding of CD47 to SIRP ⁇ .
  • the ability of macrophages to phagocytose tumor cells is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% in the presence of a CD47 antibody of the invention %, at least 70%, at least 80%, at least 90% or at least 99%.
  • bispecific antibodies or antigen-binding fragments of the invention are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the bispecific antibodies or antigen-binding fragments of the invention inhibit one or more activities of CD47, eg, resulting in increased phagocytic capacity of tumor-infiltrating macrophages and/or decreased immune evasion of cancer cells.
  • the bispecific antibody or antigen-binding fragment is capable of inhibiting the proliferation of tumors that are immune escaped tumors.
  • the tumor is a gastrointestinal tumor (eg, cancer), eg, colon cancer.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a heavy chain variable region b (VHb) that specifically binds CD47, and a light chain variable region b (VLb).
  • VHb contains 3 CDRs (VHb CDR1, VHb CDR2, VHb CDR3)
  • VLb contains 3 CDRs (VLb CDR1, VLb CDR2, VLb CDR3).
  • the CDRs of the VHb are selected from heavy chain CDRs in Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio), or IBI188 (Innovative Bio), and the like.
  • the VHb is selected from the heavy chain variable region of Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio), or IBI188 (Innovative Bio), among others.
  • the CDRs of the VLb are selected from the light chain CDRs of Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio), or IBI188 (Innovent Bio), and the like.
  • the VLb is selected from the light chain variable regions of Magrolimab, AO-176 (Arch Oncology), TJC4 (Tianjing Bio), AK117 (Kangfang Bio), or IBI188 (Innovent Bio), and the like.
  • VHb CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 21-23;
  • VHb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 24-28;
  • VHb CDR3 Contains the amino acid sequence shown in any one of SEQ ID NOs: 29-35.
  • VHb CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21;
  • VHb CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24;
  • VHb CDR3 comprises the amino acid sequence set forth in SEQ ID NO:29 .
  • VLb CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-40; VLb CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-44; VLb CDR3 Contains the amino acid sequence shown in any one of SEQ ID NOs: 45-48.
  • VLb CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36; VLb CDR2 comprises the amino acid sequence set forth in SEQ ID NO:41; VLb CDR3 comprises the amino acid sequence set forth in SEQ ID NO:45 .
  • VHb CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21; VHb CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; VHb CDR3 comprises the amino acid sequence set forth in SEQ ID NO:29 VLb CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36; VLb CDR2 comprises the amino acid sequence shown in SEQ ID NO: 41; VLb CDR3 comprises the amino acid sequence shown in SEQ ID NO: 45.
  • the VHb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-74; in some embodiments, the VLb comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75-80 sequence.
  • the bispecific antibody or antigen-binding fragment of the invention comprises a variable region b consisting of a VHb having the amino acid sequence set forth in SEQ ID NO:64 and a VLb having the amino acid sequence set forth in SEQ ID NO:75.
  • the innate immune system is the first nonspecific line of defense against infection and malignant cell transformation.
  • monocytes, macrophages, and dendritic cells function as antigen-presenting cells (APCs) through phagocytosis.
  • APCs antigen-presenting cells
  • the ability of APCs to engulf tumor cells by phagocytosis is an indispensable bridge between innate and adaptive immunity. Decreased T cell activation due to reduced uptake of APCs by tumor cells, see, e.g., Avice MN et al., Role of CD47 in the induction of human naive T cell anergy, Journal of Immunology, 167(5):2459- 2468 (2001).
  • PD-L1 and CD47 proteins are both regulated by the transcription factor MYC and overexpressed on tumor cells, see, for example, Stephanie C et al., MYC regulates the antitumor immune response through CD47 and PD-L1 , Science, 352(6282):227-31 (2016).
  • MYC regulates the antitumor immune response through CD47 and PD-L1 , Science, 352(6282):227-31 (2016).
  • PD-L1/PD1 and CD47/SIRP ⁇ pathways tumor cells escape the monitoring of the innate immune system and acquire adaptive immune tolerance.
  • the present invention provides anti-PD-L1/CD47 bispecific antibodies or antigen-binding fragments that specifically bind PD-L1 and CD47.
  • the antibodies or fragments thereof of the invention bind mammalian PD-L1 and CD47, such as human PD-L1 and CD47.
  • antibody molecules specifically bind to epitopes (eg, linear or conformational epitopes) on PD-L1 and CD47.
  • the antibody molecule binds to one or more extracellular domains of PD-L1 and CD47.
  • the anti-PD-L1/CD47 bispecific antibodies or antigen-binding fragments of the invention have one or more of the following properties:
  • the anti-PD-L1/CD47 bispecific antibody of the present invention is capable of binding to both PD-L1 and CD47 while maintaining the affinity constant of the parental antibody, thereby blocking PD1/PD- L1 signaling pathway and blocking SIRP ⁇ /CD47 signaling pathway;
  • the selective binding of the anti-PD-L1/CD47 bispecific antibodies of the invention to tumor cells is facilitated by specific binding to PD-L1 on tumor cells , avoids binding with CD47 expressed in many normal tissues and reduces side effects; when the affinity with CD47 is much lower than that with PD-L1, the effective dose of the anti-PD-L1/CD47 bispecific antibody of the present invention can be significantly expanded scope;
  • the anti-PD-L1/CD47 bispecific antibodies of the invention are designed with a common light chain that avoids pairing of unrelated heavy and light chains;
  • the anti-PD-L1/CD47 bispecific antibodies of the present invention are designed with amino acid residues that stabilize the quadribody structure and facilitate proper coupling or pairing between the individual chains.
  • the respective Fc domains of the bispecific antibodies of the present invention comprise Y349C and S354C, respectively, or S354C and Y349C, respectively;
  • the respective Fc domains of the bispecific antibodies of the present invention comprise protrusions (“rods ( Knob)”) or cavity (“hole”), and the protrusion or hole in the Fc domain of the first polypeptide chain can be placed in the Fc domain of the third polypeptide chain, respectively.
  • the first polypeptide chain and the third polypeptide chain form a stable "knob-in-hole" association with each other; wherein the first polypeptide chain comprises the structure VHa-CHa can specifically recognize and bind PD-L1; the third polypeptide chain contains the structure VHb-CHb, which can specifically recognize and bind CD47.
  • the anti-PD-L1/CD47 bispecific antibody of the present invention is easy to be stably expressed in cultured cells in vitro, has good thermal stability, high antibody yield and purity, and does not require complex production Process and other advantages;
  • an anti-PD-L1/CD47 bispecific antibody or fragment thereof of the invention binds to cells expressing human PD-L1, in some embodiments, for example, at less than or equal to about 2nM, 1nM, 0.9nM, 0.8nM, EC50 of 0.7nM, 0.6nM, 0.5nM binds cells expressing human PD-L1.
  • the binding is determined by flow cytometry (eg, FACS).
  • the cells expressing human PD-L1 are CHO-S cells expressing human PD-L1.
  • an anti-PD-L1/CD47 bispecific antibody or fragment thereof of the invention blocks the related activity of PD-L1/PD-1, e.g., with an EC50 of less than or equal to about 0.3 ⁇ g/ml, Or block PD-L1/PD-1 related activity with an EC50 of approximately 0.1-3 ⁇ g/ml.
  • the related activity of PD-L1/PD-1 refers to the activation of inhibitory signals in the intracellular domain of PD1 after the binding of PD-L1 to PD-1.
  • an anti-PD-L1/CD47 bispecific antibody or fragment thereof of the invention inhibits PD with an EC50 of less than or equal to about less than or equal to about 0.3 ⁇ g/ml or with an EC50 of about 0.1-3 ⁇ g/ml in a MOA assay - Binding of L1 to PD-1.
  • the cells are CHO cells that overexpress human PD-L1.
  • the anti-PD-L1/CD47 bispecific antibody of the present invention does not cause significant cell agglutination, eg, the anti-PD-L1/CD47 bispecific antibody of the present invention does not cause significant red blood cell hemagglutination.
  • the level of agglutination in the presence of the anti-PD-L1/CD47 bispecific of the invention is reduced by at least 5%, at least 10%, At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, indicating the anti-PD-L1/CD47 bispecific antibody of the present invention No significant levels of agglutination resulted.
  • the anti-PD-L1/CD47 bispecific antibodies of the invention do not cause significant cell agglutination at antibody concentrations between 400 pM and 800 nM.
  • the anti-PD-L1/CD47 bispecific antibody of the present invention does not bind to human erythrocytes.
  • the level of human erythrocyte binding in the presence of an anti-PD-L1/CD47 bispecific antibody of the invention is reduced by at least 5% if compared to the level of human erythrocyte binding in the presence of the anti-CD47 positive control antibody Hu5F9-G4 , at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%, then the anti-PD-L1 of the present invention /CD47 bispecific antibody did not bind significantly to human erythrocytes.
  • the anti-PD-L1/CD47 bispecific antibodies of the invention do not bind significantly to human erythrocytes at antibody concentrations between 200 pM and 100 nM.
  • the anti-PD-L1/CD47 bispecific antibodies of the invention are blocking antibodies that block the binding of CD47 to SIRP ⁇ .
  • the ability of macrophages to phagocytose tumor cells is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40% in the presence of an anti-PD-L1/CD47 bispecific antibody of the invention , at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.
  • the anti-PD-L1/CD47 bispecific antibodies or fragments thereof of the invention are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the anti-PD-L1/CD47 bispecific antibody or fragment thereof of the invention has a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the anti-PD-L1/CD47 bispecific antibody or fragment thereof of the invention is incapable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the anti-PD-L1/CD47 bispecific antibody or fragment thereof of the present invention synergistically inhibits one or more activities of PD-L1 and CD47.
  • the anti-PD-L1/CD47 bispecific antibody is capable of inhibiting tumor proliferation, and the tumor is immune escape from the tumor.
  • the tumor is a gastrointestinal tumor (eg, cancer), eg, colon cancer.
  • the anti-PD-L1/CD47 bispecific antibodies of the invention have four peptide chains, two heavy chains and two light chains, respectively.
  • One heavy chain includes VHa
  • the other heavy chain includes VHb
  • one light chain includes VLa
  • the other light chain includes VLb
  • the variable region a composed of VHa and VLa specifically binds to PD-L1
  • Region b binds specifically to CD47.
  • the two light chains are the same light chain.
  • the bispecific antibodies of the invention use the "Knobs-into-Holes" technique (see, eg, John B.B. Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering, 9(7): p.617-21 (1996); Shane Atwell et al., Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol, 270: p. 26-35 (1997); Paul Carter, Bispecific human IgG by design, Journal of Immunological Methods, 248, 7-15 (2001); patent US8216805B2).
  • This technique can engineer the interface between the different chains of the bispecific antibodies of the invention to facilitate proper association of the individual chains of the bispecific antibodies of the invention.
  • this technique involves the introduction of "bumps" ("knobs") at the interface of one chain and corresponding "holes” ("holes") at the interface of the other chain with which it is to be paired , so that the protrusion can be placed in the cavity.
  • Bulges can be constructed by replacing amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (such as amino acid substitution T366W (Eu numbering)). By replacing large amino acid side chains with smaller side chains (e.g.
  • an interface with the CH3 domain of the heavy chain constant domain of the other chain to be paired is constructed with a convex Compensatory cavities of the same or similar size.
  • one heavy chain Fc domain comprises Y349C, T366S, L368A and Y407V
  • the other heavy chain Fc domain comprises S354C and T366W, forming a "knob-in-hole" stabilization associate.
  • the Fc region of a bispecific antibody of the invention comprises a modification of binding affinity for an Fc receptor.
  • the Fc receptor is an Fc ⁇ receptor, particularly a human Fc ⁇ receptor.
  • the Fc receptor is an activating Fc receptor.
  • the modification reduces the effector function of the bispecific antibodies of the invention.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the modification is within the Fc region of the immunoglobulin molecule, particularly within its CH2 region.
  • the immunoglobulin molecule comprises an amino acid substitution at position 297 (Eu numbering) of an immunoglobulin heavy chain.
  • the amino acid substitution is N297A (see, e.g., J. Lund et al., Oligosaccharide-protein interactions in IgG can modulate recognition by Fc gamma receptors, FASEB. J. 9, 115-119 (1995)).
  • the heavy chain of the anti-PD-L1/CD47 bispecific antibody of the invention further comprises a signal peptide sequence, eg, MEFGLSWVFLVAILKGVQC (SEQ ID NO:90).
  • the light chain of the anti-PD-L1/CD47 bispecific antibody of the invention further comprises a signal peptide sequence, eg, MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 91).
  • the anti-PD-L1/CD47 bispecific antibody comprises a VHa as set forth in any one of SEQ ID NOs: 49-63, a VHb as set forth in any one of SEQ ID NOs: 64-74, and VLa and VLb as set forth in any of SEQ ID NOs: 75-80.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:49, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:50, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:51, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:52, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:53, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:75 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:76 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:77 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:78 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:79 and VLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises VHa as set forth in SEQ ID NO:54, VHb as set forth in SEQ ID NO:64, and VLa as set forth in SEQ ID NO:80 and VLb.
  • the heavy chain constant region is of the IgGl subtype (SEQ ID NO:81). In some embodiments, the heavy chain constant region comprises one or more of the following amino acid mutations: N297A, Y349C, S354C, T366W, T366S, L368A, and Y407V.
  • the anti-PD-L1/CD47 bispecific antibody comprises CHa as set forth in SEQ ID NO:83 and CHb as set forth in SEQ ID NO:84; and CLa as set forth in SEQ ID NO:82 and CLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises CHa as set forth in SEQ ID NO:84 and CHb as set forth in SEQ ID NO:83; and CLa as set forth in SEQ ID NO:82 and CLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises CHa as set forth in SEQ ID NO:85 and CHb as set forth in SEQ ID NO:86; and CLa as set forth in SEQ ID NO:82 and CLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises CHa as set forth in SEQ ID NO:86 and CHb as set forth in SEQ ID NO:85; and CLa as set forth in SEQ ID NO:82 and CLb.
  • the anti-PD-L1/CD47 bispecific antibody comprises two different heavy chains and two identical light chains, heavy chain a as set forth in SEQ ID NO: 92 and heavy chain b as set forth in SEQ ID NO: 93, the light chain is shown in SEQ ID NO:96.
  • the anti-PD-L1/CD47 bispecific antibody comprises two different heavy chains and two identical light chains, heavy chain a as set forth in SEQ ID NO:94 and heavy chain b as set forth in SEQ ID NO: 95, the light chain is shown in SEQ ID NO:96.
  • the sequences of the antibodies or antigen-binding fragments disclosed in the present invention may be replaced, and the amino acid sequences thereof differ from the naturally occurring amino acid sequences of the antibody after replacement.
  • the substituted amino acid sequence can be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence, such as it can be about 80%, about 85%, about 90% identical to the starting sequence , about 95%, about 98%, or about 99%, or a range between any two of these values (including the endpoint), or any value therein.
  • the antibody comprises an amino acid sequence with one or more modifying groups.
  • the bispecific antibodies disclosed herein can be modified to add functional groups (eg, PEG, drugs, toxins, or tags).
  • antibodies and antigen-binding fragments disclosed herein include derivatives that are modified, ie, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope.
  • antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by existing techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies can be conjugated or fused to therapeutic agents, which can include cells with detectable labels (eg, radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutics, diagnostics, drugs, or toxins Toxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • detectable labels eg, radiolabels
  • immunomodulators e.g, hormones, enzymes, oligonucleotides, photosensitizing therapeutics, diagnostics, drugs, or toxins
  • Toxic agents e.g, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescent labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalate esters.
  • the present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention.
  • the polynucleotides disclosed in the present invention can encode VHa, VHb, VLa, VLb, CHa, CHb, CLa, CLb, Fc region, heavy chain a, heavy chain b, light chain and the like. Methods of making antibodies are well known in the art and described in the present invention.
  • the antibodies and antigen-binding fragments of the present disclosure comprise variable and constant regions that are fully human. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and described herein.
  • fully human antibodies directed against a particular antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to challenge with the antigen.
  • Exemplary techniques that can be used to make such antibodies are found in US Pat. Nos. 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference.
  • the bispecific antibody in the present invention is a fusion of fragments that specifically bind to antigen a and antigen b.
  • fragments of the bispecific antibody refer to the above-mentioned preparation method of an antibody that binds to a single antigen.
  • the antibodies produced do not elicit an adverse immune response in the animal (eg, human) to be treated.
  • the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, eg, residues that improve antigen binding.
  • framework substitutions can be identified by methods well known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues that are important for antigen binding and by sequence alignment to identify framework residues that are aberrant at specific positions. (See US Pat. No. 5,585,089; the entire contents of which are incorporated herein by reference).
  • Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596; ), and rearrangement of chains (US Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering an antibody to modify T cell epitopes (see eg WO/9852976 A1 and WO/0034317 A2).
  • the heavy and light chain variable region sequences from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated, showing the epitopes relative to complementarity determining regions (CDRs) and the positions of other key residues within the sequence.
  • CDRs complementarity determining regions
  • a series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed, and these sequences are subsequently incorporated into a series of binding polypeptides.
  • the genes containing the modified variable and human constant regions of the intact heavy and light chains are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies.
  • the optimal antibodies are then identified by comparing the antibodies in appropriate biochemical and biological experiments.
  • binding specificity of the bispecific antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFvs in the bispecific antibodies of the invention can be found in techniques for producing single chain units (US Pat. No. 4,694,778).
  • the heavy and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit, resulting in a single-chain fusion peptide.
  • Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).
  • Yeast can be used to display the scFv
  • the yeast display vector can be pYD1 vector (Addgene)
  • the host Saccharomyces cerevisiae can be Saccharomyces cerevisiae EBY100 (Invitrogen).
  • scFvs single chain Fvs
  • antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498.
  • Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, eg, antibodies having the variable regions of murine monoclonal antibodies and the constant regions of human immunoglobulins. Methods of producing chimeric antibodies are known in the art, see US Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire contents of the patents are incorporated herein by reference.
  • Fully human antibodies that recognize selective epitopes can be produced using a technique known as "guided selection.”
  • selected non-human monoclonal antibodies eg, mouse antibodies
  • guided selection is used to guide the screening of fully human antibodies recognizing the same epitope.
  • DNA encoding the desired monoclonal antibody can be isolated and subjected to Sequencing.
  • Isolated and subcloned hybridoma cells can serve as a source of such DNA.
  • the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that does not produce other immunoglobulins in cells.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of antibodies, as described in US Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Pat. No. 5,658,570.
  • one or more CDRs of the antibodies of the invention can be inserted into framework regions, eg, into human framework regions, to construct humanized, non-fully human antibodies.
  • Framework regions can be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a list of human framework regions).
  • Some polynucleotides may encode an antibody that specifically binds to at least one epitope of an antigen of interest resulting from a combination of framework regions and CDRs.
  • One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected to improve binding of the antibody to its antigen.
  • substitution or deletion of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed using this method, resulting in antibody molecules lacking one or more interchain disulfide bonds.
  • Other changes to polynucleotides that are within the skill in the art are also encompassed by the present invention.
  • Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, etc. can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, which in their entirety include Supplementary content is incorporated by reference in its entirety.
  • DNA encoding the antibody can be designed and synthesized according to the amino acid sequences of the antibodies described herein according to conventional methods, placed in an expression vector, then transfected into host cells, and the transfected host cells are grown in culture to produce Antibody.
  • the DNA expressing the heavy chain and light chain of the antibody can be placed in the same vector or in different vectors; if placed in different vectors, the vector expressing the heavy chain of the antibody and the vector of the light chain can be transfected into host cells (such as Tihomir) in an appropriate ratio.
  • the expression antibody vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal and a polyA tail.
  • Efficient transcription can be achieved by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and also Some other cellular promoters such as the actin promoter may be used.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+ /-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc.
  • Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, and mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain a selectable marker.
  • selectable markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening isolation of successful cells.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in selective medium, the successfully transfected cells grow in large quantities to produce the desired target protein.
  • Bispecific antibody preparation methods are widely documented in the literature, such as Qiong Wang, et al., Design and Production of Bispecific Antibodies, Antibodies, 8, 43 (2019); Zhuang Zuo, et al., An efficient route to the production of an IgG-like bispecific antibody, Protein Engineering, Design and Selection, 13(5):361–367(2000); Matthias Mack, et al., A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell Cytotoxicity, Proc.Natl.Acad.Sci., 92:7021-7025, (1995); Rodrigo Vazquez-Lombardi, et al., Transient expression of human antibodies in mammalian cells, Nature Protocols 13(1):99-117( 2018); Elisa Corsiero, Monoclonal Antibodies: Expression and Purification in a Basic Research Laboratory, Mater Methods, 6:1481 (2016).
  • mutations can be introduced in the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those of skill in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions.
  • Variants include derivatives
  • substitution of amino acids, substitution of less than 4 amino acids, substitution of less than 3 amino acids, or substitution of less than 2 amino acids can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • substitutions described herein are conservative amino acid substitutions.
  • the present invention also provides methods and uses of treatment.
  • methods are provided for the treatment or amelioration of various types of cancers, tumors, or infections related diseases, the methods comprising administering to a patient an effective dose of the bispecific antibody.
  • the use of the bispecific antibody in the treatment or amelioration of related diseases such as cancer, tumor or infection is provided.
  • the application of the bispecific antibody in the preparation of a medicament for treating or ameliorating related diseases such as cancer, tumor or infection is provided.
  • the present invention relates to a method for treating related diseases with PD-L1 and/or CD47 as therapeutic targets; it relates to ameliorating, slowing, reducing, and reducing the binding of PD-L1 to PD1 and/or the binding of CD47 to SIRP ⁇ by eliminating, inhibiting or reducing the binding of PD-L1 and PD1.
  • a method of inhibiting or preventing any disease or condition involving providing a method of treating a cancer or tumor in a subject, a method of alleviating a symptom of a cancer or tumor in a subject, and a method of avoiding the recurrence of a tumor or cancer in a subject, the method comprising applying The subject is administered an effective amount of any anti-PD-L1/CD47 bispecific antibody or fragment thereof described herein.
  • the antibodies and antigen-binding fragments thereof provided by the present invention and pharmaceutical compositions comprising the same can be used as therapeutic agents for diagnosing, prognosing, monitoring, treating, alleviating and/or preventing abnormal PD-L1 and/or CD47 in a subject Diseases and disorders related to expression, activity and/or signaling.
  • the monoclonal antibodies, bispecific antibodies and antigen binding thereof disclosed herein can be administered by identifying the presence of diseases and disorders associated with aberrant PD-L1 or CD47 expression, activity and/or signaling in a subject using standard methods Fragments and pharmaceutical compositions containing the same.
  • the anti-PD-L1/CD47 bispecific antibodies disclosed in the present application meet this need, which can not only effectively promote phagocytosis, but not cause significant erythrocyte agglutination, and more preferably, do not significantly bind to human erythrocytes.
  • the anti-PD-L1/CD47 bispecific antibodies of the present invention are capable of binding to PD-L1 with ultra-high affinity, and to CD47 with high or medium affinity; through specificity for PD-L1 on tumor cells Sexual binding promotes the selective binding of the anti-PD-L1/CD47 bispecific antibody of the present invention to tumor cells, avoids binding to CD47 expressed in many normal tissues, and reduces side effects;
  • the affinity of -L1 can significantly expand the effective dose range of the anti-PD-L1/CD47 bispecific antibody of the present invention.
  • cancers treated and/or prevented with the antibodies described herein include, but are not limited to, solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastases sexual lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignancies, eg, sarcomas and carcinomas of multiple organ systems, eg, involving the lung, breast, ovary, lymphoid, gastrointestinal (eg, colon), anus, genitals, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, neural or glial cells), head and neck, skin (eg, melanoma), nasopharyngeal (eg, differentiated or undifferentiated metastases) nasopharyngeal carcinoma) and those of the pancreas.
  • Cancer can be in early, intermediate or advanced stages or metastatic.
  • the tumor is tumor immune escape.
  • the tumor is a gastrointestinal tumor (eg, cancer), eg, colon cancer.
  • the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody or derivative used, the patient's age and weight, general health, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are left to the judgment of health care professionals, including those within the purview of those of ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the effect desired.
  • the dose to be used can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, an effective dose ranges from about 0.01 mg/kg to about 100 mg/kg, which may be, for example, twice a week (BIW) or to once a month.
  • Methods of administration of the antibody or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection.
  • the pharmaceutical compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelia or mucocutaneous (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.
  • compositions containing the antibodies of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (eg, by powder, ointment, drops or transdermal patch), oral administration, or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration can be systemic or topical. Furthermore, it may be desirable to introduce the antibodies of the invention into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injection; intracerebroventricular injection may be accomplished via an intraventricular catheter connected, for example, to a reservoir (which may be an Ommaya reservoir). Assisted injection. Pulmonary administration is also possible, eg, by the use of an inhaler or nebulizer, as well as the use of aerosolized formulations.
  • Antibodies of the invention may be administered topically to the area in need of treatment; by, but not limited to, by local infusion during surgery, such as topical application in combination with post-surgical wound dressings, by injection, by catheter, by suppository, or by implant
  • the implant is a porous, non-porous or gel-like material, including membranes (eg, silicone rubber membranes) or fibers.
  • membranes eg, silicone rubber membranes
  • Methods for treating disease are typically tested in vitro, comprising administration of an antibody or derivative of the invention, followed by in vivo testing for the desired therapeutic or prophylactic activity in an acceptable animal model, followed by administration to humans.
  • Suitable animal models including transgenic animals, are known to those of ordinary skill in the art.
  • in vitro assays for demonstrating the therapeutic use of the antibodies, antigen-binding fragments of the invention include the effect of the antibody on a cell line or patient tissue sample.
  • the effect of antibodies on cell lines and/or tissue samples can be detected using techniques known to those of skill in the art, such as those disclosed elsewhere herein.
  • In vitro assays that can be used to determine whether to administer a specific antibody in accordance with the present invention include in vitro cell culture experiments in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound and the effect of such compound is observed on The effect of tissue samples.
  • Various known delivery systems can be used to administer the antibodies or derivatives of the invention or polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated Endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviral or other vectors, and the like.
  • the antibodies of the invention may be used in conjunction with or in combination with other therapeutic or prophylactic regimens, including administration of one or more antibodies of the invention and one or more other therapeutic agents or methods.
  • the antibody and other therapeutic agents may be administered simultaneously or separately.
  • the antibody of the invention may be administered before or after administration of the other other therapeutic agent.
  • the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein may also be administered to the patient in combination with one or more other therapies, eg, treatment modalities and/or other therapeutic agents (eg, chemotherapeutic agents, radiotherapeutic agents, or biomacromolecular drugs).
  • therapies eg, treatment modalities and/or other therapeutic agents (eg, chemotherapeutic agents, radiotherapeutic agents, or biomacromolecular drugs).
  • Such combination therapy encompasses combined administration (in which two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case other therapies, such as therapeutic modality and Administration of the antibodies of the invention occurs before, concurrently with, and/or after the therapeutic agent.
  • Antibody molecules and/or other therapies eg, therapeutic agents or treatment modalities, can be administered during active disease or during remission or less active disease.
  • Antibody molecules can be administered before, concurrently with, after, or during remission of the disease.
  • the bispecific antibodies of the invention are administered in combination with a chemotherapeutic agent.
  • chemotherapeutic agents that can be administered with the anti-PD-L1/CD47 bispecific antibodies of the invention include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and radioactivity Bacteriocin D), antiestrogens (eg, tamoxifen), antimetabolites (eg, fluorouracil, 5-FU, methotrexate, floxuridine, interferon alfa-2b, glutamate, mithranil urea, mercaptopurine and 6-thioguanine), cytotoxic agents (such as carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, methylphenidate procarbazine, mitomycin, busulfan, cisplatin, and vincris
  • antibiotic derivatives e
  • melphalan chlorambucil, dichloromethyldiethylammonium (nitrogen mustard) and thiotepa) , steroids and combinations thereof (eg betamethasone sodium phosphate), and other compounds (eg dazolamide, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
  • steroids and combinations thereof eg betamethasone sodium phosphate
  • other compounds eg dazolamide, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide.
  • the antibodies of the invention are administered in combination with cytokines.
  • Cytokines that can be administered with the antibodies of the invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, and IL-15, etc.
  • the bispecific antibodies of the invention are administered in combination with a biomacromolecule drug.
  • biomacromolecular drugs include immunotherapeutics, including, but not limited to, therapeutic antibodies suitable for treating patients.
  • therapeutic antibodies include sintuzumab, abavolumab, adekatumumab, afutuzumab, alemtuzumab, atumomab, amatuximab Antibiotics, Anatumumab, Asimumab, Bavetuximab, Bettumumab, Bevacizumab, Bivazumab, Brenatumumab, Brentuximab monoclonal antibody, cantuzumab, catumazumab, cetuximab, cetuzumab, cetuximab, crivatuzumab, kentuzumab, Latuzumab, Jochituzumab, Doligotuzumab, Dulcetu
  • the antibodies of the invention may be used with immune checkpoint inhibitors. In some embodiments, the antibodies of the invention are administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy.
  • compositions comprise an effective dose of the antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an anticancer agent (eg, an immune checkpoint inhibitor).
  • the term "pharmaceutically acceptable” refers to a substance approved by a regulatory agency of the government or listed in a recognized pharmacopeia for use in animals, particularly in humans.
  • pharmaceutically acceptable carrier generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid and the like.
  • carrier refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc.
  • the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates, if desired.
  • Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the composition can be formulated as a suppository with traditional binders and carriers such as triglycerides.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to the patient.
  • the formulation should be suitable for the mode of administration.
  • the parental formulation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • compositions are formulated according to conventional procedures into pharmaceutical compositions suitable for intravenous injection into humans.
  • Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffer.
  • the composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied in unit dosage form either individually or mixed together, eg, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container (eg, an ampule or sachet) indicative of the amount of active agent.
  • the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • ampoules of sterile water for injection or saline can be used so that the active ingredient can be mixed prior to administration.
  • the compounds of the present invention may be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include those derived from anions such as hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those derived from anions such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamine Salts formed from cations of aminoethanol, histidine, procaine, etc.
  • CD47-His antigen preparation Add 8 ⁇ HIS tag (SEQ ID NO:3) to the C-terminus of human CD47 extracellular domain (ECD) protein (SEQ ID NO:1) to construct a CD47 recombinant protein.
  • ECD extracellular domain
  • the ECD region of human CD47 is genetically synthesized and subcloned into a mammalian expression vector (eg, pcDNA3.1(+) expression vector). After transient transfection of HEK293F cells, nickel-based immobilized metal affinity chromatography-purified (GE Healthcare) CD47-His antigen was obtained.
  • PD-L1-His antigen preparation Add 8 ⁇ HIS tag (SEQ ID NO:3) to the C-terminal of human PD-L1 extracellular domain (ECD) protein (SEQ ID NO:2) to construct PD-L1 recombinant protein .
  • ECD extracellular domain
  • the ECD region of human PD-L1 was gene-synthesized and subcloned into mammalian expression vectors (eg, pcDNA3.1(+) expression vector). After transient transfection of HEK293F cells, nickel-based immobilized metal affinity chromatography-purified (GE Healthcare) PD-L1-His antigen was obtained.
  • CD47-ECD SEQ ID NO: 1
  • the nucleic acid sequence encoding the scFv fragment of the anti-PD-L1 antibody (including the variable region of the heavy chain, the variable region of the light chain and the linker (G 4 S) 3 of the variable region of the heavy and light chains) was synthesized, inserted into a vector, and Positive yeast clones were obtained by electroporation into the host Saccharomyces cerevisiae. The positive yeast clones obtained above were picked out and monoclonally cultured.
  • variable region of the heavy chain and the variable region of the light chain in the scFv expressed on the surface of the ATE positive yeast clone are consistent with Atezolizumab
  • the scFv expressed on the surface of other positive yeast clones are shown in Table 1, and the corresponding sequences of each VH SEQ ID NO: are as follows: As shown in Table 2, the corresponding CDRs are shown in Table 3.
  • the amino acid sequence of the linker is GGGGSGGGGSGGGGS, and the nucleic acid sequence thereof is ggtggaggcggttcaggcggaggtggctctggcggtggatcg.
  • VH SEQ ID NO: VL SEQ ID NO: CP11-27 49 75 CP11-36 50 75 CP11-46 51 75 CP11-47 52 75 CP11-55 53 75 CP11-71 54 75 CP14-16 55 75 CP14-80 56 75 CP21-8 57 75 CP21-47 58 75 CP21-59 59 75 CP22-34 60 75 CP22-40 61 75 CP22-44 62 75 CP22-45 63 75
  • VH and VL of 11 kinds of IgG1 whole antibodies is shown in Table 4; VH and CH constitute the heavy chain of the antibody, and VL and CL constitute the light chain of the antibody; the sequence of CH is such as SED ID NO: 81, and the sequence of CL is such as SED ID NO : 82; represented by "antibody” + antibody number, such as antibody 17 (the heavy chain of the antibody consists of VH as shown in SED ID NO: 65 and CH as shown in SED ID NO: 81, and the light chain of the antibody consists of as shown in SED ID NO: 81). Composed of VL as shown in SED ID NO:98 and CL as shown in SED ID NO:82).
  • X-VL stands for 3-3-VL, 3-6-VL, 3-VL, 6-VL, 10-VL, 16-VL, 18-VL, 24-VL, 25-VL
  • Hu5F9-G4 Magneticrolimab
  • the assembly scheme of the antibody is shown in Table 7.
  • the variable region assembly scheme is "VH+VL"
  • the sequence of CH is shown in SED ID NO: 81
  • the sequence of CL is shown in SED ID NO: 82.
  • the light chain variable region sequences are shown in Table 8, and the light chain variable region CDRs are shown in Table 9.
  • the gene encoding the heavy chain of the antibody and the gene of the light chain of the antibody are cloned into expression vectors, respectively, to obtain a heavy chain expression vector and a light chain expression vector.
  • the heavy chain expression vector and light chain expression vector were then transiently transfected into HEK293F cells. After the cells were expressed, the culture medium was purified by immobilized metal affinity chromatography (IMAC) using a ProteinA column to obtain the antibody, which was consistent with the expected sequence after sequencing.
  • IMAC immobilized metal affinity chromatography
  • the schematic diagram of the structure of the anti-PD-L1/CD47 bispecific antibody is shown in Figure 1, and the amino acid sequences of the variable regions of the five antibodies are shown in Table 10. It can be seen from the schematic diagram of Fig. 1 that the anti-PD-L1/CD47 bispecific antibody is composed of 4 polypeptide chains.
  • the C-terminus of the VH of anti-PD-L1 is connected to the N-terminus of the constant region (SEQ ID NO: 83) derived from human IgG1, and its Fc region comprises "Rod into hole” mutation for stable association with anti-CD47 heavy chain; light chain is linked by R2-4 (SEQ ID NO:75) and human kappa light chain constant region (SEQ ID NO:82) to form a common light chain
  • the C-terminus of the VH of anti-CD47 is linked to the N-terminus of a constant region (SEQ ID NO: 84) derived from human IgG1, and its Fc region contains a "rod-in-hole” mutation for stable association with the anti-PD-L1 heavy chain.
  • Antibody BsAb-71-N297A is on BsAb-71, and the Fc region is mutated with N297A to reduce the ADCC effect of the antibody. That is, the anti-PD-L1 heavy chain of BsAb-71-N297A is shown in SEQ ID NO:92, the anti-CD47 heavy chain is shown in SEQ ID NO:93, and the light chain is shown in SEQ ID NO:96.
  • the nucleotide sequence encoding the anti-PD-L1/CD47 bispecific antibody constructed in step 2.3.1 was constructed into an expression vector, and the anti-PD-L1/CD47 antibody was obtained by expressing and purifying it in HEK293F cells, as follows:
  • the gene encoding the heavy chain of anti-PD-L1 (take BsAb-71-N297A as an example, the gene sequence is as shown in SEQ ID NO: 99) and the gene of the heavy chain of anti-CD47 (take BsAb-71-N297A as an example, The gene sequences are shown in SEQ ID NO: 100) and cloned into expression vectors respectively to obtain anti-PD-L1 heavy chain plasmids and anti-CD47 heavy chain plasmids; the genes encoding the light chain of anti-PD-L1/CD47 antibodies (with BsAb-71-N297A is taken as an example, the gene sequence is shown in SEQ ID NO: 101) and cloned into an expression vector to obtain a light chain plasmid.
  • anti-PD-L1 heavy chain plasmid, anti-CD47 heavy chain plasmid and light chain plasmid were transiently transfected into HEK293F cells in a molar ratio of 1:1:2. After cell expression, the culture medium is subjected to affinity chromatography and ion exchange chromatography to obtain an anti-PD-L1/CD47 antibody (eg BsAb-71-N297A). Antibody sequences were confirmed after sequencing.
  • SEC size exclusion chromatography
  • Anti-PD-L1 heavy chain nucleotide sequence SEQ ID NO: 99 is as follows:
  • the nucleic acid sequence of its Fc is (SEQ ID NO:87):
  • the anti-CD47 heavy chain nucleotide sequence SEQ ID NO: 100 is as follows:
  • the nucleic acid sequence of its Fc is (SEQ ID NO:88):
  • the light chain nucleotide sequence SEQ ID NO: 101 is as follows:
  • the nucleic acid sequence of its CL is (SEQ ID NO: 89):
  • yeast-displayed anti-PD-L1 scFv to the antigenic protein PD-L1-His-Biotin was detected by flow cytometry.
  • the positive yeast clones in step 2.1 of Example 2 were incubated with 1 nM PD-L1-His-Biotin antigen protein for 1 h at room temperature, washed three times with PBS with pH 7.4, and then mixed with Streptavidin-PE fluorescent secondary antibody at room temperature. Incubate for 30 min, wash with PBS at pH 7.4 for 3 times, add new PBS, and prepare for on-board detection.
  • PD-L1-His-Biotin The purified PD-L1-His antigen protein was added with 26.6 ⁇ l of biotin (Sigma-Aldrich, B4501-1G) prepared with 10 nM DMSO per 1 mg of protein, and incubated at room temperature for 2 h, then incubated with pH 7 .4 PBS for dialysis.
  • biotin Sigma-Aldrich, B4501-1G
  • the binding force of the anti-PD-L1 scFv (CP11-36, CP11-46, CP11-71, CP14-16 and CP14-80) of the present invention to the PD-L1-His-Biotin antigen protein is significantly better than Positive control ATE scFV; compared with positive control ATE scFV, the anti-PD-L1 scFv of the present invention (CP-11-47, CP-11-55, CP21-8, CP21-47 and CP22-34) and PD-L1-
  • the binding capacity of His-Biotin antigen protein is basically the same.
  • the bound SIRP ⁇ -Fc-Bio reacts with the HRP-conjugated streptavidin secondary antibody to generate a chemiluminescence reaction, and the OD450 value is detected by a plate reader, and the IC50 of the antibody is converted according to the OD450 value, thereby judging the SIRP ⁇ blocking of the antibody active.
  • the results are shown in Table 11. The results showed that the above-mentioned anti-CD47 antibodies could inhibit the binding of SIRPa to CD47 on the cell surface.
  • Example 5 Affinity of anti-PD-L1 antibodies and blocking activity against PD-1/PD-L1
  • the equilibrium dissociation constant (KD) of the antibody in step 2.2.2 of Example 2 of the present invention bound to the corresponding antigen was determined by kinetic binding assay using Biacore system (GE Company). According to the method of the user manual, 5 ⁇ g of the antigenic protein PD-L1-His was immobilized on the chip, and the anti-PD-L1 antibody concentration of up to 100nM (1:2 dilution, 5 gradients) was used as the mobile phase for affinity determination. The results were analyzed with Biacore T200 Evaluation Software. Among them, the positive control is ATE antibody (the amino acid sequence of ATE antibody is the same as that of Atezolizumab, which is expressed by HEK293F cells).
  • Table 12 shows the KD data of the exemplary antibodies, it can be seen that the antibodies of the invention L1-R2-4-36, L1-R2-4-46, L1-R2-4-47, L1-R2-4 compared to the ATE antibodies -55 and L1-R2-4-71 have higher affinity for PD-L1 antigen.
  • the biological activity detection method of the anti-PD-L1 antibody detects the inhibitory effect of the anti-PD-L1 antibody constructed above on the binding of PD-1/PD-L1.
  • Anti-PD-1/PD-L1 antibody can relieve the inhibition of downstream NFAT signaling pathway by blocking the binding of PD-1 and PD-L1.
  • MOA Mechanisms of Action, MOA
  • detection system PD-1/PD-L1 Blockade Bioassay, Propagation Model, Catalog J1252
  • the NFAT signal was reflected by detecting the expression of the fluorescent reporter gene according to the method provided in the instructions. Therefore, the inhibitory effect of the antibody in step 2.2.2 of Example 2 on PD-1/PD-L1 binding was detected.
  • the negative control antibody IgG-Isotype was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (catalog number: HG1K).
  • Plating CHO-PD-L1 cells (from the MOA assay system above) one day before the activity assay: Passage 1-2 days before plating CHO-PD-L1, discard the culture supernatant, and wash the cells with sterile PBS. An appropriate amount of Trypsin (Gibco) was added and digested in a 37°C, 5% CO2 incubator for 3-5min. Digestion was terminated by adding fresh medium, and cells were transferred to 50 ml centrifuge tubes and counted. Take the required volume of cells and centrifuge at 900 rpm for 5 min. DMEM-F12 medium (Gibco) was added and the cells were resuspended to 4 ⁇ 10 5 cells/mL. Cells were added to 96-well white cell culture plates (Corning) at 100 ⁇ l/well. Cells were cultured overnight in a 37°C, 5% CO2 incubator.
  • Jurkat-PD1 cells (from the above MOA detection system) were processed on the day of detection: after counting, cells of the required volume were taken and centrifuged at 900 rpm for 5 min. Cells were resuspended to 1.25 x 106 cells/ml with assay buffer (1640 medium (Gibco) + 1% FBS) until use. Aspirate the medium supernatant in the CHO-PD-L1 cell culture plate, discard 95 ⁇ l/well, and add 40 ⁇ l/well of the test sample (the above-mentioned anti-PD-L1 antibody, ATE antibody and negative control HG1K antibody, the maximum concentration is 20 ⁇ g/well).
  • ONE-Glo TM Luciferase Assay System purchased from Promega, E6120 was thawed in advance. After 6 hours, add ONE-Glo TM reagent, 50 ⁇ l/well. Place at room temperature for 5-10min and read.
  • the antibodies of the present invention L1-R2-4-36, L1-R2-4-46, L1-R2-4-47, L1-R2-4-55 and L1-R2-4-71 Both can effectively block the interaction of PD1/PD-L1; compared with the control antibody ATE, the antibodies (L1-R2-4-36 and L1-R2-4-46) have stronger blocking activity.
  • the dissociation constants of the anti-PD-L1 antibody and the anti-CD47 antibody in step 2.2.3 of Example 2 were determined by using the method of Biacore in Example 5 to detect the affinity dissociation constant (KD). The results are shown in Table 13.
  • the different light chains of the present invention can simultaneously form monoclonal antibodies with anti-PD-L1 and anti-CD47 heavy chains, and bind to human PD-L1 and human CD47 proteins in solution respectively.
  • Example 5 The MOA method of Example 5 was used to detect the blocking activity of the anti-PD-L1 antibody in step 2.2.3 of Example 2 on PD-1/PD-L1.
  • the experimental results are shown in Figure 5.
  • the anti-PD-L1 antibodies of different light chains of the present invention PD-L1 antibodies can effectively block the interaction of PD1/PD-L1.
  • Specific detection method collect fresh human blood, wash it three times with normal saline and prepare a 2% human erythrocyte suspension, mix the 2% human erythrocyte suspension with the test antibody (the highest final concentration is 800nM, twice serially diluted, a total of 11 1 diluted concentration) were mixed in equal volumes, incubated at 37°C for 4 hours, and then assessed for the agglutination of antibody erythrocytes, tilt the 96-well U-shaped plate at 45°, and observe the flow direction of the erythrocyte mass. Agglutination occurs. In experiments performed as described in the assay above, the results of the hemagglutination reaction are shown in Figure 6 .
  • step 2.2.3 An in vitro phagocytosis assay was performed to assess whether the anti-CD47 antibody of Example 2, step 2.2.3, enhanced phagocytosis of CD47-expressing target cells by macrophages. Briefly, RAW264.7 macrophages (2 ⁇ 10 5 cells/mL) were mixed with CFSE (Invitrogen, catalog number: C34554 )-labeled Raji cells (4 ⁇ 105/mL) were plated in a 1 :2 ratio into a 24-well bottom plate and incubated for 2 hours at 37°C in the dark.
  • CFSE Invitrogen, catalog number: C34554
  • phagocytosis index number of CFSE-F4/80 double-positive macrophages (ie, the number of macrophages that phagocytosed tumor cells)/per 5000 macrophages.
  • phagocytosis index number of CFSE-F4/80 double-positive macrophages (ie, the number of macrophages that phagocytosed tumor cells)/per 5000 macrophages.
  • Tm values of the exemplified bispecific antibodies and the reference antibody were determined by DSC (differential scanning calorimeter, Malvern Panalytical, MicroCal VP-Capillary), and according to the This preliminary judgment exemplifies the thermal stability of the bispecific antibody.
  • the sample protein was prepared to a concentration of 2 mg/mL in 1 ⁇ PBS buffer (pH 7.4). Starting at 40°C, the specific heat capacity (Cp) of the sample or blank buffer was scanned at a rate of 180°C/hr. The results of the sample scan were deducted from the results of the corresponding buffer solution, and the obtained Cp value was used to plot the temperature, wherein the temperature corresponding to the peak of the significantly increased Cp value was the Tm value of the sample.
  • Cp specific heat capacity
  • the exemplified bispecific antibody and the L1-R2-4-71 reference sample both showed significant Tm values, including the dissolution temperature of CH2 at around 70°C and the dissolution of CH3 at around 85°C. temperature.
  • the Tm value of the bispecific antibody is slightly lower than that of L1-R2-4-71, but the difference is not obvious. It can be preliminarily determined that the bispecific antibody is similar to the monoclonal antibody and has good thermal stability.
  • the binding capacity of exemplary bispecific antibodies to PD-L1 and CD47 was determined by kinetic binding assays using the Octet system (ForteBio).
  • the ProA Biosensor AHC sensor Pall, 1506091 was soaked in PBS buffer for 10 minutes at room temperature to equilibrate.
  • the experimental method was the same as above, but the binding order of the antibody to the two antigens was reversed from the above experiment, and the ProA biosensor AHC was immersed in the wells respectively containing the antibody solutions for 120 seconds at room temperature. Loading. The sensors were then washed in PBS buffer to baseline, then immersed in wells containing 100 ⁇ l of CD47-His antigen solution, then the sensors were washed in PBS buffer to baseline, then immersed in 100 ⁇ l of PD-L1-His In the wells of the antigen solution, the association of the antibody with the antigen is monitored. The results are shown in Figure 8B.
  • the experimental method was the same as above, but the bispecific antibody was BsAb-71-N297A, and the binding activity of the bispecific antibody BsAb-71-N297A to two antigens was detected.
  • the results are shown in Figure 8C, indicating that BsAb-71-N297A can simultaneously bind to PD-L1 and CD47 proteins in solution.
  • bispecific antibodies exemplified in the present invention can simultaneously bind to PD-L1 and CD47 proteins in solution, and will not interfere with each other when binding to different epitopes.
  • Example 9 Dissociation constant determination of anti-PD-L1/CD47 bispecific antibody
  • Table 15 Dissociation constants (KD) of exemplary antibodies to human PD-L1-His or human CD47-His antigens, respectively, determined by Biacore kinetic binding assay
  • the bispecific antibody exemplified in the present invention can bind to human PD-L1-His in solution using the fitting method of 1:1 Binding, and maintains the affinity constant of the parent antibody; the bispecific antibody exemplified in the present invention can bind to human PD-L1-His in solution. It can bind to human CD47-His protein in solution.
  • the affinity constant of the antibody is weaker than that of the parent antibody, and it has a moderate anti-CD47 affinity.
  • Example 10 Binding analysis of anti-PD-L1/CD47 bispecific antibodies to CHO cells overexpressing PD-L1 or CD47
  • human PD-L1 or human CD47 cDNA was inserted into an expression vector and transfected into Chinese hamster ovary cancer cells (CHO, Invitrogen) to generate CHO cells (PD-L1 or CD47) overexpressing human PD-L1 or human CD47.
  • CHO Chinese hamster ovary cancer cells
  • -L1-CHO cells or CD47-CHO cells overexpressing human PD-L1 or human CD47.
  • PD-L1-CHO cells or CD47-CHO cells were mixed with different serial dilutions of exemplified bispecific antibodies, and incubated on ice for 1 h; cells were washed twice with PBS, and then PE-labeled anti-human Fc antibody (Invitrogen, 12-4998-82) fluorescent secondary antibody, incubate on ice for 30 minutes in the dark; after washing the cells twice with PBS, resuspend the cells in PBS, and detect the binding of the antibody to the cells by FACS.
  • FIG. 9A and 9B The results are shown in Figures 9A and 9B. It can be seen from FIG.
  • the anti-PD-L1/CD47 bispecific antibody BsAb-71-N297A was assayed for binding to CHO cells overexpressing PD-L1 or CD47 according to the methods described above.
  • the results are shown in Figures 9C and 9D. It can be seen from Figure 9C that BsAb-71-N297A can bind to PD-L1 expressed on the cell surface and maintain the binding EC50 of the parental antibody. Meanwhile, it can be seen from Fig. 9D that BsAb-71-N297A can bind to CD47 expressed on the cell surface.
  • CD47 protein is expressed in various normal tissues of human beings to different degrees, which brings certain obstacles to the therapeutic application of anti-CD47 antibodies.
  • the present invention by strategically reducing the affinity and binding force of the bispecific antibody to human CD47, it is foreseen that the side effects caused by the antibody can be reduced in clinical treatment, and it can be widely used in the treatment of various cancers.
  • Example 11 Detection of anti-PD-L1 activity of bispecific antibodies exemplified in the present invention based on MOA method
  • the anti-PD-L1 activity of the bispecific antibodies (BsAb-36, BsAb-46, BsAb-47, BsAb-71 and BsAb-71-N297A) exemplified in the present invention was detected.
  • the results are shown in Figure 10A and Figure 10B, the bispecific antibody of the present invention can relieve the inhibitory effect of PD-1/PD-L1 interaction on NFAT signaling pathway, and its activity is better than that of L1-R2- anti-PD-L1 antibody.
  • the 4-71 antibody was used alone.
  • Example 12 Detection of anti-PD-L1/CD47 bispecific antibody promoting hemagglutination activity
  • the present invention further studies the hemagglutination effect of the anti-PD-L1/CD47 bispecific antibody.
  • the results are shown in Fig. 11 , the bispecific antibody exemplified in the present invention did not cause erythrocyte agglutination, and its activity of promoting erythrocyte agglutination was significantly lower than that of the control group Hu5F9-G4, which was consistent with the parent antibody 47-R2-4.
  • Example 13 Detection of the ability of bispecific antibodies to promote macrophage phagocytosis of tumor cells (phagocytosis map)
  • bispecific antibodies of the invention The ability of exemplified bispecific antibodies of the invention to promote phagocytosis of tumor cells by macrophages was measured in a flow cytometry-based assay.
  • the bispecific antibody exemplified in the present invention was evaluated in vitro to promote the phagocytosis of target cells by macrophages. It can be seen from Figure 12 that the anti-PD-L1/CD47 bispecific antibody can effectively induce macrophages to phagocytose target cells, and its induction activity is similar to that of the anti-CD47 monoclonal antibody.
  • the bispecific antibody of the present invention has only weak binding to human erythrocytes, which is significantly weaker than that of the parent antibody 47-R2-4 and the positive control antibody Hu5F9-G4 and human erythrocytes. Red blood cell binding; differentiated red blood cell binding properties do not affect the binding of the bispecific antibody and the parent antibody to tumor cells, therefore, the bispecific antibody of the present invention has higher clinical application safety and wider therapeutic adaptability.
  • Example 15 Effect of anti-PD-L1/CD47 bispecific antibody on IL2 cytokine release from human peripheral blood mononuclear cells (PBMC) under exogenous stimulation
  • PBMC peripheral blood mononuclear cells
  • the example antibodies were incubated with PBMCs cultured in vitro and derived from different donors, and the IL2 expression in the system was detected to reflect the activation effect of different antibodies on T cells.
  • PBMC separation Take fresh anticoagulated whole blood from donors (Donor1 and Donor2), dilute the whole blood with an equal volume of PBS at a ratio of 1:1; add 7mL of human peripheral blood lymphocyte separation solution to a 15mL centrifuge tube (Dayou, 7912011) , slowly add 7mL of the diluted blood sample to the top of the liquid level of the human peripheral blood lymphocyte separation solution (Dayou, 7912011) to keep the interface between the two liquid surfaces clear.
  • the blood sample should float above the separation solution and cannot break through the interface; at room temperature, Centrifuge the horizontal rotor at 800g for 30min, set the acceleration of acceleration to 1, and set the acceleration of deceleration to 0; after the centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation liquid, the top layer is the plasma/tissue homogenate layer, the plasma layer and the separation Between the liquid layers is a thin and dense tunica albuginea, the PBMC cell layer. Carefully aspirate the buffy coat layer into a new centrifuge tube; dilute the aspirated PBMC to a certain volume with PBS, and mix by inversion. Room temperature, horizontal rotor, centrifugation at 250g for 10min, discarding the supernatant, and washing twice; resuspend the cells with PBS for counting.
  • Antibody preparation prepare antibodies BsAb-71-N297A (5 ⁇ g/ml), 47-R2-4 (5 ⁇ g/ml), L1-R2-4-71 (5 ⁇ g/ml), 47-R2-4 ( 5 ⁇ g/ml) + L1-R2-4-71 (5 ⁇ g/ml).
  • PBMC stimulation Take an appropriate amount of PBMC and resuspend it with PBMC medium to a cell density of 1 ⁇ 10 6 cells/ml; add SEE (Toxin Technology, ET404) to the above cell suspension to make the SEE concentration 100ng/ml; take one piece In a 96-well cell culture plate, the PBMC cells added with SEE were evenly distributed into wells, 100 ⁇ l per well. Add the prepared antibody solution to the plate, add 100 ⁇ L to each well, and mix well. At this time, the final concentration of SEE is 50 ng/ml, and the final concentration of antibody is 2.5 ⁇ g/ml. There are a total of 100,000 PBMC cells in each well. Antibodies were co-cultured with cells in a carbon dioxide incubator at 37°C for 4 days.
  • IL-2 detection coat the prepared anti-IL-2 antibody on a 96-well plate ( Assay Plate), add 100 ⁇ l of anti-IL-2 antibody to each well, incubate overnight at 4°C; wash the plate twice with a microwell plate washer, and set 300 ⁇ l of washing solution each time. Gently pat dry on clean paper, add 200 ⁇ l of blocking solution to each well, and incubate at 37°C for 2 hours; add 75 ⁇ l of experimental diluent and 25 ⁇ l of PBMC supernatant incubated with antibodies for 4 days to each well, add The diluted IL-2 standard (Human IL-2 ELISA development kit (HRP), Mabtech) was incubated at 37° C.
  • HRP Human IL-2 ELISA development kit
  • the anti-PD-L1/CD47 bispecific antibody BsAb-71-N297A exemplified in the present invention can effectively activate T cells in vitro.
  • Example 16 In vivo anti-tumor effect of the anti-PD-L1/CD47 bispecific antibody of the present invention
  • MC38 cells expressing human CD47 (MC38-hCD47(Tg), Jiangsu JiCui Yaokang Biotechnology Co., Ltd.) were used to measure the anti-PD-L1/CD47 bispecific antibodies of the present invention in hSIRP ⁇ transgenic C57BL/6-hSIRP ⁇ mice tumor effect.
  • Human SIRP ⁇ transgenic mice female C57BL/6-hSIRP ⁇ mice (5-8 weeks old) (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).
  • Experimental cells collect mouse colon cancer cells MC38-hCD47(Tg) in logarithmic growth phase, remove the culture medium and wash twice with PBS, then inoculate; inoculation volume: 1 ⁇ 10 6 /100 ⁇ l/cell; inoculation location: right abdomen Regional location.
  • Group administration When the average tumor volume is about 50-100 mm 3 , the tumor-bearing mice are randomly divided into groups; the inclusion criteria: the CV value of the tumor volume is less than 30%; the day of inoculation is defined as D0 day, and on the day of grouping, according to the experiment Protocol design to start dosing. The doses and modes of administration are shown in Table 16. Following cell inoculation, tumor effects on the animals' normal behavior were routinely monitored weekly. Specific content: activity of experimental animals, food and water intake, weight gain or loss, eyes, coat and other abnormalities. Clinical symptoms observed during the trial were recorded in the raw data. After initiation of dosing, body weights were weighed twice a week.
  • TGI% (1-average relative tumor volume of treatment group/average relative tumor volume of vehicle group) ⁇ 100%.
  • the results of tumor inhibition rate are shown in Figure 15 and Table 17: on the 33rd day after inoculation, compared with the Isotype IgG1 group, the tumor inhibition rate of 5mg/kg L1-R2-4-71 single drug was 32.25%; 5mg/kg 47- The tumor inhibition rate of R2-4 single drug was 18.90%; the tumor inhibition rate of 5mg/kg L1-R2-4-71 combined with 5mg/kg47-R2-4 was 76.73%; 10mg/kg anti-PD-L1/CD47 dual The tumor inhibition rate of the specific antibody BsAb-71-N297A was 68.70%, which was significantly better than that of the single drug, and there was no statistical difference compared with the combined administration group (p>0.05). There was no significant difference in the body weight of mice in each group.
  • TGI Tumor Inhibition Rate

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Abstract

双特异抗体及其应用，该双特异抗体或抗原结合片段可以结合两个或更多个抗原，或同一抗原的两个或更多个表位，可以用于治疗各种疾病，比如炎性疾病、自身免疫性疾病、癌症或脊髓损伤，也可以用于相关疾病的诊断和预后。

Description

双特异抗体及其应用 技术领域
本发明属于生物医药领域，尤其涉及双特异抗体及其应用。
背景技术
癌症免疫疗法是近年来癌症治疗上的革命性突破，是生物科学领域的研究热潮，随着更多的抗体新药批准上市，已将癌症治疗的前景从传统疗法的补充要素转变为中心和标准的癌症治疗方案。在以免疫检查点(Checkpoint)为靶点，激活T细胞功能，从而提高获得性免疫系统能力来攻克癌症这一道路上，围绕PD-L1/PD-1途径开发抗体疗法，已成为本领域的热点。当机体发生炎症或是受到感染时，程序性死亡配体1(Programmed death-ligand 1，PD-L1)能够被诱导表达在造血、内皮或上皮细胞上，通过结合到表达于T细胞、B细胞和单核细胞的上的程序性死亡受体1(Programmed death 1，PD-1)来抑制免疫应答。PD-1胞质区含有两个基于酪氨酸的信号转导结构域ITIM(免疫受体酪氨酸抑制基序)和ITSM(免疫受体酪氨酸转换基序)。PD-L1在许多癌症中过表达，所述癌症包括多种多样的实体瘤以及血液瘤，诸如膀胱癌、乳腺癌、结肠癌、肺癌、黑色素瘤、卵巢癌、胃癌、甲状腺癌、原发性纵膈大B细胞淋巴瘤和典型霍奇金淋巴瘤。过表达在肿瘤细胞上的PD-L1，与T细胞上的PD-1结合，激活PD-1的ITIM结构域，导致T细胞功能障碍和衰竭，阻止细胞毒性T细胞有效地靶向肿瘤细胞，向免疫系统发出“don’t find me”信号，使肿瘤细胞获得适应性免疫耐受，从而促进肿瘤侵袭，造成不良的预后。
固有免疫系统是抵御感染和恶性细胞转化的第一道非特异性防线。在固有免疫系统中，单核细胞、巨噬细胞以及树突状细胞通过吞噬作用(phagocytosis)，起抗原提呈细胞(antigen presenting cells，APCs)的作用。而APCs通过吞噬作用吞没肿瘤细胞的能力是连接先天免疫和适应性免疫不可或缺的桥梁。靶向调节吞噬作用的检查点(Phagocytosis checkpoints，如CD47-SIRPα)，提供了一条开发癌症免疫疗法的新途径。CD47(integrin associated protein，IAP，CD47)在正常细胞表面广泛表达，通过与巨噬细胞表面的SIRPα(Signal regulatory protein α，SIRPα)结合，释放一种“don’t eatme”的信号，从而保护健康细胞不被巨噬细胞“吃掉”。癌细胞利用这一机制，在其表面过量表达CD47，使巨噬细胞把它们当作“正常细胞”，从而逃脱固有免疫的监 控，逃避巨噬细胞介导的吞噬攻击。已有研究证实，几乎所有的肿瘤细胞都高表达CD47，诸如甲状腺癌、卵巢癌、前列腺癌、子宫颈癌、膀胱癌、头颈癌、胃癌、急性髓细胞性白血病、B细胞和T细胞急性白血病、非霍奇金淋巴瘤等，且CD47过表达与临床预后差有关。
单抗免疫疗法为部分患者带来了持久的生存益处，实现了患者治愈的希望。随着对癌症和其他多种疾病的深入研究，认识到疾病的发生和发展由多种信号通路和机制参与，而单抗单一的刺激或抑制无法达到彻底杀伤肿瘤的效果，使得单药治疗响应率有限。双特异抗体(bispecific monoclonal antibody，BsAb)能够同时结合两个抗原或一个抗原的两个不同表位，阻断或激活不同的信号通路，更有效地介导免疫细胞对肿瘤细胞杀伤，因此可能会获得比单抗甚至联合用药更好的治疗效果。随着抗体药物研发技术的飞速发展，双特异抗体也迎来研发热潮，已有多个双特异抗体处于临床开发阶段，有3个双特异抗体已获批上市。2009年，欧盟批准了第一个治疗性双特异抗体——Catumaxomab(靶向CD3和EpCAM)，其结构为Triomab，用于治疗癌性腹水。与单抗相比，双特异抗体具有特异性高、靶向性强、用量少等优点，不仅在肿瘤的临床治疗中意义重大，在炎症性疾病治疗中也取得突破，具有非常广阔的应用前景。
根据已有知识可知，PD-L1与CD47蛋白同时过表达在肿瘤细胞上，通过PD-L1/PD1与CD47/SIRPα通路，逃避固有免疫系统监控，获得适应性免疫耐受。因此，同时阻抗PD-L1/PD1和CD47/SIRPα通路，激活固有免疫和适应性免疫系统，将可能发挥更好的抑制肿瘤效果。
发明内容
本发明提供了多价和多特异抗体或抗原结合片段及其应用。本发明提供了新的与PD-L1的结合更优且成药性好的抗PD-L1抗体。此外，本发明提供了新的不与人红细胞结合，不引起人红细胞凝集，具有良好的靶点特异性，且副作用较小的抗CD47抗体。此外，本发明采用了具有共同轻链的“杆入臼”(Knobs-into-holes)对称IgG样双特异抗体结构，实现不同重链以及轻链的正确装配，增加双特异抗体的产率，易于在体外的培养细胞中稳定表达，不需要复杂的生产工艺。此外，本发明采用的双特异抗体结构能够保持各抗原结合位点与相应的不同表位结合的亲和力，且在结合不同表位的时候不会互相干扰结合，具有好的成药性。进一步地，本发明的双特异抗体具有稳定的物理和生物学性质，这允许该抗体具有更好的生产性和可开发性。
在一些实施方案中，本发明提供了双特异抗体或抗原结合片段。本发明提供的抗体或抗原结合片段可以结合两个或更多个抗原，或同一抗原的两个或更多个表位，或 同一表位的两个或更多个拷贝。
在一些实施方案中，本发明提供了一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含特异性结合PD-L1的可变区a；其中，所述可变区a包含(a)-(f)中一种或多种氨基酸序列：
(a)VHa CDR1，其包含如SEQ ID NO:4-8中任一项所示的氨基酸序列，或与SEQ ID NO:4-8任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(b)VHa CDR2，其包含如SEQ ID NO:9-18中任一项所示的氨基酸序列，或与SEQ ID NO:9-14中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(c)VHa CDR3，其包含如SEQ ID NO:19或20所示的氨基酸序列，或与SEQ ID NO:19或20所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(d)VLa CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列，或与SEQ ID NO:36-40中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(e)VLa CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列，或与SEQ ID NO:41-44中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(f)VLa CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列，或与SEQ ID NO:45-48中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区a包含重链可变区a(VHa)，所述重链可变区a包含VHa CDR1、VHa CDR2、VHa CDR3；其中VHa CDR1包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19或20所示的氨基酸序列。
在一些实施方案中，所述可变区a包含轻链可变区a(VLa)，所述轻链可变区a包含VLa CDR1、VLa CDR2和VLa CDR3；其中VLa CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述可变区a包含VHa和VLa；所述VHa包含VHa CDR1、VHa CDR2、VHa CDR3；所述VLa包含VLa CDR1、VLa CDR2和VLa CDR3。其中，VHa CDR1包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19或20所示的氨基酸序列；VLa CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或 与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:9-14中任一项所示的氨基酸序列，或与SEQ ID NO:9-14中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa包含如SEQ ID NO:49-63中任一项所示的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区a包含VHa和VLa。所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，本发明提供的一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段还包含轻链恒定区a(CLa)，以及重链恒定区a(CHa)。
在一些实施方案中，所述CHa包含如SEQ ID NO:81、83-86中任一项所示的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述CLa包含如SEQ ID NO:82所示的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，本发明提供的一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含一条本文所述重链a、一条重链x以及一条轻链a和一条轻链x；所述重链a和所述轻链a配对形成PD-L1抗原结合位点，所述重链x和所述轻链x配对形成其它抗原结合位点。在一些实施方案中，所述重链a包含本文所述的VHa CDR1、VHa CDR2、VHa CDR3。在一些实施方案中，所述重链a包含本文所述的VHa。在一些实施方案中，所述重链a包含本文所述的VHa及CHa。在一些实施方案中，所述轻链a包含本文所述的VLa CDR1、VLa CDR2和VLa CDR3。在一些实施方案中，所述轻链a包含本文所述的VLa。在一些实施方案中，所述轻链a包含本文所述的VLa及CLa。在一些实施方案中，所述重链x与本文所述重链b相同。在一些实施方案中，所述轻链x与所述轻链a相同。
在一些实施方式中，所述其它抗原为CD47、LAG3、TGFβ、CTLA-4、4-1BB、PD1、TIGIT、KIR、c-Met、VISTA或BCMA。在一些实施方式中，所述其它抗原为CD47。
在一些实施方式中，所述重链x的CDR选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的重链CDR。在一些实施方式中，所述重链x的可变区选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的重链可变区。在一些实施方式中，所述轻链x的CDR选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的轻链CDR。在一些实施方式中，所述轻链x的可变区选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的轻链可变区。在一些实施方案中，所述轻链x与所述轻链a相同。
在一些实施方案中，所述重链a包含如SEQ ID NO:92或94所示的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与PD-L1的亲和力指数KD≤10nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与PD-L1的亲和力指数KD≤1nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与PD-L1的亲和力指数KD≤0.5nM。
另一方面，本发明提供了一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含特异性结合CD47的可变区b；其中，所述可变区b包含(g)-(l)中一种或多种氨基酸序列：
(g)VHb CDR1，其包含如SEQ ID NO:21-23中任一项所示的氨基酸序列，或与SEQ ID NO:21-23中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(h)VHb CDR2，其包含如SEQ ID NO:24-28中任一项所示的氨基酸序列，或与SEQ ID NO:24-28中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(i)VHb CDR3，其包含如SEQ ID NO:29-35中任一项所示的氨基酸序列，或与SEQ ID NO:29-35中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(j)VLb CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列，或与SEQ ID NO:36-40中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(k)VLb CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列，或与SEQ ID NO:41-44中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(l)VLb CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列，或与SEQ ID NO:45-48中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区b包含重链可变区b(VHb)，所述重链可变区b包含VHb CDR1、VHb CDR2、VHb CDR3；其中VHb CDR1包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29-35中任一项所示的氨基酸序列。
在一些实施方案中，所述可变区b包含轻链可变区b(VLb)，所述轻链可变区b包含VLb CDR1、VLb CDR2和VLb CDR3；其中VLb CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述可变区b包含VHb和VLb；所述VHb包含VHb CDR1、 VHb CDR2、VHb CDR3；所述VLb包含VLb CDR1、VLb CDR2和VLb CDR3。其中，VHb CDR1包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29-35中任一项所示的氨基酸序列；VLb CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列，或与SEQ ID NO:29所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列，或与SEQ ID NO:29所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHb包含如SEQ ID NO:64-74中任一项所示的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLb包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列相比具有一个或多个保守氨基 酸取代的氨基酸序列。
在一些实施方案中，所述VLb包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区b包含VHb和VLb。所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLb包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，本发明提供的一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段还包含轻链恒定区b(CLb)，以及重链恒定区b(CHb)。
在一些实施方案中，所述CHb包含如SEQ ID NO:81、83-86中任一项所示的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述CLb包含如SEQ ID NO:82所示的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，本发明提供的一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含一条本文所述重链b、一条重链y以及一条本文所述轻链b和一条轻链y；所述重链b和所述轻链b配对形成CD47抗原结合位点，所述重链y和所述轻链y配对形成其它抗原结合位点。在一些实施方案中，所述重链b包含本文所述的VHb CDR1、VHb CDR2、VHb CDR3。在一些实施方案中，所述重链b包含本文所述的VHb。在一些实施方案中，所述重链b包含本文所述的VHb及CHb。在一些实施方案中，所述轻链b包含本文所述的VLb CDR1、VLb CDR2和VLb CDR3。在一些实施方案中，所述轻链b包含本文所述的VLb。在一些实施方案中，所述轻链b包含本文所述的VLb及CLb。在一些实施方案中，所述轻链y与所述轻链b相同。
在一些实施方式中，所述其它抗原为PD-L1、PD-1、BCMA、MSLN、CD19、CD20或VEGF。在一些实施方式中，所述其它抗原为PD-L1。
在一些实施方式中，所述重链y的CDR选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的重链CDR。在一些实施方式中，所述重链y的可变区选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的重链可变区。在一些实施方式中，所述轻链y的CDR选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的轻链CDR。在一些实施方式中，所述轻链的可变区选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的轻链可变区。在一些实施方案中，所述重链y与本文所述重链a相同。在一些实施方案中，所述轻链y与所述轻链b相同。
在一些实施方案中，所述重链b包含如SEQ ID NO:93或95所示的氨基酸序列， 或与SEQ ID NO:93或95所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:93或95所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤30nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤10nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤5nM。
在一些实施方式中，本发明提供的双特异抗体或抗原结合片段不引起红细胞凝集。
另一方面，本发明提供一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含特异性结合PD-L1的可变区a，以及特异性结合CD47的可变区b。
其中，所述可变区a包含(a)-(f)中一种或多种氨基酸序列：
(a)VHa CDR1，其包含如SEQ ID NO:4-8中任一项所示的氨基酸序列，或与SEQ ID NO:4-8中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(b)VHa CDR2，其包含如SEQ ID NO:9-18中任一项所示的氨基酸序列，或与SEQ ID NO:9-18中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(c)VHa CDR3，其包含如SEQ ID NO:19或20所示的氨基酸序列，或与SEQ ID NO:19或20所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(d)VLa CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列，或与SEQ ID NO:36-40中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(e)VLa CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列，或与SEQ ID NO:41-44中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(f)VLa CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列，或与SEQ ID NO:45-48中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
所述可变区b包含(g)-(l)中一种或多种氨基酸序列：
(g)VHb CDR1，其包含如SEQ ID NO:21-23中任一项所示的氨基酸序列，或与SEQ ID NO:21-23中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(h)VHb CDR2，其包含如SEQ ID NO:24-28中任一项所示的氨基酸序列，或与SEQ ID NO:24-28中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(i)VHb CDR3，其包含如SEQ ID NO:29-35中任一项所示的氨基酸序列，或与SEQ ID NO:29-35中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(j)VLb CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列，或与SEQ ID NO:36-40中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(k)VLb CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列，或与SEQ ID NO:41-44中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
(l)VLb CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列，或与SEQ ID NO:45-48中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区a包含重链可变区a(VHa)，所述重链可变区a包含VHa CDR1、VHa CDR2、VHa CDR3；其中VHa CDR1包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19或20所示的氨基酸序列；所述可变区b包含重链可变区b(VHb)，所述重链可变区b包含VHb CDR1、VHb CDR2、VHb CDR3；其中VHb CDR1包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29-35中任一项所示的氨基酸序列。
在一些实施方案中，所述可变区a包含轻链可变区a(VLa)，所述轻链可变区a包含VLa CDR1、VLa CDR2和VLa CDR3；其中VLa CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列；所述可变区b包含轻链可变区b(VLb)，所述轻链可变区b包含VLb CDR1、VLb CDR2和VLb CDR3；其中VLb CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述可变区a包含VHa和VLa；所述可变区b包含VHb和VLb。所述VHa包含VHa CDR1、VHa CDR2、VHa CDR3；所述VLa包含VLa CDR1、VLa CDR2和VLa CDR3。其中，VHa CDR1包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19或20所示的氨基酸序列。所述VHb包含VHb CDR1、VHb CDR2、VHb CDR3；所述VLb包含VLb CDR1、VLb CDR2和VLb CDR3。其中，VHb CDR1包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29-35中任一项所示的氨基酸序列；VLb CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。VLa CDR1和VLb CDR1包含独立的选自如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLa CDR2和VLb CDR2包含独立的选自如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLa CDR3和VLb CDR3包含独立的选自如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:9-14中任一项所示的氨基酸序列，或与SEQ ID NO:9-14中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列，或与SEQ ID NO:29所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列，或与SEQ ID NO:29所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa CDR1或VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR2或VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR3或VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示的氨基酸序列相比具有一个或多个保守氨基酸取代 的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列，或与SEQ ID NO:29所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。所述VLa CDR1或VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR2或VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VLa CDR3或VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa CDR1与所述VLb CDR1包含相同的氨基酸序列。
在一些实施方案中，所述VLa CDR2与所述VLb CDR2包含相同的氨基酸序列。
在一些实施方案中，所述VLa CDR3与所述VLb CDR3包含相同的氨基酸序列。
在一些实施方案中，所述VLa CDR1与所述VLb CDR1包含相同的氨基酸序列，所述VLa CDR2与所述VLb CDR2包含相同的氨基酸序列，以及所述VLa CDR3与所述VLb CDR3包含相同的氨基酸序列。
在一些实施方案中，所述可变区a包含重链可变区VHa和轻链可变区VLa；其中，所述VHa包含如SEQ ID NO:49-63中任一项所示的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或所述VLa包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述可变区a包含重链可变区VHa和轻链可变区VLa；其中，所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或所述VLa包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述可变区b包含重链可变区VHb和轻链可变区VLb；其中，所述VHb包含如SEQ ID NO:64-74中任一项所示的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或所述VLb包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述可变区b包含重链可变区VHb和轻链可变区VLb；其中，所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或所述VLb包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述VHa包含如SEQ ID NO:49-63中任一项所示的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb包含如SEQ ID NO:64-74中任一项所示的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa和VLb包含独立的选自如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa和VLb包含独立的选自如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述可变区a包含VHa和VLa；所述可变区b包含VHb和VLb。所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。所述VLa或VLb包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VLa与所述VLb包含相同的氨基酸序列。
在一些实施方案中，本文所述抗体或抗原结合片段还包含轻链恒定区a(CLa)，以及重链恒定区a(CHa)，轻链恒定区b(CLb)，以及重链恒定区b(CHb)。
在一些实施方案中，所述CHa为IgG1亚型。在一些实施方案中，所述CHb为IgG1亚型。
在一些实施方案中，所述CHa和/或CHb包含如下一种或多种氨基酸突变的IgG1亚型：Y349C、S354C、T366W、T366S、L368A以及Y407V，其中氨基酸位置为Eu编号。
在一些实施方案中，所述CHa和/或CHb包含如下氨基酸突变的IgG1亚型：N297A，其中氨基酸位置为Eu编号。
在一些实施方案中，所述CHa和CHb中的一个重链恒定区包含如下一种或多种氨基酸突变：N297A、Y349C、T366S、L368A以及Y407V。
在一些实施方案中，所述CHa和CHb中的另一个重链恒定区包含如下一种或多种氨基酸突变：N297A、S354C以及T366W。
在一些实施方案中，所述CHa包含如SEQ ID NO:81、83-86中任一项所示的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列至少有90％同源性的 氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，所述CHb包含如SEQ ID NO:81、83-86中任一项所示的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:81、83-86中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述CHa包含如SEQ ID NO:81、83或85所示的氨基酸序列，或与SEQ ID NO:81、83或85所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:81、83或85所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述CHb包含如SEQ ID NO:81、84或86所示的氨基酸序列，或与SEQ ID NO:81、84或86所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:81、84或86所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述CHa包含如SEQ ID NO:83所示的氨基酸序列，所述CHb包含如SEQ ID NO:84所示的氨基酸序列。在一些实施方案中，所述CHa包含如SEQ ID NO:85所示的氨基酸序列，所述CHb包含如SEQ ID NO:86所示的氨基酸序列。在一些实施方案中，所述CHa包含如SEQ ID NO:84所示的氨基酸序列，所述CHb包含如SEQ ID NO:83所示的氨基酸序列。在一些实施方案中，所述CHa包含如SEQ ID NO:86所示的氨基酸序列，所述CHb包含如SEQ ID NO:85所示的氨基酸序列。
在一些实施方案中，所述CLa包含如SEQ ID NO:82所示的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，所述CLb包含如SEQ ID NO:82所示的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链a包含如SEQ ID NO:92或94所示的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述重链b包含如SEQ ID NO:93或95所示的氨基酸序列，或与SEQ ID NO:93或95所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:93或95所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，所述轻链a和轻链b均包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与PD-L1的亲和力指数KD≤10nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与PD-L1的亲和力指数KD≤1nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤30nM。在一些实施方式中，本发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤10nM。在一些实施方式中，本 发明提供的双特异抗体或抗原结合片段与CD47的亲和力指数KD≤5nM。
另一方面，本发明提供了一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含特异性结合PD-L1的可变区a，特异性结合CD47的可变区b，轻链恒定区，以及重链恒定区；所述重链恒定区为IgG1亚型，或包含如下一种或多种氨基酸突变的IgG1亚型：Y349C、S354C、T366W、T366S、L368A以及Y407V，其中氨基酸位置为Eu编号。
在一些实施方案中，所述重链恒定区还包含如下氨基酸突变的IgG1亚型，其中氨基酸位置为Eu编号：N297A。
在一些实施方案中，所述重链恒定区包含第一重链恒定区和第二重链恒定区，所述第一重链恒定区包含如下一种或多种氨基酸突变：
N297A、S354C以及T366W；和/或
所述第二重链恒定区包含如下一种或多种氨基酸突变：
N297A、Y349C、T366S、L368A以及Y407V。
在一些实施方案中，所述第一重链恒定区包含如SEQ ID NO:81、83或85所示的氨基酸序列，或与SEQ ID NO:81、83或85所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或所述第二重链恒定区包含如SEQ ID NO:81、84或86所示的氨基酸序列，或与SEQ ID NO:81、84或86所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述可变区a中的VHa与所述第一重链恒定区连接，所述可变区b中的VHb与所述第二重链恒定区连接；或，所述可变区a中的VHa与所述第二重链恒定区连接，所述可变区b中的VHb与所述第一重链恒定区连接。
在一些实施方案中，所述轻链恒定区包含如SEQ ID NO:82所示的氨基酸序列，或与SEQ ID NO:82所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段包含本文所述的可变区a，和本文所述的可变区b。
另一方面，本发明提供了一种双特异抗体或抗原结合片段，所述抗体或抗原结合片段包含一条重链a、一条重链b以及两条相同的轻链。
在一些实施方案中，所述轻链的可变区(VL)包含VL CDR1、VL CDR2和VL CDR3；其中VL CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VL CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VL CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
在一些实施方案中，所述VL CDR1包含如SEQ ID NO:36所示的氨基酸序列，或与SEQ ID NO:36所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VL CDR2包含如SEQ ID NO:41所示的氨基酸序列，或与SEQ ID NO:41所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述VL CDR3包含如SEQ ID NO:45所示的氨基酸序列，或与SEQ ID NO:45所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VL包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序 列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述VL包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述轻链包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列至少有90％同源性的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链a和一条所述轻链配对形成PD-L1抗原结合位点，所述重链b和另一条所述轻链配对形成CD47、LAG3、TGFβ、CTLA-4、4-1BB、PD-1、TIGIT、KIR、c-Met、VISTA或BCMA抗原结合位点。在一些实施方案中，所述重链a和一条所述轻链配对形成PD-L1、PD-1、BCMA、MSLN、CD19、CD20或VEGF抗原结合位点，所述重链b和另一条所述轻链配对形成CD47抗原结合位点。
在一些实施方案中，所述重链a和一条所述轻链配对形成PD-L1抗原结合位点，所述重链b和另一条所述轻链配对形成CD47抗原结合位点。
在一些实施方案中，所述重链a包含如SEQ ID NO:92或94所示的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列至少有90％同源性的氨基酸序列；所述重链b包含如SEQ ID NO:93或95所示的氨基酸序列，或与SEQ ID NO:93或95所示的氨基酸序列至少有90％同源性的氨基酸序列；所述轻链包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列至少有90％同源性的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段与PD-L1的亲和力指数KD≤1nM。
在一些实施方案中，所述抗体或抗原结合片段与CD47的亲和力指数KD≤10nM。
在一些实施方案中，所述抗体或抗原结合片段与CD47的亲和力指数KD≤30nM。
另一方面，本发明提供了一种多聚核苷酸，所述多聚核苷酸编码上述的抗体或抗原结合片段。在一些实施方案中，所述多聚核苷酸为分离的多聚核苷酸。
另一方面，本发明提供了一种表达载体，所述表达载体包含如上述的多聚核苷酸。在一些实施方案中，所述表达载体为分离的表达载体。
另一方面，本发明提供了一种宿主细胞，所述细胞包含上述的多聚核苷酸，或上述的表达载体。在一些实施方案中，所述宿主细胞为分离的宿主细胞。在一些实施方案中，所述细胞为CHO细胞、HEK细胞(如HEK293F细胞)、BHK细胞、Cos1细胞、Cos7细胞、CV1细胞或鼠L细胞。
另一方面，本发明提供了制备本文所述的抗体或抗原结合片段的方法，包含在培养基中培养上述宿主细胞以产生抗体或抗原结合片段。在一些实施方案中，所述方法还包括从所述宿主细胞或所述培养基回收所述抗体或抗原结合片段。
另一方面，本发明提供了一种组合物，所述组合物包含如上述的抗体或抗原结合片段、上述的多聚核苷酸，或上述的表达载体、或上述的细胞，以及药学上可接受的载体。
另一方面，本发明提供了上述的抗体或抗原结合片段、上述的多聚核苷酸、或上 述的表达载体、上述的细胞或上述的组合物在制备治疗疾病的药物中的应用。
在一些实施方案中，所述疾病是自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、癌症。在一些实施方案中，所述疾病是与PD-L1和CD47相关的病症。在一些实施方案中，所述疾病是与PD-L1和CD47相关的病症包括但不限于各种血液病和实体瘤，如急性骨髓性白血病(AML)、慢性骨髓性白血病、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤和其他实体瘤。在一个实施方案中，所述癌症是胃肠道癌，如结肠癌。
另一方面，本发明提供了一种治疗疾病的方法，包括向需要治疗的患者施用有效剂量的上述的双特异抗体或抗原结合片段、上述的多聚核苷酸、或上述的表达载体、上述的细胞或上述的组合物。
在一些实施方案中，所述疾病是自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、癌症。在一些实施方案中，所述疾病是与PD-L1和CD47相关的病症。在一些实施方案中，所述疾病是与PD-L1和CD47相关的病症包括但不限于各种血液病和实体瘤，如急性骨髓性白血病(AML)、慢性骨髓性白血病、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤和其他实体瘤。在一个实施方案中，所述癌症是胃肠道癌，如结肠癌。
在一些实施方案中，本发明涉及试剂盒或制品，其包含上述的双特异抗体或抗原结合片段、上述的多聚核苷酸或上述的表达载体。
本发明提供了双特异抗体或抗原结合片段及其应用，本发明的双特异抗体或抗原结合片段可以结合两个或更多个抗原，或同一抗原的两个或更多个表位。本发明抗体或抗原结合片段可以用于治疗或预防各种疾病，比如自身免疫病、急性和慢性炎性疾病、感染性疾病、癌症，也可以用于相关疾病的诊断和预后。
附图说明
图1示例本发明双特异抗体的结构。
图2为组图，图2A和图2B分别显示了SEC检测的两种抗PD-L1/CD47双特异抗体BsAb-46和BsAb-71的纯度。
图3抗PD-L1 scFv与PD-L1-His-Biotin抗原蛋白的结合力，其中克隆编号表示表达相应的抗PD-L1 scFv的阳性酵母克隆。
图4显示了基于MOA法检测的以R2-4为轻链，与不同抗PD-L1的VH组成的抗PD-L1抗体，以及作为阴性对照的IgG1抗体(IgG-Isotype)对PD-1/PD-L1信号转导通路的影响。
图5显示了基于MOA法检测的不同轻链的抗PD-L1抗体以及作为阴性对照的IgG1抗体(IgG-Isotype)对PD-1/PD-L1信号转导通路的影响。
图6显示了不同轻链的抗CD47抗体、阳性对照抗体Hu5F9-G4和IgG1对照抗体(IgG-Isotype)对红细胞凝集的影响；其中Hu5F9代表Hu5F9-G4。外观显示红细胞团流成一条线的为不引起血凝反应，轻微凝聚的为一个点，凝集明显的为整个孔模糊一团。
图7显示了抗CD47抗体以及作为阳性对照抗体Hu5F9-G4促进巨噬细胞吞噬肿瘤细胞的能力；其中Hu5F9代表Hu5F9-G4。
图8为组图，分别显示了抗PD-L1/CD47双特异抗体与PDL1和CD47的结合能力。图8A先结合PD-L1-His，后结合CD47-His；图8B先结合CD47-His，后结合PD-L1-His；图8C显示了抗PD-L1/CD47双特异抗体BsAb-71-N297A的结合能力，上部分图为先结合PD-L1-His，后结合CD47-His，下部分图为先结合CD47-His，后结合PD-L1-His；图中hPDL1-His代表PD-L1-His，hCD47-His代表CD47-His。
图9为组图，图9A和图9C显示了通过FACS检测的示例的抗PD-L1/CD47双特异抗体、作为亲本的抗PD-L1抗体，以及作为阴性对照的抗CD47抗体47-R2-4与过量表达PD-L1的CHO细胞的结合。图9B和图9D显示了通过FACS检测的示例的抗PD-L1/CD47双特异抗体、作为亲本的抗CD47抗体47-R2-4、以及作为阴性对照的抗PD-L1抗体L1-R2-4-71与过量表达CD47的CHO细胞的结合。图中横轴表示抗体浓度、纵轴表示平均荧光强度(MFI)。
图10为组图，显示了基于MOA法检测的本发明示例的抗PD-L1/CD47双特异抗体，作为亲本的抗PD-L1抗体L1-R2-4-71以及作为阴性对照的IgG1抗体(IgG-Isotype)对PD-1/PD-L1信号转导通路的影响。图10A为抗PD-L1/CD47双特异抗体BsAb-36、BsAb-46、BsAb-47、BsAb-71的结果，图10B为抗PD-L1/CD47双特异抗体BsAb-71-N297A的结果。
图11显示了示例的抗PD-L1/CD47双特异抗体、抗CD47的阳性抗体Hu5F9-G4、抗CD47的亲本抗体47-R2-4和IgG1对照抗体(IgG-Isotype)对红细胞凝集的影响；其中Hu5F9代表Hu5F9-G4。
图12显示了示例的抗PD-L1/CD47双特异抗体、抗CD47的亲本抗体47-R2-4和IgG1对照抗体(IgG-Isotype)促进巨噬细胞吞噬肿瘤细胞的能力。
图13为组图，图13A显示了通过FACS检测的示例的抗PD-L1/CD47双特异抗体、抗CD47的阳性抗体Hu5F9-G4、作为亲本的抗CD47抗体47-R2-4、以及作为阴性对照的抗PD-L1抗体L1-R2-4-71与人红细胞细胞的结合。图13B显示了通过FACS 检测的示例的抗PD-L1/CD47双特异抗体、抗CD47的阳性抗体Hu5F9-G4、作为亲本的抗CD47抗体47-R2-4、以及作为阴性对照的抗PD-L1抗体L1-R2-4-71与人T淋巴细胞白血病Jurkat细胞的结合。图中横轴表示抗体浓度、纵轴表示平均荧光强度(MFI)。
图14抗PD-L1/CD47双特异抗体对外源刺激下的PBMC释放IL2细胞因子的影响。
图15显示了，与IgG1对照抗体相比，抗PD-L1抗体L1-R2-4-71、抗CD47抗体47-R2-4、抗PD-L1抗体L1-R2-4-71与抗CD47抗体47-R2-4联合用药、抗PD-L1/CD47双特异抗体BsAb-71-N297A在MC38-hCD47(Tg)/C57BL/6-hSIRPα小鼠模型中的肿瘤抑制活性。
具体实施方式
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。除非本文中另外说明，否则恒定区中的氨基酸残基的编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
“约”或“大约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到的“约”或“大约”指所描述的数值以及其±10％、±5％、±1％或±0.1％的范围。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体组成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组 成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：Ala，一字母代码：A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、 约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
“至少90％同一性”为约90％同一性、约91％同一性、约92％同一性、约93％同一性、约95％同一性、约96％同一性、约97％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性或序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对和同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
多聚核苷酸是由四个核苷酸碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(VL CDR1-3)和重链的CDR区(VH CDR1-3)。本发明所述抗体或抗原结合片段为双特异抗体，包含特异性结合抗原a、抗原b的抗体片段。在一些实施方案中，第一多肽链包含结构VHa-CHa，第二多肽链包含结构VLa-CLa，第三多肽链包含结构VHb-CHb，第四多肽链包含结构VLb-CLb。在一些实施方案中，第二多肽链与第四多肽链的氨基酸序列相同。
术语“抗体片段”或“抗原结合片段”指抗体的一部分，本发明抗体片段的组成形式可类似于单特异性抗体片段中的F(ab') 2、F(ab) 2、Fab'、Fab、Fv、scFv等。不管其结构如何，抗体片段与被完整抗体识别的同一抗原结合。术语“抗体片段”包括适体、镜像异构体和双价抗体。术语“抗原结合片段”还包括通过与特定抗原结合形成复合物起抗体作用的任何合成或基因工程蛋白质。
“单链可变片段”或“scFv”是指免疫球蛋白的重链(VH)和轻链(VL)的可变区的融合蛋白。在一些方面，这些区域与10个至约25个氨基酸的短接头肽连接。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。ScFv分子通常是本领域中已知的，例如在美国专利5,892,019中有相关描述。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
本发明公开的抗体、抗原结合片段或衍生物包括但不限于多克隆、单克隆、多特 异性、全人源、人源化、灵长类化、嵌合抗体、单链抗体、表位结合片段(例如类Fab、类Fab'和类F(ab') 2)、类单链Fvs(scFv)。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为V λ。
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链(VL)和重链(VH)部分的可变区决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域分别包含重链和轻链的羧基端。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.,etal.,U.S.Department of Health and Human Services,Sequences of Proteins of Immunological Interest,(1983)和Chothia and Lesk,J.Mol.Biol.,196:901-917(1987))。
同一抗体的可变区的CDR的边界，根据不同的指派系统，可能有所差异。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体：其可变区序列包含本发明的CDR序列，但是由于应用了不同的方案而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在 “Sequence of Proteins of Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。
本发明公开的抗体可以来源于任何动物，包括鸟类和哺乳动物。较佳地，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。
重链恒定区包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包括源自IgG1分子的CH1结构域和源自IgG3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG1分子和部分源自IgG3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG1分子和部分源自IgG4分子的嵌合铰链区。
“轻链恒定区”包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。
“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，“CH1结构域”包括免疫球蛋白重链的第一个恒定区。CH2结构域不与其它结构域紧密配对，而是在完整的天然IgG分子的两个CH2结构域之间插入两个N-连接的分支碳水化合物链。CH3结构域从CH2结构域开始延伸到IgG分子的C-末端，大约包含108个残基。“铰链区”包括连接CH1结构域和CH2结构域的部分重链区域。所述铰链区包含约25个残基并且是有韧性的，从而使得两个N端抗原结合区能够独立移动。铰链区可以被细分为三个不同的结构域：上、中和下铰链结构域(Rouxetal.,J.Immunol 161:4083(1998))。
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接。
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。
“特异性结合”通常是指抗体或抗原结合片段与特定抗原通过其抗原结合结构域与表位互补性结合形成相对稳定的复合物。“特异性”可以用抗体或抗原结合片段与特定抗原或表位结合的相对亲和力表达。例如，如果抗体“A”比抗体“B”与同一抗原的相对亲和力大，可以认为抗体“A”比抗体“B”对该抗原具有更高的特异性。特异性结合可以用平衡解离常数(KD)来描述，较小的KD意味着较紧密的结合。确定两个分子是否 特异性结合的方法是本领域内众所周知的，并包括例如平衡透析、表面等离子共振、生物膜层光学干涉测量法等。“特异性结合”抗原a的抗体包括与抗原a平衡解离常数KD小于或等于约100nM、小于或等于约10nM、小于或等于约5nM、小于或等于约1nM或小于或等于约0.5nM的抗体。
“EC 50”即半最大效应浓度(concentration for 50％of maximal effect,EC 50)是指能引起50％最大效应的浓度。
“双特异”抗体指具有两个抗原结合位点的抗体，所述两个抗原结合位点可以是相同抗原的不同表位，也可以是不同抗原的不同表位。
术语“共同轻链”指的是该轻链能够同时与不同的重链组装成具有相应功能的完全抗体；该轻链可以用于表达双特异抗体中，也可以用于表达含有两种抗体的混合物中。
术语“效应子功能”指归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括：C1q结合和补体依赖的细胞毒性(CDC)、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、Fc受体结合作用、免疫复合物介导的抗原呈递细胞摄取抗原、下调细胞表面受体(例如B细胞受体)和B细胞活化。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。
“患者”指需要诊断、预防、预后或治疗的任何哺乳动物，包括人类、狗、猫、兔子、鼠、马、牛等。
双特异抗体
双特异抗体与单克隆抗体相比具备更强特异性、靶向肿瘤细胞和降低脱靶毒性等显著优势。双特异抗体与单克隆抗体相比增加了一个特异性抗原结合位点，在治疗方面表现出了以下优势：(1)两个抗原结合位点分别可以结合肿瘤细胞和免疫细胞，将T免疫细胞聚集到肿瘤细胞周围，增强对肿瘤的杀伤力；(2)可以同时阻断两种不同介质通路而发挥独特的或重叠的功能，介导多种免疫信号通路从而增强细胞杀伤毒性；(3)两种不同的细胞表面抗原结合后，相对而言可能潜在地增加结合特异性，降低脱 靶等副作用。因此，双特异抗体在肿瘤免疫治疗和炎症治疗中展现了广阔的应用前景。
由于在肿瘤免疫中，靶向多个靶点的治疗方案能够相互协同，对于防止肿瘤的免疫逃逸是有利的，目前靶向肿瘤免疫中的不同靶点的抗体的共施用也进入了临床试验中。但是，共施用需要注射两个独立的抗体产品或需要单次注射两种不同抗体的联合制剂。尽管两次注射允许给药量和时程的灵活性，但是它造成了患者不便依从和疼痛。另外，尽管联合制剂可能提供在给药量方面的某种灵活性，但通常难以找到在溶液中允许两种抗体的化学和物理稳定性的配制条件，原因在于两种抗体的分子特征不同。何况共施用和联合制剂两种不同抗体的疗法可能增加患者和/或付款人的额外花费，因此，需要治疗肿瘤的备选免疫疗法，并且优选地这类备选免疫疗法涉及双特异抗体。
1)双特异抗体中与PD-L1结合的部分
在一些实施方案中，本发明的双特异抗体或抗原结合片段可特异性结合PD-L1。在一些实施方案中，本发明的双特异抗体或抗原结合片段可特异性结合哺乳动物PD-L1。在一些实施方案中，PD-L1为人PD-L1。在一些实施方案中，抗体分子与PD-L1的一个或多个胞外结构域结合。
在一些实施方案中，抗体结合亲和力是使用表面等离子共振技术(例如Biacore亲和测量)进行测定的。
在一些实施方案中，本发明的双特异抗体或抗原结合片段具有以下一种或多种性质：
(a)本发明的双特异抗体或抗原结合片段以高亲和力结合PD-L1(如人PD-L1)，例如，以以下平衡解离常数(KD)与PD-L1结合，所述KD小于约12nM，如小于或等于约10nM，小于或等于约5nM，小于或等于约4nM、3nM、2nM或1nM。在一些实施方案中，所述KD小于或等于约0.9nM、或0.5nM。
(b)本发明的双特异抗体或抗原结合片段结合表达人PD-L1的细胞，在一些实施方案中，例如，其EC50小于或等于大约2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、或0.5nM。在一些实施方案中，所述结合用流式细胞术(例如FACS)测定。在一些实施方案中，表达人PD-L1的细胞为表达人PD-L1的CHO细胞。
(c)在一些实施方案中，本发明的双特异抗体或抗原结合片段阻断PD-L1/PD-1的相关活性，例如其EC50小于或等于大约6μg/ml、5μg/ml、4μg/ml、3μg/ml、2μg/ml、或1μg/ml。在一些实施方案中，PD-L1的相关活性是PD-L1与PD-1的结合。在一些实施方案中，本发明的抗体或其片段在MOA测定中以小于或等于大约小于或等于大约6μg/ml、5μg/ml、4μg/ml、3μg/ml、2μg/ml、或1μg/ml的EC50，或以大约0.1-3μg/ml、0.1-0.4μg/ml或0.1-0.3μg/ml的EC50抑制PD-L1与PD-1的结合。在一些实施方案中，细胞为过表达人PD-L1的CHO细胞。
(d)本发明的双特异抗体或抗原结合片段能够诱发抗体依赖性细胞介导的细胞毒性 (ADCC)。
(e)本发明的双特异抗体或抗原结合片段抑制PD-L1的一种或多种活性，例如，导致以下一者或多者：肿瘤浸润型淋巴细胞增加、T细胞受体介导的增殖增加、或癌细胞的免疫逃避减少。在一些实施方案中，双特异抗体或抗原结合片段能够抑制肿瘤的增殖，肿瘤是肿瘤免疫逃逸。在一些实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。
在一些实施方案中，本发明的双特异抗体或抗原结合片段包含特异性结合PD-L1的重链可变区a(VHa)，和轻链可变区a(VLa)。其中VHa包含3个CDR(VHa CDR1、VHa CDR2、VHa CDR3)，VLa包含3个CDR(VLa CDR1、VLa CDR2、VLa CDR3)。在一些实施方式中，VHa的CDR选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的重链CDR。在一些实施方式中，VHa选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的重链可变区。在一些实施方式中，VLa的CDR选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的轻链CDR。在一些实施方式中，VLa选自Atezolizumab、Avelumab、Durvalumab、Envafolimab或Cosibelimab中的轻链可变区。
在一些实施方式中，VHa CDR1包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19或20所示的氨基酸序列。在一些实施方式中，VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:9-14中任一项所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列。在一些实施方式中，VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列。
在一些实施方式中，VLa CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。在一些实施方式中，VLa CDR1包含如SEQ ID NO:36所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45所示的氨基酸序列。
在一些实施方式中，VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列；VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列；VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列；VLa CDR1包含如SEQ ID NO:36所示的氨基酸序列；VLa CDR2包含如SEQ ID NO:41所示的氨基酸序列；VLa CDR3包含如SEQ ID NO:45所示的氨基酸序列。
在一些实施方式中，VHa包含如SEQ ID NO:49-63中任一项所示的氨基酸序列；在一些实施方式中，VLa包含如SEQ ID NO:75-80中任一项所示的氨基酸序列。
在一些实施方式中，本发明的双特异抗体或抗原结合片段包括由氨基酸序列如 SEQ ID NO:50所示的VHa和氨基酸序列如SEQ ID NO:75所示的VLa组成的可变区a。
在一些实施方式中，本发明的双特异抗体或抗原结合片段包括由氨基酸序列如SEQ ID NO:51所示的VHa和氨基酸序列如SEQ ID NO:75所示的VLa组成的可变区a。
在一些实施方式中，本发明的双特异抗体或抗原结合片段包括由氨基酸序列如SEQ ID NO:52所示的VHa和氨基酸序列如SEQ ID NO:75所示的VLa组成的可变区a。
在一些实施方式中，本发明的双特异抗体或抗原结合片段包括由氨基酸序列如SEQ ID NO:54所示的VHa和氨基酸序列如SEQ ID NO:75所示的VLa组成的可变区a。
2)双特异抗体中与CD47结合的部分
在一些实施方案中，本发明的双特异抗体或抗原结合片段可特异性结合CD47。在一些实施方案中，本发明的双特异抗体或抗原结合片段可特异性结合哺乳动物CD47。在一些实施方案中，CD47为人CD47。在一些实施方案中，抗体分子与CD47的一个或多个胞外结构域结合。
在一些实施方案中，本发明的双特异抗体或抗原结合片段具有以下一种或多种性质：
(a)本发明的双特异抗体或抗原结合片段以高亲和力结合CD47(例如人CD47)，例如，以以下平衡解离常数(KD)与CD47结合，所述KD小于约700nM，如小于或等于约30nM，小于或等于约10nM，小于或等于约5nM。
(b)本发明的双特异抗体或抗原结合片段不会导致显著的细胞凝集，例如本发明的双特异抗体或抗原结合片段不会导致显著的血红细胞血凝反应。在一些实施方案中，如果与抗CD47阳性对照抗体Hu5F9-G4存在时的凝集水平相比，本发明的双特异抗体或抗原结合片段存在时的凝集水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，则说明本发明的双特异抗体或抗原结合片段未导致显著的凝集水平。在抗体浓度介于400pM和800nM之间时，本发明的双特异抗体或抗原结合片段不会导致显著的细胞凝集。
(c)本发明的双特异抗体或抗原结合片段不会与人红细胞结合。在一个实施方案中，如果与抗CD47阳性对照抗体Hu5F9-G4存在时的人红细胞结合水平相比，本发明的双特异抗体或抗原结合片段存在时的人红细胞结合水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，则说明本发明的双特异抗体或抗原结合片段未明显 地与人红细胞结合。优选地，在抗体浓度介于200pM和100nM之间时，本发明的双特异抗体或抗原结合片段不会明显地与人红细胞结合。
(d)在一些实施方案中，本发明的双特异抗体或抗原结合片段是阻断性抗体，阻断CD47与SIRPα的结合。在一些实施方案中，本发明的CD47抗体存在时巨噬细胞吞噬肿瘤细胞的能力升高了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％。
(e)本发明的双特异抗体或抗原结合片段能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)。
(f)本发明的双特异抗体或抗原结合片段抑制CD47的一种或多种活性，例如，导致肿瘤浸润型巨噬细胞的吞噬能力增加和/或癌细胞的免疫逃避减少。在一些实施方案中，双特异抗体或抗原结合片段能够抑制肿瘤的增殖，肿瘤是肿瘤免疫逃逸。在一些实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。
在一些实施方案中，本发明的双特异抗体或抗原结合片段包含特异性结合CD47的重链可变区b(VHb)，和轻链可变区b(VLb)。其中VHb包含3个CDR(VHb CDR1、VHb CDR2、VHb CDR3)，VLb包含3个CDR(VLb CDR1、VLb CDR2、VLb CDR3)。在一些实施方式中，VHb的CDR选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的重链CDR。在一些实施方式中，VHb选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的重链可变区。在一些实施方式中，VLb的CDR选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的轻链CDR。在一些实施方式中，VLb选自Magrolimab、AO-176(Arch Oncology)、TJC4(天境生物)、AK117(康方生物)或IBI188(信达生物)等中的轻链可变区。
在一些实施方式中，VHb CDR1包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29-35中任一项所示的氨基酸序列。在一些实施方式中，VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列。
在一些实施方式中，VLb CDR1包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。在一些实施方式中，VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列。
在一些实施方式中，VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列；VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列；VHb CDR3包含如SEQ ID NO:29 所示的氨基酸序列；VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列；VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列；VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列。
在一些实施方式中，VHb包含如SEQ ID NO:64-74中任一项所示的氨基酸序列；在一些实施方式中，VLb包含如SEQ ID NO:75-80中任一项所示的氨基酸序列。
在一些实施方式中，本发明的双特异抗体或抗原结合片段包括由氨基酸序列如SEQ ID NO:64所示的VHb和氨基酸序列如SEQ ID NO:75所示的VLb组成的可变区b。
3)抗PD-L1/CD47双特异抗体
固有免疫系统是抵御感染和恶性细胞转化的第一道非特异性防线。在固有免疫系统中，单核细胞、巨噬细胞以及树突状细胞通过吞噬作用，起抗原提呈细胞(APCs)的作用。而APCs通过吞噬作用吞没肿瘤细胞的能力是连接先天免疫和适应性免疫不可或缺的桥梁。由于肿瘤细胞被APCs的摄取减少，间接导致了T细胞活化下降，参见，例如，Avice MN等人，Role of CD47 in the induction of human naive T cell anergy，Journal of Immunology，167(5):2459-2468(2001)。已有研究证实，PD-L1与CD47蛋白的表达，都受到转录因子MYC的调控，同时过表达在肿瘤细胞上，参见，例如，Stephanie C等，MYC regulates the antitumor immune response through CD47 and PD-L1，Science，352(6282):227-31(2016)。肿瘤细胞通过PD-L1/PD1与CD47/SIRPα通路，逃避固有免疫系统监控，获得适应性免疫耐受。
在一些实施方案中，本发明提供抗PD-L1/CD47双特异抗体或抗原结合片段，其可特异性结合PD-L1和CD47。在一些实施方案中，本发明的抗体或其片段结合哺乳动物PD-L1和CD47，如人PD-L1和CD47。例如，抗体分子与PD-L1和CD47上的表位(例如，线性或构象表位)特异性结合。在一些实施方案中，抗体分子与PD-L1和CD47的一个或多个胞外结构域结合。
在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体或抗原结合片段具有以下一种或多种性质：
(a)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体能够同时与PD-L1和CD47结合，且维持了亲本抗体的亲和力常数，由此，能够阻断PD1/PD-L1信号传导通路和阻断SIRPα/CD47信号传导通路；
(b)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体能够以超高的亲和力(例如，KD＝0.174nM)与PD-L1结合，以高亲和力(例如，KD＝3.78nM)或中等亲和力(例如，KD＝27.8nM)与CD47结合；通过与肿瘤细胞上PD-L1的特异性结合促进了本发明的抗PD-L1/CD47双特异抗体对肿瘤细胞的选择性结合，避免了与许多正常组织中表达的CD47结合，减少副作用；通过当与CD47的亲和力远低 于与PD-L1的亲和力，可显著扩大本发明的抗PD-L1/CD47双特异抗体的有效剂量范围；
(c)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体，设计了能够避免非相关重链和轻链配对的共同轻链；
(d)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体，设计了能够稳定四链抗体结构和有利于各条链之间的正确偶合或配对的氨基酸残基。在一些实施方案中，本发明的双特异抗体各自的Fc结构域中分别包含Y349C和S354C或者分别包含S354C和Y349C；本发明的双特异抗体各自的Fc结构域中分别包含凸起(“杆(knob)”)或空穴(“臼(hole)”)，并且第一多肽链Fc结构域中的所述凸起或空穴可分别置于第三多肽链Fc结构域中的所述空穴或凸起中，由此所述第一多肽链和第三多肽链彼此形成“杆入臼(knob-in-hole)”的稳定缔合；其中，第一多肽链包含结构VHa-CHa，可特异性识别并结合PD-L1；第三多肽链包含结构VHb-CHb，可特异性识别并结合CD47。
(e)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体，具有易于在体外的培养细胞中稳定表达，热稳定性好，抗体产率和纯度高，不需要复杂的生产工艺等优点；
(f)本发明的抗PD-L1/CD47双特异抗体或其片段结合表达人PD-L1的细胞，在一些实施方案中，例如，以小于或等于大约2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM的EC50结合表达人PD-L1的细胞。在一些实施方案中，所述结合用流式细胞术(例如FACS)测定。在一些实施方案中，表达人PD-L1的细胞为表达人PD-L1的CHO-S细胞。
(g)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体或其片段阻断PD-L1/PD-1的相关活性，例如以小于或等于大约0.3μg/ml的EC50，或以大约0.1-3μg/ml的EC50阻断PD-L1/PD-1的相关活性。在一些实施方案中，PD-L1/PD-1的相关活性，是指PD-L1与PD-1结合后，激活PD1胞内区的抑制信号。在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体或其片段在MOA测定中以小于或等于大约小于或等于大约0.3μg/ml或以大约0.1-3μg/ml的EC50抑制PD-L1与PD-1的结合。在一些实施方案中，细胞为过表达人PD-L1的CHO细胞。
(h)本发明的抗PD-L1/CD47双特异抗体不会导致显著的细胞凝集，例如本发明的抗PD-L1/CD47双特异抗体不会导致显著的血红细胞血凝反应。在一些实施方案中，如果与抗CD47阳性对照抗体Hu5F9-G4存在时的凝集水平相比，本发明的抗PD-L1/CD47双特异存在时的凝集水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，则说明本发明的抗PD-L1/CD47双特异抗体未导致显著的凝集水平。优选地，在抗体浓度介于400pM和800nM之间时，本发明的抗PD-L1/CD47双特异 抗体不会导致显著的细胞凝集。
(i)本发明的抗PD-L1/CD47双特异抗体不会与人红细胞结合。在一个实施方案中，如果与抗CD47阳性对照抗体Hu5F9-G4存在时的人红细胞结合水平相比，本发明的抗PD-L1/CD47双特异抗体存在时的人红细胞结合水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，则说明本发明的抗PD-L1/CD47双特异抗体未明显地与人红细胞结合。优选地，在抗体浓度介于200pM和100nM之间时，本发明的抗PD-L1/CD47双特异抗体不会明显地与人红细胞结合。
(j)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体是阻断性抗体，阻断CD47与SIRPα的结合。在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体存在时巨噬细胞吞噬肿瘤细胞的能力升高了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％。
(k)在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体或其片段能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)。在一个实施方案中，本发明的抗PD-L1/CD47双特异抗体或其片段具有减少的诱发抗体依赖性细胞介导的细胞毒性(ADCC)的能力。在一个实施方案中，本发明的抗PD-L1/CD47双特异抗体或其片段不能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)。
(l)本发明的抗PD-L1/CD47双特异抗体或其片段协同抑制PD-L1和CD47的一种或多种活性。在一些实施方案中，抗PD-L1/CD47双特异抗体能够抑制肿瘤的增殖，肿瘤是肿瘤免疫逃逸。在一些实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。
在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体具有四条肽链，分别为两条重链和两条轻链。一条重链包含VHa，另一条重链包含VHb，一条轻链包含VLa，另一条轻链包含VLb；其中VHa、VLa构成的可变区a特异性结合PD-L1，VHb、VLb构成的可变区b特异性结合CD47。在一些实施方案中，两条轻链为相同的轻链。
在一些实施方案中，本发明的双特异抗体使用了“杆入臼”(Knobs-into-Holes)技术(参见，例如，John B.B.Ridgway等人，‘Knobs-into-holes’engineering of antibody CH3domains for heavy chain heterodimerization,Protein Engineering,9(7):p.617-21(1996)；Shane Atwell等人，Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library，J.Mol.Biol,270:p.26-35(1997)；Paul Carter,Bispecific human IgG by design,Journal of Immunological Methods,248,7-15(2001)；专利US8216805B2)。该技术可在本发明双特异抗体的不同链之间改造界面，以促进本发明双特异抗体的各条链正确缔合。通常，该技术涉及在一条链的界面引入“凸起”(“杆(knobs)”)，在欲与之配对的另一条链的界面引入相应的“空穴”(“臼(holes)”)，使得凸起可置于空穴中。可通过将来自一条链的重链恒定结构域的CH3结构域的界面的氨基酸 侧链替换为较大的侧链(如氨基酸置换T366W(Eu编号))来构建凸起。通过将大氨基酸侧链替换为较小的侧链(例如氨基酸置换T366S、L368A和Y407V(Eu编号))，在欲配对的另一条链的重链恒定结构域的CH3结构域的界面构建与凸起相同或相似大小的补偿性空穴。在一些实施方案中，一条重链Fc结构域中包含Y349C,T366S,L368A和Y407V，另一条重链Fc结构域中包含S354C和T366W，形成“杆入臼(knob-in-hole)”的稳定缔合。
在一些实施方案中，本发明双特异抗体的Fc区包含对Fc受体的结合亲和力的修饰。在一些实施方案中，所述Fc受体是Fcγ受体，特别地是人Fcγ受体。在一个实施方案中，所述Fc受体是活化性Fc受体。在一些实施方案中，所述修饰减少本发明双特异抗体的效应子功能。在一些实施方案中，所述效应子功能是抗体依赖的细胞介导的细胞毒性(ADCC)。在一些实施方案中，所述修饰在所述免疫球蛋白分子Fc区内，特别地在其CH2区内。在一些实施方案中，所述免疫球蛋白分子包含在免疫球蛋白重链第297位置(Eu编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是N297A(参见，例如，J.Lund等，Oligosaccharide-protein interactions in IgG can modulate recognition by Fc gamma receptors,FASEB.J.9,115-119(1995))。
在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体的重链还包含信号肽序列，例如MEFGLSWVFLVAILKGVQC(SEQ ID NO:90)。在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体的轻链还包含信号肽序列，例如MDMRVLAQLLGLLLLCFPGARC(SEQ ID NO:91)。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49-63中任一项所示的VHa，如SEQ ID NO:64-74中任一项所示的VHb，以及如SEQ ID NO:75-80中任一项所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:49所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa， 如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:50所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:51所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:52所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:53所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:75所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:76所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:77所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:78所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:79所示的VLa和VLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:54所示的VHa，如SEQ ID NO:64所示的VHb，以及如SEQ ID NO:80所示的VLa和VLb。
在一些实施方案中，重链恒定区为IgG1亚型(SEQ ID NO:81)。在一些实施方案中，重链恒定区包含如下一种或多种氨基酸突变：N297A、Y349C、S354C、T366W、T366S、L368A以及Y407V。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:83所示的CHa和如SEQ ID NO:84所示的CHb；以及如SEQ ID NO:82所示的CLa和CLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:84所示的CHa和如SEQ ID NO:83所示的CHb；以及如SEQ ID NO:82所示的CLa和CLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:85所示的CHa和如SEQ ID NO:86所示的CHb；以及如SEQ ID NO:82所示的CLa和CLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含如SEQ ID NO:86所示的CHa和如SEQ ID NO:85所示的CHb；以及如SEQ ID NO:82所示的CLa和CLb。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含两条不同的重链和两条相同的轻链，重链a如SEQ ID NO:92所示，重链b如SEQ ID NO:93所示，轻链如SEQ ID NO:96所示。
在一些实施方案中，抗PD-L1/CD47双特异抗体包含两条不同的重链和两条相同的轻链，重链a如SEQ ID NO:94所示，重链b如SEQ ID NO:95所示，轻链如SEQ ID NO:96所示。
恒定区及全长序列如下：
Figure PCTCN2021129045-appb-000001
Figure PCTCN2021129045-appb-000002
Figure PCTCN2021129045-appb-000003
本领域普通技术人员还应当理解，本发明所公开抗体或抗原结合片段序列是可以被替换的，替换后其氨基酸序列不同于该抗体的天然存在的氨基酸序列。例如，替换后的氨基酸序列可以是与起始序列相似的，比如与起始序列具有一定比例的同一性，比如它可以与起始序列的同一性是约80％、约85％、约90％、约95％、约98％或约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在某些实施方案中，抗体包含的氨基酸序列具有一个或多个修饰基团。例如，本 发明公开的双特异抗体可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。
本发明公开的抗体、抗原结合片段包括被修饰的衍生物，即通过任何类型的分子与抗体的共价连接进行修饰，其中共价连接不会阻止抗体与表位结合。包括但不限制以下实例，抗体可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。
在一些实施方案中，抗体可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG缀合。
抗体可以与治疗剂缀合或融合，所述治疗剂可包括可检测标记(如放射性标记)、免疫调节剂、激素、酶、寡核苷酸、光敏治疗剂、诊断剂、药物或毒素的细胞毒性剂、超声增强剂、非放射性标记物及其组合物，和本领域已知的其它此类试剂。
抗体可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。
抗体和编码抗体的多聚核苷酸的制备方法
本发明还公开了编码本发明所述抗体、抗原结合片段、及其衍生物的多聚核苷酸或核酸分子。本发明公开的多聚核苷酸可以编码VHa、VHb、VLa、VLb、CHa、CHb、CLa、CLb、Fc区、重链a、重链b、轻链等。制备抗体的方法是本领域公知的并且在本发明中有所描述。在某些实施方案中，本发明公开的抗体、抗原结合片段包括的可变区和恒定区都是全人源的。全人源抗体和抗原结合片段可以使用本领域中公开的技术和本发明所述的技术制备。例如，针对特定抗原的全人源抗体可以通过将抗原施用于转基因动物中来制备，所述转基因动物已经被改良过以响应抗原攻击而产生全人源抗体。可用于制备这类抗体的示例性技术参见美国专利6,458,592；6,420,140，其全部内容通过引用并入本文。本发明中所述双特异抗体是将特异性结合抗原a、抗原b的片段进行融合，双特异抗体中部分片段可参见上述结合单个抗原的抗体的制备方法。
在一些实施方案中，制备的抗体不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一实施方案中，本发明公开的抗体、抗原结合片段、或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通 过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基。(参考美国专利5,585,089；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400；WO 91/09967；美国专利5,225,539,5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596；)，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO/9852976 A1和WO/0034317 A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。
本发明公开的双特异抗体或抗原结合片段的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。
或者，本发明双特异抗体中scFv可参见生产单链单元的技术(美国专利4,694,778)。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.,Science 242:1038-1041(1988))。可以采用酵母展示scFv，酵母展示载体可以为pYD1载体(Addgene)，宿主酿酒酵母可以为酿酒酵母EBY100(Invitrogen)。
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见美国专利5,807,715、4,816,567和4,816,397， 其全部内容通过引用并入本文。
此外，在Newman,Biotechnology 10:1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在共同转让的美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。
抗体可以通过本领域已知的多种方法制备，包括使用来自免疫球蛋白序列的抗体文库进行的噬菌体展示方法。也可参考美国专利4,444,887和4,716,111，以及PCT公布文本WO 98/46645、WO 98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735和WO 91/10741，每个专利的全部内容通过引用并入本文。
识别选择性表位的全人源抗体可以使用被称为“引导选择”的技术来生产。在该方法中，使用选择的非人单克隆抗体(例如小鼠抗体)来引导识别相同表位的全人源抗体的筛选(参见美国专利5,565,332，其全部内容通过引用并入本文)。
在另一实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以分离编码所需单克隆抗体的DNA并对其进行测序。分离的和亚克隆的杂交瘤细胞可以作为此类DNA的来源。一旦分离出来，DNA可以被置于表达载体中，然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不产生其他免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。
此外，使用常规重组DNA技术，可将本发明的抗体的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.,J.Mol.Biol.278:457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内可以进行一个或多个氨基酸取代，可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。
抗体可以通过使用常规重组DNA技术制备。使用本领域技术人员公知的技术可 以选择、构建和培养生产抗体的载体及细胞系等。这些技术在各种实验室手册和主要出版物中均有描述，例如Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells，D.L.Hacker,F.M.Wurm,in Reference Module in Life Sciences,2017，其全部内容包括补充内容通过引用并入全文。
在一些实施方案中，可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生抗体。表达抗体重链轻链的DNA可以置入同一载体中或置入不同载体中；若置入不同载体中，表达抗体重链的载体和轻链的载体可以以适当比例转染宿主细胞(如Tihomir S.Dodev et al.,A tool kit for rapid cloning and expression of recombinant antibodies,Scientific Reports volume 4,Article number:5885(2014)；Stefan Schlatter et al.,On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells,Biotechnol Progress,21:122-133(2005)；Hadi Bayat et al.,Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells,Preparative Biochemistry & Biotechnology,48(8):822-829(2018)。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
双特异抗体制备方法在文献中有广泛记载，例如Qiong Wang,et al.,Design and Production of Bispecific Antibodies,Antibodies,8,43(2019)；Zhuang Zuo,et al.,An efficient route to the production of an IgG-like bispecific antibody,Protein Engineering,Design and Selection,13(5):361–367(2000)；Matthias Mack,et al.,A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity,Proc.Natl.Acad.Sci.,92:7021-7025,(1995)；Rodrigo Vazquez-Lombardi,et al.,Transient expression of human antibodies in mammalian cells,Nature Protocols  13(1):99-117(2018)；Elisa Corsiero,Monoclonal Antibodies:Expression and Purification in a Basic Research Laboratory,Mater Methods,6:1481(2016)。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区VH CDR1、VH CDR2、VH CDR3和轻链可变区VL CDR1、VL CDR2或VL CDR3来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
在一些实施方案中，本文所述取代为保守氨基酸取代。
治疗方法
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善各种类型的癌症、肿瘤或感染等相关疾病的方法，所述方法包括向患者施用有效剂量的所述的双特异抗体。在一些实施方案中，提供了所述的双特异抗体在用于治疗或改善癌症、肿瘤或感染等相关疾病中的应用。在一些实施方案中，提供了所述的双特异抗体在制备用于治疗或改善癌症、肿瘤或感染等相关疾病的药物中的应用。
本发明涉及治疗以PD-L1和/或CD47为治疗靶点的相关疾病的方法；涉及可以通过消除、抑制或降低PD-L1与PD1的结合和/或CD47与SIRPα的结合而改善、减缓、抑制或预防的任何疾病或病症的方法；涉及提供治疗受试者癌症或者肿瘤的方法、缓解受试者癌症或者肿瘤症状的方法、避免受试者肿瘤或者癌症复发的方法，所述方法包括向受试者施用有效量的本文所述的任何抗PD-L1/CD47双特异抗体或其片段。
本发明提供的抗体及其抗原结合片段和包含其的药物组合物可以用作治疗剂，用于诊断、预后、监控、治疗、缓解和/或预防受试者中异常PD-L1和/或CD47的表达、活性和/或信号传递相关的疾病和病症。通过使用标准方法鉴定受试者中存在异常PD-L1或CD47的表达、活性和/或信号传递相关的疾病和病症时，可以给药本发明公开的单克隆抗体、双特异抗体及其抗原结合片段和包含其的药物组合物。
鉴于现有技术中公开的多数CD47抗体能够引起人红细胞的血凝反应，目前仍然迫切需要获得不仅能够有效促进巨噬细胞的吞噬作用且不导致细胞凝集的新的抗CD47抗体。本申请公开的抗PD-L1/CD47双特异抗体满足了这方面的需求，其不仅可 以有效促进吞噬作用，不会导致显著的红细胞凝集，更优地，不会明显地与人红细胞结合。
在一些实施方案中，本发明的抗PD-L1/CD47双特异抗体能够以超高的亲和力与PD-L1结合，以高亲和力或中亲和力与CD47结合；通过与肿瘤细胞上PD-L1的特异性结合促进了本发明的抗PD-L1/CD47双特异抗体对肿瘤细胞的选择性结合，避免了与许多正常组织中表达的CD47结合，减少副作用；通过当与CD47的亲和力远低于与PD-L1的亲和力，可显著扩大本发明的抗PD-L1/CD47双特异抗体的有效剂量范围。
在一些实施方案中，用本发明所述的抗体治疗和/或预防的癌症包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)及转移性病灶。在一个实施方案中，癌是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌，如侵袭肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、CNS(例如，脑、神经的或神经胶质细胞)、头颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰腺的那些癌。癌症可以处于早期、中期或晚期或是转移性癌。在一个实施方案中，肿瘤是肿瘤免疫逃逸。在一些实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。在一些实施方案中，有效剂量的范围为从约0.01mg/kg到约100mg/kg，可以是例如每周两次(BIW)或至每月一次。
抗体或衍生物的施用方法包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外和口服注射。药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，含有本发明抗体的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和关节内注射和输注的施用方式。
施用方式可以是全身施用或局部施用。此外，可能需要通过任何合适的途径将本发明的抗体引入中枢神经系统，包括脑室内和鞘内注射；脑室内注射可以通过脑室内导管连接到如贮液囊(可以是Ommaya贮液囊)来辅助注射。也可以通过肺部给药，例如通过使用吸入器或喷雾器，以及使用雾化的制剂。
本发明抗体可以局部施用于需要治疗的区域；可以通过但不限于以下方式：手术期间局部输注，例如与手术后伤口敷料联合的局部应用，通过注射，通过导管，借助栓剂或借助植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜)或纤维。优选地，当施用本发明的蛋白质(包括抗体)时，必须注意使用不吸收蛋白质的材料。
通常在进行体外测试用于治疗疾病的方法，包括施用本发明所述抗体或衍生物，然后在可接受的动物模型中体内测试期望的治疗性或预防性活性，最后施用于人体。合适的动物模型(包括转基因动物)是本领域普通技术人员所公知的。例如，用于证明本发明所述抗体、抗原结合片段的治疗用途的体外测定包括抗体对细胞系或患者组织样品的影响。抗体对细胞系和/或组织样品的作用可以利用本领域技术人员已知的技术进行检测，例如本发明其他部分公开的技术。根据本发明的内容，可用于确定是否施用特异性抗体的体外测定实验包括体外细胞培养实验，其中患者组织样品在培养物中培养，并暴露于或以其他方式施用化合物，并观察这种化合物对组织样品的影响。
各种已知输送系统可用于施用本发明抗体或衍生物或编码其的多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细胞、受体介导的内吞作用(参见例如Wu and Wu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
联合疗法
在一些实施方案中，本发明抗体可以结合其它治疗或预防方案，包括施用一种或多种本发明抗体以及一种或多种其它治疗剂或方法一起使用或组合使用。对于组合治疗，抗体可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明抗体。
在一些实施方案中，在向患者施用本发明的双特异抗体时，还可以向患者联合施用本文公开的抗体分子或药物组合物或免疫缀合物，以及一种或多种其它疗法，例如治疗方式和/或其它治疗剂(例如化疗剂、放疗剂或生物大分子药物)。
此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的疗法，例如治疗方式和/或 治疗剂之前，同时，和/或之后发生本发明的抗体的施用。抗体分子和/或其他疗法，例如治疗剂或治疗方式可以在活动性疾病期间或在缓解或活动度更小的疾病期间施用。抗体分子可以在其他治疗前、与其他治疗同时、治疗后或在疾病缓解期间施用。
在一些实施方案中，本发明双特异抗体与化疗剂组合施用。在一些实施方案中，可与本发明的抗PD-L1/CD47双特异抗体一起施用的化疗剂包括但不限于抗生素衍生物(例如阿霉素、博来霉素、柔红霉素和放线菌素D)、抗雌激素药(如他莫昔芬)、抗代谢物(如氟尿嘧啶、5-FU、甲氨蝶呤、氟尿苷、干扰素α-2b、谷氨酸、光神霉素，巯基嘌呤和6-硫基鸟嘌呤)、细胞毒性剂(如卡莫司汀、BCNU、洛莫司汀、CCNU、阿糖胞苷、环磷酰胺、雌莫司汀、羟基脲、甲基苄肼、丝裂霉素、白消安、顺铂和硫酸长春新碱)、激素(如甲羟孕酮、雌莫司汀磷酸钠、炔雌醇、雌二醇、醋酸甲地孕酮、甲睾酮、己烯雌酚二磷酸、氯烯雌醚和睾内酯)、氮芥衍生物(例美法仑、苯丁酸氮芥、二氯甲基二乙铵(氮芥)和噻替哌)、类固醇及其组合(如倍他米松磷酸钠)，以及其它化合物(如氮烯唑胺、天冬酰胺酶、米托坦、硫酸长春新碱、硫酸长春碱和依托泊苷)。
在一些实施方案中，本发明抗体与细胞因子联合施用。可以与本发明抗体一起施用的细胞因子包括但不限于IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-10、IL-12、IL-13、和IL-15等。
在一些实施方案中，本发明双特异抗体与生物大分子药物联合施用。生物大分子药物的实例包括免疫治疗剂，包括但不限于适用于治疗患者的治疗性抗体。治疗性抗体的一些实例包括辛妥珠单抗、阿巴伏单抗、阿德卡妥姆单抗、阿富妥珠单抗、阿仑单抗、阿妥莫单抗、阿马妥昔单抗、阿纳妥姆单抗、阿西莫单抗、巴维妥昔单抗、贝妥莫单抗、贝伐单抗、比伐单抗、布莱纳妥姆单抗、布伦妥昔单抗、坎妥珠单抗、卡妥马索单抗、西妥昔单抗、西妥珠单抗、西妥木单抗、克里瓦妥珠单抗、克那妥姆单抗、达拉妥姆单抗、乔奇妥单抗、杜利戈妥单抗、杜西吉妥单抗、地莫单抗、达塞妥珠单抗、达洛妥珠单抗、埃克洛莫昔单抗、埃洛妥珠单抗、恩西妥昔单抗、厄妥马索单抗、伊他拉昔珠单抗、法勒妥珠单抗、菲可拉妥珠单抗、菲吉妥姆单抗、弗兰沃妥单抗、富妥昔单抗、加尼妥单抗、吉妥珠单抗、吉伦妥昔单抗、格兰巴妥姆单抗、替伊莫单抗、伊戈伏单抗、伊姆加妥珠单抗、英达妥昔单抗、伊诺妥珠单抗、因替妥姆单抗、伊匹单抗、伊拉妥姆单抗、拉贝妥珠单抗、来沙木单抗、林妥珠单抗、洛沃妥珠单抗、卢卡妥姆单抗、玛帕妥姆单抗、玛妥珠单抗、米拉妥珠单抗、明瑞莫单抗、米妥姆单抗、莫斜妥姆单抗、纳那妥单抗、纳妥姆单抗、内吉妥姆单抗、尼莫妥珠单抗、诺费妥单抗、奥卡妥珠单抗、奥法木单抗、奥拉妥珠单抗、奥那妥珠单抗、奥泼妥珠单抗、奥戈伏单抗、帕尼单抗、帕萨妥珠单抗、帕崔妥单抗、彭妥姆单抗、帕妥珠单抗、平妥姆单抗、普拖木单抗、拉蔻妥姆单抗、拉吉妥姆单抗、里洛妥姆单抗、 利妥昔单抗、洛巴妥姆单抗、沙妥莫单抗、思布妥珠单抗、思妥昔单抗、索力图单抗、塔卡妥珠单抗、塔普利妥珠单抗、特纳妥姆单抗、特普洛妥姆单抗、提咖妥珠单抗、拖西莫单抗、曲妥珠单抗、图库图珠单抗、尤不理妥昔单抗、维尔妥珠单抗、沃思妥珠单抗、伏妥莫单抗和扎鲁妥姆单抗等。
在一些实施例中，本发明抗体可以与免疫检查点抑制剂一起使用。在一些实施方案中，本发明抗体与其它治疗或预防方案联合施用，例如放射性疗法。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗体或抗原结合片段以及药学上可接受的载体。在一些实施方案中，药物组合物还包含抗癌剂(例如免疫检查点抑制剂)。
在一些实施方案中，术语“药学上可接受的”是指由政府的监管机构批准的或公认药典中列出的用于动物，特别是用于人类的物质。此外，“药学上可接受的载体”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“载体”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右旋葡萄糖也是可以预见的。这些组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合片段，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。亲本制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
在一些实施方案中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物 组合物。用于静脉内给药的组合物通常是在无菌等渗水性缓冲液中的溶液。组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有药用级无菌水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括衍生自如盐酸、磷酸、乙酸、草酸、酒石酸等的阴离子形成的盐，以及衍生自如钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的阳离子形成的盐。
实施例
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
实施例1：抗原的制备
CD47-His抗原制备：在人CD47细胞外结构域(ECD)蛋白质(SEQ ID NO:1)的C-末端添加8×HIS标签(SEQ ID NO:3)构建成CD47重组蛋白。人CD47的ECD区通过基因合成并亚克隆到哺乳动物的表达载体(如pcDNA3.1(+)表达载体)中。在瞬时转染HEK293F细胞之后，获得基于镍的固定化金属亲和层析纯化(GE Healthcare)的CD47-His抗原。
PD-L1-His抗原制备：在人PD-L1细胞外结构域(ECD)蛋白质(SEQ ID NO:2)C-末端添加8×HIS标签(SEQ ID NO:3)构建成PD-L1重组蛋白。人PD-L1的ECD区通过基因合成并亚克隆到哺乳动物的表达载体(如pcDNA3.1(+)表达载体)中。在瞬时转染HEK293F细胞之后，获得基于镍的固定化金属亲和层析纯化(GE Healthcare)的PD-L1-His抗原。
CD47-ECD(SEQ ID NO:1)：
Figure PCTCN2021129045-appb-000004
PD-L1-ECD(SEQ ID NO:2)：
Figure PCTCN2021129045-appb-000005
8×HIS(SEQ ID NO:3)：
Figure PCTCN2021129045-appb-000006
实施例2：抗体制备
2.1酵母展示的抗PD-L1 scFv
合成编码抗PD-L1抗体的scFv片段(包括重链可变区、轻链可变区以及重轻链可变区的连接子(G 4S) 3)的核酸序列，插入至载体中，并通过电转仪转进宿主酿酒酵母中获得阳性酵母克隆。将以上获得的阳性酵母克隆挑出，进行单克隆培养。其中，ATE阳性酵母克隆表面表达的scFv中重链可变区、轻链可变区与Atezolizumab是一致的，其他阳性酵母克隆表面表达的scFv见表1，各VH SEQ ID NO:相应的序列如表2所示，对应的CDR如表3所示。连接子的氨基酸序列为GGGGSGGGGSGGGGS，其核酸序列为ggtggaggcggttcaggcggaggtggctctggcggtggcggatcg。
表1：阳性酵母克隆展示的scFv
编号 VH SEQ ID NO: VL SEQ ID NO:
CP11-27 49 75
CP11-36 50 75
CP11-46 51 75
CP11-47 52 75
CP11-55 53 75
CP11-71 54 75
CP14-16 55 75
CP14-80 56 75
CP21-8 57 75
CP21-47 58 75
CP21-59 59 75
CP22-34 60 75
CP22-40 61 75
CP22-44 62 75
CP22-45 63 75
表2：抗PD-L1抗体重链可变区序列
Figure PCTCN2021129045-appb-000007
Figure PCTCN2021129045-appb-000008
表3：抗PD-L1抗体重链可变区CDR序列
Figure PCTCN2021129045-appb-000009
Figure PCTCN2021129045-appb-000010
2.2抗CD47抗体、抗PD-L1抗体及其制备
2.2.1抗CD47抗体
11种IgG1全抗的VH和VL的组合见表4；VH与CH组成抗体的重链，VL和CL组成抗体的轻链；CH的序列如SED ID NO:81，CL的序列如SED ID NO:82所示；用“抗体”+抗体编号表示，如抗体17(抗体重链由如SED ID NO:65所示的VH和如SED ID NO:81所示的CH组成，抗体轻链由如SED ID NO:98所示的VL和如SED ID NO:82所示的CL组成)。
表4对应的VH和VL序列见表5，其中，重链CDR见表6。
表4：抗CD47抗体的可变区组合
Figure PCTCN2021129045-appb-000011
表5：抗体可变区序列
Figure PCTCN2021129045-appb-000012
Figure PCTCN2021129045-appb-000013
注：X-VL代表3-3-VL、3-6-VL、3-VL、6-VL、10-VL、16-VL、18-VL、24-VL、25-VL
表6：抗体VH CDR
Figure PCTCN2021129045-appb-000014
抗CD47抗体的阳性对照Hu5F9-G4(Magrolimab)是在HEK293F细胞中瞬时表达的人CD47抗体，其序列与美国专利US2015/0183874A1中的抗体“Hu5F9”的序列相同。
2.2.2抗PD-L1抗体
选取CP11-36、CP11-46、CP11-47、CP11-55、CP11-71制备成IgG1全抗，其中VH和VL如表1所示，CH的序列如SED ID NO:81所示，CL的序列如SED ID NO:82所示；并将上述5个抗PD-L1抗体分别命名为：L1-R2-4-36、L1-R2-4-46、L1-R2-4-47、L1-R2-4-55以及L1-R2-4-71。
2.2.3具有共同轻链的抗CD47抗体和抗PD-L1抗体
抗体的组装方案如表7所示。可变区组装方案为“VH+VL”，CH的序列如SED ID NO:81，CL的序列如SED ID NO:82所示。轻链可变区序列如表8所示，轻链可变区CDR如表9所示。
表7：抗体编号、组装方案
Figure PCTCN2021129045-appb-000015
Figure PCTCN2021129045-appb-000016
表8：抗体轻链可变区序列
Figure PCTCN2021129045-appb-000017
表9：抗体轻链可变区CDR序列
Figure PCTCN2021129045-appb-000018
Figure PCTCN2021129045-appb-000019
2.2.4抗体的制备
将编码抗体重链的基因和抗体轻链的基因分别克隆至表达载体中，得到重链表达载体和轻链表达载体。再将重链表达载体和轻链表达载体瞬时转染HEK293F细胞，细胞表达后培养液使用ProteinA柱经由固定化金属亲和层析(IMAC)纯化得到抗体，测序后与预期序列一致。
2.3抗PD-L1/CD47双特异抗体及其制备
2.3.1抗PD-L1/CD47双特异抗体
抗PD-L1/CD47双特异抗体结构示意图如图1所示，5种抗体可变区氨基酸序列见表10。从图1的结构示意图可见，抗PD-L1/CD47双特异抗体由4条多肽链组成。
抗体BsAb-36、BsAb-46、BsAb-47、BsAb-71中，抗PD-L1的VH的C端连接到衍生于人IgG1的恒定区(SEQ ID NO:83)N端，其Fc区包含“杆入臼”突变，以与抗CD47重链稳定缔合；轻链由R2-4(SEQ ID NO:75)和人κ轻链恒定区(SEQ ID NO:82)连接，组成共同轻链；抗CD47的VH的C端连接到衍生于人IgG1的恒定区(SEQ ID NO:84)N端，其Fc区包含“杆入臼”突变，以与抗PD-L1重链稳定缔合。
抗体BsAb-71-N297A是在BsAb-71上，Fc区进行N297A突变，以减弱抗体的ADCC效应。即BsAb-71-N297A的抗PD-L1重链如SEQ ID NO:92所示，抗CD47重链如SEQ ID NO:93所示，轻链如SEQ ID NO:96所示。
表10：抗体编号，组装方案
Figure PCTCN2021129045-appb-000020
2.3.2抗PD-L1/CD47双特异抗体的制备
将编码步骤2.3.1中构建的抗PD-L1/CD47双特异抗体核苷酸序列构建到表达载体中，在HEK293F细胞中进行表达和纯化得到抗PD-L1/CD47抗体，具体如下：
将编码抗PD-L1的重链的基因(以BsAb-71-N297A为例，基因序列如SEQ ID NO:99所示)和抗CD47的重链的基因(以BsAb-71-N297A为例，基因序列如SEQ ID NO:100所示)分别克隆至表达载体中，分别得到抗PD-L1重链质粒和抗CD47重链质粒；将编码抗PD-L1/CD47抗体的轻链的基因(以BsAb-71-N297A为例，基因序列如SEQ ID NO:101所示)克隆至表达载体中，得到轻链质粒。将以上构建正确的抗PD-L1重链质粒、抗CD47重链质粒和轻链质粒按照摩尔比1:1:2的比例瞬时转染HEK293F细胞。细胞表达后培养液经由亲和层析和离子交换层析后，获得抗PD-L1/CD47抗体(例如BsAb-71-N297A)。测序后证实抗体序列。
利用分子排阻色谱法(size exclusion chromatography；SEC)检测收集的样品纯度。部分示例抗体的SEC结果如图2A和图2B所示，双特异抗体BsAb-46纯度为96.30％，BsAb-71纯度为97.05％。
抗PD-L1重链核苷酸序列SEQ ID NO:99如下：
Figure PCTCN2021129045-appb-000021
其Fc的核酸序列为(SEQ ID NO:87)：
Figure PCTCN2021129045-appb-000022
Figure PCTCN2021129045-appb-000023
抗CD47重链核苷酸序列SEQ ID NO:100如下：
Figure PCTCN2021129045-appb-000024
其Fc的核酸序列为(SEQ ID NO:88)：
Figure PCTCN2021129045-appb-000025
Figure PCTCN2021129045-appb-000026
轻链核苷酸序列SEQ ID NO:101如下：
Figure PCTCN2021129045-appb-000027
其CL的核酸序列为(SEQ ID NO:89)：
Figure PCTCN2021129045-appb-000028
实施例3：抗PD-L1 scFv与抗原蛋白PD-L1-His-Biotin的结合力
采用流式细胞仪检测酵母展示的抗PD-L1 scFv与抗原蛋白PD-L1-His-Biotin的结合力。将实施例2步骤2.1的阳性酵母克隆分别与1nM的PD-L1-His-Biotin抗原蛋白在室温孵育1h，再用pH7.4的PBS洗涤3次，然后再与Streptavidin-PE荧光二抗在室温孵育30min，再用pH7.4的PBS洗涤3次后，加入新的PBS，准备上机检测。
PD-L1-His-Biotin制备：将纯化得到的PD-L1-His抗原蛋白，按照每1mg蛋白加入26.6μl 10nM DMSO配置的biotin(Sigma-Aldrich，B4501-1G)，室温孵育2h后，用pH7.4的PBS进行透析。
如图3所示，本发明抗PD-L1 scFv(CP11-36、CP11-46、CP11-71、CP14-16和CP14-80)与PD-L1-His-Biotin抗原蛋白的结合力明显优于阳性对照ATE scFV；与阳 性对照ATE scFV相比，本发明抗PD-L1 scFv(CP-11-47、CP-11-55、CP21-8、CP21-47和CP22-34)与PD-L1-His-Biotin抗原蛋白的结合力的基本相同。
实施例4：抗CD47抗体的SIRPα阻断活性
采用ELISA竞争法，将11种抗体(抗体L12-6、抗体17、抗体3、抗体3-3、抗体3-6、抗体6、抗体10、抗体16、抗体18、抗体24、抗体25)和阳性对照(抗体Hu5F9-G4)稀释成不同浓度(12、6、3、2、1.5、1.2、0.75、0.375、0.188、0.094μg/ml)，与包被在ELISA板上的CD47-His(2μg/ml)抗原室温孵育1小时，PBST洗四遍，再加入SIRPα-Fc-Bio(0.1μg/ml)(参考Thermo Scientific
Figure PCTCN2021129045-appb-000029
NHS-Biotin Reagents试剂盒制备)配体与CD47-His抗原室温孵育1小时，PBST洗四遍。结合的SIRPα-Fc-Bio与HRP-缀合的链霉亲和素二抗产生化学发光反应，并通过读板机检测OD450值，根据OD450值换算出抗体的IC50，从而判断抗体的SIRPα阻断活性。结果如表11所示。结果表明上述抗CD47抗体均能抑制SIRPa结合细胞表面CD47。
表11：抗CD47抗体IC50值
Figure PCTCN2021129045-appb-000030
实施例5：抗PD-L1抗体的亲和力和对PD-1/PD-L1的阻断活性
Biacore检测亲和力解离常数(KD)
采用Biacore系统(GE公司)通过动力学结合测定法确定本发明实施例2步骤2.2.2的抗体结合相应抗原的平衡解离常数(KD)。按照使用手册的方法，将5μg的抗原蛋白PD-L1-His固定到芯片上，以最高100nM(按照1:2稀释，5个梯度)的抗PD-L1抗体浓度为流动相，进行亲和力测定，并用Biacore T200 Evaluation Software分析结果。其中，阳性对照为ATE抗体(ATE抗体的氨基酸序列与Atezolizumab是一致的，其由HEK293F细胞表达)。表12显示了示例抗体的KD数据，可见，与ATE抗体相比，本发明抗体L1-R2-4-36、L1-R2-4-46、L1-R2-4-47、L1-R2-4-55和L1-R2-4-71对PD-L1抗原的亲和力更高。
表12：通过Biacore动力学结合测定法确定的示例抗体的解离常数(KD*)
抗体编号 KD(M)
L1-R2-4-36 3.41×10 -10
L1-R2-4-46 2.82×10 -10
L1-R2-4-47 2.90×10 -10
L1-R2-4-55 3.71×10 -10
L1-R2-4-71 2.82×10 -10
ATE 1.84×10 -9
注：*拟合方式为：1:1Binding。
抗PD-L1抗体的生物活性检测方法，对以上所述构建的抗PD-L1抗体对PD-1/PD-L1结合的抑制作用进行检测。
MOA法检测抗PD-L1抗体对PD-1/PD-L1的阻断活性
抗PD-1/PD-L1抗体能够通过阻断PD-1和PD-L1的结合，从而解除对下游NFAT信号通路的抑制作用。采用Promega公司提供的MOA(Mechanisms of Action，MOA)检测系统(PD-1/PD-L1 Blockade Bioassay，Propagation Model，Catalog J1252)，根据说明书提供的方法，通过检测荧光报告基因的表达反应出NFAT信号的激活情况，从而检测实施例2步骤2.2.2的抗体对PD-1/PD-L1结合的抑制作用。其中，阴性对照抗体IgG-Isotype购自北京义翘神州科技股份有限公司(目录号：HG1K)。
活性检测前一天铺种CHO-PD-L1细胞(来自上述MOA检测系统)：铺CHO-PD-L1前1-2天传代，丢弃培养上清，用无菌PBS洗一遍细胞。加入适量Trypsin(Gibco)于37℃、5％CO2培养箱中消化3-5min。加入新鲜培养基终止消化，转移细胞至50ml离心管并计数。取所需体积细胞，900rpm离心5min。加入DMEM-F12培养基(Gibco)，重悬细胞至4×10 5个细胞/mL。将细胞加入96孔白色细胞培养板(Corning)，100μl/孔。细胞于37℃、5％CO 2培养箱中培养过夜。
检测当天处理Jurkat-PD1细胞(来自上述MOA检测系统)：计数后取所需体积细胞，900rpm离心5min。用测定缓冲液(1640培养基(Gibco)+1％FBS)重悬细胞至1.25×10 6个细胞/ml，待用。将CHO-PD-L1细胞培养板中的培养基上清，吸弃95μl/孔，加入40μl/孔的测试样品(上述抗PD-L1抗体、ATE抗体以及阴性对照HG1K抗体，最高浓度为20μg/ml，2倍稀释在测定缓冲液中，总共9个梯度)，再加入40μl Jurkat-PD1细胞，轻微震荡混匀，放置于37℃、5％CO2培养箱中培养6小时。检测：提前将ONE-Glo TMLuciferase Assay System(购自Promega，E6120)融化。6小时后，加入ONE-Glo TM试剂，50μl/孔。室温放置5-10min，读数。实验结果如图4所示，本发明抗体L1-R2-4-36、L1-R2-4-46、L1-R2-4-47、L1-R2-4-55和L1-R2-4-71均可以有效阻断PD1/PD-L1的相互作用；与对照抗体ATE相比，抗体(L1-R2-4-36和L1-R2-4-46)具有更强的阻断活性。
实施例6：共同轻链抗体亲和力及活性
Biacore检测亲和力解离常数(KD)
采用实施例5的Biacore检测亲和力解离常数(KD)的方法测定实施例2步骤2.2.3的抗PD-L1抗体和抗CD47抗体的解离常数。结果如表13所示。
表13：通过Biacore动力学结合测定法确定的示例抗体的解离常数(KD*)
Figure PCTCN2021129045-appb-000031
注：*拟合方式为：1:1Binding。
通过以上数据可见，本发明的不同轻链，能够同时分别与抗PD-L1和抗CD47的重链组成单克隆抗体，且分别与溶液中的人PD-L1和人CD47蛋白结合。
MOA法检测抗PD-L1抗体对PD-1/PD-L1的阻断活性
采用实施例5的MOA法检测实施例2步骤2.2.3的抗PD-L1抗体对PD-1/PD-L1的阻断活性，实验结果如图5所示，本发明的不同轻链的抗PD-L1抗体，均可以有效阻断PD1/PD-L1的相互作用。
抗CD47抗体红细胞凝集实验
已知大多数的抗CD47抗体具有促进红细胞凝集的副作用，因而这些抗CD47抗体的治疗应用受到限制。因此，进一步检测本发明实施例2步骤2.2.3的抗CD47抗体的红细胞凝集作用。具体检测方法：采集新鲜人类血液，用生理盐水清洗三次后制备成2％人红细胞悬液，将2％人红细胞悬液与受试抗体(最高终浓度为800nM，两倍系列稀释，总共获得11个稀释后浓度)等体积混合，在37℃孵育4小时，随后评估抗体红细胞凝集情况，将96孔U型板倾斜45°，观察红细胞团的流向，如果呈“一字型”，说明红细胞没有发生凝集。在如以上测定法所述进行的实验中，红细胞凝集反应结果如图6所示。在高浓度情况下，47-R2-89和对照抗体Hu5F9-G4引起红细胞凝集，其它示例抗体不会引起红细胞凝集。可见，本发明的大部分抗CD47抗体，显著地降低红细胞凝集作用，因此在临床治疗中具有显著降低的副作用，可以广泛应用于多种癌症的治疗中。
抗CD47抗体促进吞噬作用实验
进行体外吞噬作用测定以评估实施例2步骤2.2.3的抗CD47抗体是否增强巨噬细胞对表达CD47的靶细胞的吞噬。简而言之，在抗CD47抗体(0.7nM)的存在下，将RAW264.7巨噬细胞(2×10 5个/mL)与CFSE(Invitrogen，目录号：C34554)标记的Raji细胞(4×10 5个/mL)以1:2比率铺板到24-孔底板中，37℃下避光孵育2小时。孵育完成时，用PBS洗涤细胞两次后，再加入100μlAPC-F4/80荧光抗体(eBiosciences)，冰上(避光)孵育30分钟，PBS缓冲液洗涤两次并通过流式细胞术进行分析。分析吞噬指数，吞噬指数计算方式为：吞噬指数＝CFSE-F4/80双阳性的巨噬细胞数(即吞噬了肿瘤细胞的巨噬细胞数)/每5000个巨噬细胞。如图7可见，本发明的抗CD47抗体和Hu5F9-G4都可以有效诱导巨噬细胞对靶细胞发挥吞噬作用。
实施例7：抗PD-L1/CD47双特异抗体的Tm值的测定
采用DSC(差式扫描热量仪，Malvern Panalytical，MicroCal VP-Capillary)的方法测定示例的双特异抗体以及参考抗体(此处用L1-R2-4-71作为参比品)的Tm值，并据此初步判断示例的双特异抗体的热稳定性。
样品蛋白于1×PBS缓冲液(pH7.4)中，制备成2mg/mL浓度的溶液。自40℃开始，以180℃/hr的速率对样品或空白缓冲液的比热容(Cp)进行扫描。将样品扫描的结果分别扣除相应缓冲液的结果，利用得到的Cp值对温度作图，其中，Cp值明显升高的峰值所对应的温度即为样品的Tm值。
由表14可知，与传统抗体相似，示例的双特异抗体以及L1-R2-4-71参比样品均显示明显的Tm值，包括70℃左右的CH2溶解温度以及位于85℃左右的CH3的溶解温度。同时可以看出，和L1-R2-4-71相比，双特异抗体的Tm值略低，但差别并不明显，可以初步判定双特异抗体与单克隆抗体相近，具有良好的热稳定性。
表14：抗PD-L1/CD47双特异抗体的Tm值
Figure PCTCN2021129045-appb-000032
实施例8：抗PD-L1/CD47抗体与两种抗原的结合活性
使用Octet系统(ForteBio公司)通过动力学结合测定法确定示例的双特异抗体与 PD-L1和CD47的结合能力。在实验开始时，将ProA生物传感器AHC传感器(Pall,1506091)浸泡于PBS缓冲液中于室温平衡10分钟。向96孔黑色聚苯乙烯半量微孔板(Greiner)的孔中分别加入100μl双特异抗体溶液(BsAb-36、BsAb-46、BsAb-47或BsAb-71(20μg/ml))、100μl PD-L1-His抗原溶液(200nM、40nM、8nM、1.6nM、0.32nM和0)和100μl CD47-His抗原溶液(200nM、40nM、8nM、1.6nM、0.32nM和0)。将ProA生物传感器AHC浸没于分别含所述抗体溶液的孔中，在室温浸没120秒上样。随后将传感器在PBS缓冲液中洗涤至达到基线，然后浸没于含100μl PD-L1-His抗原溶液的孔中，随后将传感器在PBS缓冲液中洗涤至达到基线，然后浸没于含100μl CD47-His抗原溶液的孔中，监测抗体与抗原的缔合。转速为1000转/分钟，温度为30℃。结果如图8A所示。
在另一个实验中，实验方法同上所述，但将抗体与两种抗原的结合次序与上面的实验调转，将ProA生物传感器AHC浸没于分别含所述抗体溶液的孔中，在室温浸没120秒上样。随后将传感器在PBS缓冲液中洗涤至达到基线，然后浸没于含100μl CD47-His抗原溶液的孔中，随后将传感器在PBS缓冲液中洗涤至达到基线，然后浸没于含100μl PD-L1-His抗原溶液的孔中，监测抗体与抗原的缔合。结果如图8B所示。
在另一个实验中，实验方法同上所述，但双特异抗体为BsAb-71-N297A，检测双特异抗体BsAb-71-N297A与两种抗原的结合活性。结果图8C所示，表明BsAb-71-N297A能够同时和溶液的PD-L1和CD47蛋白结合。
通过以上结果可见，本发明示例的双特异抗体能够同时和溶液中的PD-L1和CD47蛋白结合，且在结合不同表位的时候不会互相干扰结合。
实施例9：抗PD-L1/CD47双特异抗体的解离常数测定
采用实施例5的Biacore检测亲和力解离常数(KD)的方法，对以上所述构建的抗PD-L1/CD47双特异抗体结合人PD-L1-His或人CD47-His抗原的平衡解离常数(KD)进行测定，并采用不同的拟合方式进行结果分析。表15显示了示例抗体的KD数据。
表15：通过Biacore动力学结合测定法确定的示例抗体分别对人PD-L1-His或人CD47-His抗原的解离常数(KD)
Figure PCTCN2021129045-appb-000033
注：*拟合方式为：1:1 Binding；^拟合方式为：Two State Reaction。
通过以上数据可见，采用1:1Binding的拟合方式，本发明示例的双特异抗体能够和溶液中的人PD-L1-His结合，且维持了亲本抗体的亲和力常数；本发明示例的双特异抗体能够和溶液中的人CD47-His蛋白结合，抗体的亲和力常数比亲本抗体弱，具有中等的抗CD47亲和力。
实施例10：抗PD-L1/CD47双特异抗体与过量表达PD-L1或CD47的CHO细胞的结合分析
通过流式细胞术测量本发明示例的抗PD-L1/CD47双特异抗体与过量表达人PD-L1或人CD47的CHO细胞的结合。简而言之，将人PD-L1或人CD47 cDNA插入到表达载体中，并转染至中国仓鼠卵巢癌细胞(CHO，Invitrogen)，产生过量表达人PD-L1或人CD47的CHO细胞(PD-L1-CHO细胞或CD47-CHO细胞)。将PD-L1-CHO细胞或CD47-CHO细胞分别与不同系列稀释的示例的双特异抗体混匀，冰上孵育1h；用PBS洗涤细胞两次，再加入PE标记的抗人Fc抗体(Invitrogen，12-4998-82)荧光二抗，冰上避光孵育30分钟；PBS洗涤细胞两次后，再用PBS重悬细胞，通过FACS检测抗体与细胞的结合。结果见图9A和图9B。由图9A可见，本发明示例的双特异抗体均能够与细胞表面表达的PD-L1相结合，并维持了亲本抗体的结合EC50。同时，由图9B可见，本发明示例的双特异抗体均能够与细胞表面表达的CD47相结合。
另一个实施例中，根据上述方法分析抗PD-L1/CD47双特异抗体BsAb-71-N297A与过量表达PD-L1或CD47的CHO细胞的结合。结果如图9C和图9D所示。由图9C可见，BsAb-71-N297A能够与细胞表面表达的PD-L1相结合，并维持了亲本抗体的结合EC50。同时，由图9D可见，BsAb-71-N297A能够与细胞表面表达的CD47相结合。
现有报道已知CD47蛋白在人各种正常组织中，均有不同程度的表达，因而给抗CD47抗体的治疗应用带来一定的阻碍。在本发明中，通过策略性降低双特异抗体与人CD47的亲和力和结合力，预见能够在临床治疗中降低抗体引起的副作用，可以广泛应用于多种癌症的治疗。
实施例11：基于MOA法检测本发明示例的双特异抗体的抗PD-L1活性
采用实施例5的MOA检测方法，检测本发明示例的双特异抗体(BsAb-36、BsAb-46、BsAb-47、BsAb-71以及BsAb-71-N297A)的抗PD-L1活性。结果如图10A和图10B所示，本发明的双特异抗体可以解除PD-1/PD-L1相互作用对NFAT信号通路的抑制作用，且活性优于作为抗PD-L1抗体的L1-R2-4-71抗体单独使用。
实施例12：抗PD-L1/CD47双特异抗体促进红细胞凝集活性的检测
如实施例6所述的红细胞凝集实验检测方法，本发明进一步研究抗PD-L1/CD47双特异抗体的红细胞凝集作用。结果如图11所示，本发明示例的双特异抗体不引起红 细胞凝集，其促进红细胞凝集的活性明显低于对照组Hu5F9-G4，与亲本抗体47-R2-4保持一致。
实施例13：双特异抗体促进巨噬细胞吞噬肿瘤细胞的能力检测(吞噬图)
在基于流式细胞术的测定法中测量本发明示例的双特异抗体促进巨噬细胞吞噬肿瘤细胞的能力。如实施例6所述的抗CD47抗体促进吞噬作用的实验方法，体外评估本发明示例的双特异抗体促进巨噬细胞对靶细胞的吞噬能力。从图12可见，抗PD-L1/CD47双特异抗体可以有效诱导巨噬细胞对靶细胞发挥吞噬作用，其诱导活性和抗CD47单克隆抗体相近。
实施例14：双特异抗体与人红细胞的结合分析(RBC binding)
现有技术已知只有在肿瘤细胞和红细胞上，阻断CD47-SIRPα的相互作用会引发吞噬作用。这一机制决定了抗CD47抗体的作用机制，因而治疗引起的红细胞减少造成严重贫血，限制了抗CD47抗体的治疗应用。红细胞结合差异化特性的抗CD47抗体，可以有效规避与正常红细胞结合，降低临床治疗中的副作用。本实施例采用流式细胞术，检测示例的双特异抗体对肿瘤细胞和人红细胞的选择性结合性质。具体方法：采集新鲜人类血液，用生理盐水清洗三次后制备成2％人红细胞悬液，将2％人红细胞悬液与受试抗体(最高终浓度为200nM，两倍系列稀释，总共获得11个稀释后浓度)等体积混合，冰上孵育1h；用PBS洗涤细胞两次，再加入PE标记的抗人Fc抗体(Jackson Invitrogen，12-4998-82)荧光二抗，冰上避光孵育30分钟；PBS洗涤细胞两次后，再用PBS重悬细胞，通过FACS检测抗体与细胞的结合。以上述同样的方法，制备示例的双特异抗体与人T淋巴细胞白血病Jurkat细胞的检测样品。实验结果如图13A和图13B所示，在200nM的抗体浓度下，本发明的双特异抗体与人红细胞只有弱结合，明显弱于亲本抗体47-R2-4以及阳性对照抗体Hu5F9-G4与人红细胞结合；差异化红细胞结合特性并没有影响双特异抗体以及亲本抗体与肿瘤细胞的结合，因此，本发明的双特异抗体具有更高的临床应用安全性和更广泛的治疗适应性。
实施例15：抗PD-L1/CD47双特异抗体对外源刺激下的人外周血单个核细胞(PBMC)释放IL2细胞因子的影响
本研究将示例抗体和体外培养的、来源于不同供体的PBMC共同孵育，通过检测体系中IL2的表达量，从而反应出不同抗体对T细胞的激活作用。
PBMC分离：取捐献者(Donor1和Donor2)新鲜抗凝全血，用等体积PBS以1:1比例稀释全血；在15mL离心管中加入7mL人外周血淋巴细胞分离液(达优，7912011)，将7mL稀释后的血样缓慢加到人外周血淋巴细胞分离液(达优，7912011)液面上方，保持两液面界面清晰，血样应浮在分离液上方，不能冲破界面；在室温下，水平转子 800g离心30min，加速的加速度设为1，降速的加速度设为0；离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄较致密的白膜，即PBMC细胞层。小心吸取白膜层到新的离心管中；用PBS将吸出的PBMC稀释到一定体积，颠倒混匀。室温，水平转子，250g离心10min弃上清，重复洗两次；用PBS将细胞重悬计数备用。
抗体准备：用PBMC培养液配置抗体BsAb-71-N297A(5μg/ml)、47-R2-4(5μg/ml)、L1-R2-4-71(5μg/ml)、47-R2-4(5μg/ml)+L1-R2-4-71(5μg/ml)。
PBMC刺激：取适量PBMC，用PBMC培养液重悬至细胞密度为1×10 6个/ml；往上述细胞悬液中加入SEE(Toxin Technology,ET404)，使SEE浓度为100ng/ml；取一块96孔细胞培养板，将加了SEE的PBMC细胞均匀分到孔中，每孔100μl。将准备好的抗体溶液加入板中，每孔加入100μL，混匀，此时SEE终浓度为50ng/ml，抗体终浓度为2.5μg/ml，每孔中有总数为10万个的PBMC细胞。抗体与细胞于37℃的二氧化碳培养箱中共同培养4天。
IL-2检测：将准备好的抗IL-2抗体包被96孔板(
Figure PCTCN2021129045-appb-000034
Assay Plate)，每孔加入抗IL-2抗体100μl，4℃孵育过夜；用微孔洗板机洗板2次，设置每次300μl洗涤液。在干净的纸上轻轻拍干，每孔加入200μl封闭液，于37℃电热恒温培养箱孵育2小时；每孔加入75μl实验稀释液和25μL与抗体共孵育4天的PBMC上清液，加入稀释好的IL-2标准品(Human IL-2 ELISA development kit(HRP)，Mabtech)，37℃孵育2小时；用微孔洗板机洗板5次，设置每次300μL洗涤液。在干净的纸上轻轻拍干，每孔加入100μl抗IL-2生物素化抗体，于37℃电热恒温培养箱孵育1小时；每孔加入100μl二抗溶液，于37℃电热恒温培养箱孵育1小时；用微孔洗板机洗板8次，设置每次300μl洗涤液。在干净的纸上轻轻拍干；每孔加入100μl的TMB单组分显色液，于37℃避光孵育10-15min；每孔加入50μl终止液终止显色反应，然后在多功能酶标仪中读数并分析数据，吸光值设定为450nm。
实验结果如图14所示，本发明示例的抗PD-L1/CD47双特异抗体BsAb-71-N297A可以在体外有效激活T细胞。
实施例16：本发明的抗PD-L1/CD47双特异抗体的体内抗肿瘤作用
采用表达人CD47的MC38细胞(MC38-hCD47(Tg)，江苏集萃药康生物科技有限公司)在hSIRPα转基因的C57BL/6-hSIRPα小鼠测定本发明的抗PD-L1/CD47双特异抗体的抗肿瘤作用。
人SIRPα转基因小鼠：雌性C57BL/6-hSIRPα小鼠(5-8周龄)(江苏集萃药康生物科技有限公司)。
实验细胞：收集对数生长期的小鼠结肠癌细胞MC38-hCD47(Tg)，去除培养液并用PBS洗两次后接种；接种量：1×10 6/100μl/只；接种位置：右侧腹部区域位置。
分组给药：当肿瘤平均体积在50-100mm 3左右，对荷瘤小鼠进行随机分组；入组标准：肿瘤体积CV值小于30％；接种当天定义为D0天，并于分组当天，根据实验方案设计开始给药。给药剂量和给药方式如表16所示。细胞接种后，每周常规监测肿瘤对动物正常行为的影响。具体内容：实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。试验过程中观察到的临床症状均记录在原始数据中。开始给药后，每周称量体重两次。每周测量瘤体积两次，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×a×b 2，a为肿瘤长径，b为肿瘤短径。监测至33天后结束。接种后第33天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝(1-治疗组平均相对肿瘤体积/溶媒组平均相对肿瘤体积)×100％。
肿瘤抑制率结果如图15和表17所示：在接种后第33天，与Isotype IgG1组对比，5mg/kg L1-R2-4-71单药肿瘤抑制率为32.25％；5mg/kg 47-R2-4单药肿瘤抑制率为18.90％；5mg/kg L1-R2-4-71与5mg/kg47-R2-4联合用药的肿瘤抑制率为76.73％；10mg/kg抗PD-L1/CD47双特异抗体BsAb-71-N297A的肿瘤抑制率为68.70％，明显优于单药的肿瘤抑制效果，与联合给药组相比没有统计学差异(p＞0.05)。对小鼠体重检测的结果是各组小鼠的体重之间无显著性差异。
表16：实验设计表
Figure PCTCN2021129045-appb-000035
表17：第33天肿瘤抑制率
组别 给药组 肿瘤体积(mm 3) 肿瘤抑制率(TGI％)
G1 Isotype IgG1 1480.47 N/A
G2 L1-R2-4-71 969.32 32.25％
G3 47-R2-4 1264.65 18.90％
G4 L1-R2-4-71+47-R2-4 337.41 76.37％
G5 BsAb-71-N297A 450.76 68.70％
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。

Claims (20)

  1. 一种双特异抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含特异性结合PD-L1的可变区a；其中，所述可变区a包含(a)-(f)中一种或多种氨基酸序列：
    (a)VHa CDR1，其包含如SEQ ID NO:4-8中任一项所示的氨基酸序列；
    (b)VHa CDR2，其包含如SEQ ID NO:9-18中任一项所示的氨基酸序列；
    (c)VHa CDR3，其包含如SEQ ID NO:19或20所示的氨基酸序列；
    (d)VLa CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；
    (e)VLa CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；
    (f)VLa CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
  2. 如权利要求1所述的抗体或抗原结合片段，其特征在于，所述可变区a包含所述VHa CDR1、VHa CDR2、VHa CDR3、VLa CDR1、VLa CDR2和VLa CDR3。
  3. 如权利要求1所述的抗体或抗原结合片段，其特征在于，所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:9-14中任一项所示的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列；或
    所述VHa CDR1包含如SEQ ID NO:4所示的氨基酸序列；所述VHa CDR2包含如SEQ ID NO:14所示的氨基酸序列；所述VHa CDR3包含如SEQ ID NO:19所示的氨基酸序列；所述VLa CDR1包含如SEQ ID NO:36所示的氨基酸序列；所述VLa CDR2包含如SEQ ID NO:41所示的氨基酸序列；所述VLa CDR3包含如SEQ ID NO:45所示的氨基酸序列。
  4. 如权利要求1-3中任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还包含特异性结合CD47的可变区b；其中，所述可变区b包含(g)-(l)中一种或多种氨基酸序列：
    (g)VHb CDR1，其包含如SEQ ID NO:21-23中任一项所示的氨基酸序列；
    (h)VHb CDR2，其包含如SEQ ID NO:24-28中任一项所示的氨基酸序列；
    (i)VHb CDR3，其包含如SEQ ID NO:29-35中任一项所示的氨基酸序列；
    (j)VLb CDR1，其包含如SEQ ID NO:36-40中任一项所示的氨基酸序列；
    (k)VLb CDR2，其包含如SEQ ID NO:41-44中任一项所示的氨基酸序列；
    (l)VLb CDR3，其包含如SEQ ID NO:45-48中任一项所示的氨基酸序列。
  5. 如权利要求4所述的抗体或抗原结合片段，其特征在于，所述可变区b 包含所述VHb CDR1、VHb CDR2、VHb CDR3、VLb CDR1、VLb CDR2和VLb CDR3。
  6. 如权利要求4所述的抗体或抗原结合片段，其特征在于，所述VHb CDR1包含如SEQ ID NO:21所示的氨基酸序列；所述VHb CDR2包含如SEQ ID NO:24所示的氨基酸序列；所述VHb CDR3包含如SEQ ID NO:29所示的氨基酸序列；所述VLb CDR1包含如SEQ ID NO:36所示的氨基酸序列；所述VLb CDR2包含如SEQ ID NO:41所示的氨基酸序列；所述VLb CDR3包含如SEQ ID NO:45所示的氨基酸序列。
  7. 如权利要求1-6中任一项所述的抗体或抗原结合片段，其特征在于，所述VLa CDR1与所述VLb CDR1包含相同的氨基酸序列，所述VLa CDR2与所述VLb CDR2包含相同的氨基酸序列，和/或所述VLa CDR3与所述VLb CDR3包含相同的氨基酸序列。
  8. 如权利要求1-7中任一项所述的抗体或抗原结合片段，其特征在于，所述可变区a包含重链可变区VHa和轻链可变区VLa；其中，
    所述VHa包含如SEQ ID NO:49-63中任一项所示的氨基酸序列，或与SEQ ID NO:49-63中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或
    所述VLa包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列。
  9. 如权利要求1-8中任一项所述的抗体或抗原结合片段，其特征在于，
    所述VHa包含如SEQ ID NO:54所示的氨基酸序列，或与SEQ ID NO:54所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或
    所述VLa包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列。
  10. 如权利要求1-9中任一项所述的抗体或抗原结合片段，其特征在于，所述可变区b包含重链可变区VHb和轻链可变区VLb；其中，
    所述VHb包含如SEQ ID NO:64-74中任一项所示的氨基酸序列，或与SEQ ID NO:64-74中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或
    所述VLb包含如SEQ ID NO:75-80中任一项所示的氨基酸序列，或与SEQ ID NO:75-80中任一项所示的氨基酸序列至少有90％同源性的氨基酸序列。
  11. 如权利要求1-9中任一项所述的抗体或抗原结合片段，其特征在于，所述VHb包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或
    所述VLb包含如SEQ ID NO:75所示的氨基酸序列，或与SEQ ID NO:75所示的氨基酸序列至少有90％同源性的氨基酸序列。
  12. 如权利要求1-11任一项所述的抗体或抗原结合片段，其特征在于，所述VLa与所述VLb包含相同的氨基酸序列。
  13. 如权利要求1-12任一项所述的抗体或抗原结合片段，其特征在于，所 述抗体或抗原结合片段还包含重链恒定区a和重链恒定区b，所述重链恒定区a和所述重链恒定区b分别与重链可变区a和重链可变区b连接，所述重链恒定区a和/或所述重链恒定区b包含如下一种或多种氨基酸突变，其中氨基酸位置为Eu编号：
    Y349C、S354C、T366W、T366S、L368A以及Y407V；
    可选的还包含如下氨基酸突变：N297A。
  14. 如权利要求13所述的抗体或抗原结合片段，其特征在于，所述重链恒定区a和重链恒定区b之一包含如下一种或多种氨基酸突变：
    N297A、S354C以及T366W；和/或
    另一个重链恒定区包含如下一种或多种氨基酸突变：
    N297A、Y349C、T366S、L368A以及Y407V。
  15. 如权利要求13所述的抗体或抗原结合片段，其特征在于，所述其中一个重链恒定区包含如SEQ ID NO:81、83或85所示的氨基酸序列，或与SEQ ID NO:81、83或85所示的氨基酸序列至少有90％同源性的氨基酸序列；和/或另一个重链恒定区包含如SEQ ID NO:81、84或86所示的氨基酸序列，或与SEQ ID NO:81、84或86所示的氨基酸序列至少有90％同源性的氨基酸序列。
  16. 一种双特异抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含一条重链a、一条重链b以及两条相同的轻链；所述重链a和一条所述轻链配对形成PD-L1抗原结合位点，所述重链b和另一条所述轻链配对形成CD47抗原结合位点。
  17. 如权利要求16所述的抗体或抗原结合片段，其特征在于，所述重链a包含如SEQ ID NO:92或94所示的氨基酸序列，或与SEQ ID NO:92或94所示的氨基酸序列至少有90％同源性的氨基酸序列；所述重链b包含如SEQ ID NO:93或95所示的氨基酸序列，或与SEQ ID NO:93或95所示的氨基酸序列至少有90％同源性的氨基酸序列；所述轻链包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列至少有90％同源性的氨基酸序列。
  18. 一种多聚核苷酸，其特征在于，所述多聚核苷酸编码如权利要求1-15任一项所述的抗体或抗原结合片段；或，
    一种表达载体，其特征在于，所述表达载体包含编码如权利要求1-17任一项所述的抗体或抗原结合片段的多聚核苷酸；或，
    一种细胞，其特征在于，所述细胞包含编码如权利要求1-17任一项所述的抗体或抗原结合片段的多聚核苷酸。
  19. 一种组合物，其特征在于，所述组合物包含如权利要求1-17任一项所述的抗体或抗原结合片段、如权利要求18所述的多聚核苷酸、表达载体或细胞，以及药学上可接受的载体。
  20. 权利要求1-17任一项所述的抗体或抗原结合片段、如权利要求18所述的多聚核苷酸、表达载体或细胞或如权利要求19所述的组合物在制备治疗疾病的药物中的应用。
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EP4190813A4 (en) * 2020-07-31 2025-02-12 Bio-Thera Solutions, Ltd. CD47 ANTIBODIES AND THEIR APPLICATION
EP4382540A4 (en) * 2021-08-06 2025-07-16 Bio Thera Solutions Ltd USE OF AN ANTI-PD-L1/CD47 BISPECIFIC ANTIBODY IN THE TREATMENT OF DISEASES

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KR20230129482A (ko) * 2021-01-08 2023-09-08 베이징 한미 파마슈티컬 컴퍼니 리미티드 항pd-l1/항cd47 천연항체 구조 형태의 헤테로다이머이중특이성 항체 및 그 제조방법
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