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WO2023246891A1 - 结合egfr和b7-h3的双特异性抗体 - Google Patents

结合egfr和b7-h3的双特异性抗体 Download PDF

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Publication number
WO2023246891A1
WO2023246891A1 PCT/CN2023/101807 CN2023101807W WO2023246891A1 WO 2023246891 A1 WO2023246891 A1 WO 2023246891A1 CN 2023101807 W CN2023101807 W CN 2023101807W WO 2023246891 A1 WO2023246891 A1 WO 2023246891A1
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Prior art keywords
amino acid
acid sequence
seq
region
antibody
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PCT/CN2023/101807
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English (en)
French (fr)
Inventor
何开杰
周帅祥
关剑
李亚宁
刘锡洋
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信达生物制药（新加坡）有限公司
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Publication of WO2023246891A1 publication Critical patent/WO2023246891A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates generally to the fields of immunology and antibody engineering. Specifically, the present invention relates to novel artificially designed bispecific antibody molecules, especially bispecific antibodies that simultaneously bind EGFR and B7-H3, polynucleotides encoding the antibody molecules or their respective chains, including Vectors of the polynucleotides, host cells containing the polynucleotides or vectors, immunoconjugates and pharmaceutical compositions containing the antibody molecules, and the use of the antibody molecules in the immunotherapy, prevention and/or treatment of diseases Diagnostic uses.
  • novel artificially designed bispecific antibody molecules especially bispecific antibodies that simultaneously bind EGFR and B7-H3, polynucleotides encoding the antibody molecules or their respective chains, including Vectors of the polynucleotides, host cells containing the polynucleotides or vectors, immunoconjugates and pharmaceutical compositions containing the antibody molecules, and the use of the antibody molecules in the immunotherapy, prevention and/or treatment of diseases Diagnostic uses.
  • lung cancer is one of the most common types of cancer tumors. About 80%-85% of lung cancers are non-small cell lung cancer (NSCLC) patients. NSCLC can be divided into lung adenocarcinoma, lung squamous cell carcinoma and large cell carcinoma. Common types of cell lung cancer.
  • Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and a huge transmembrane glycoprotein with a molecular weight of approximately 170KDa. It is a member of the ErbB receptor family and is the most common cancer driver gene in NSCLC.
  • NSCLC patients in Asia are caused by EGFR mutations, and about 15% of NSCLC patients in Caucasians are caused by EGFR mutations (Gower A, Wang Y, Giaccone G. Oncogenic drivers, targeted therapies, and acquired resistance in non- small-cell lung cancer.J.Mol.Med.2014;92:697-707.), in patients with other cancer tumors, such as kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, head and neck cancer and other epithelial origin cancers , all have the phenomenon of EGFR overexpression and/or abnormal mutation activation.
  • EGFR-TKI small molecule inhibitors such as gefitinib, erlotinib (Erlotinib), afatinib, dacomitinib and osimertinib ( Osimertinib), ametinib, etc.
  • biological monoclonal antibody macromolecules cetuximab, panitumumab, nimotuzumab and nimotuzumab targeted therapy.
  • EGFR-TKI small molecule inhibitors are still the standard method for the treatment of non-small cell lung cancer (NSCLC)
  • NSCLC non-small cell lung cancer
  • EGFR-TKI small molecules are mainly targeted at patients with tyrosine kinase active structural mutations, and drug resistance is often caused by target gene mutations. Ineffective, it is necessary to continuously develop new targeted drugs for new mutation sites, which greatly limits the clinical application of this type of drugs. This is also a severe challenge faced by the entire EGFR-TKI small molecule industry.
  • the EGFR activating mutation region mainly occurs in the EGFR exon 18-21 tyrosine kinase domain (Jiyeon Yun, Soo-Hwan Lee, Seok-Young Kim, et al. Antitumor activity of Amivantamab (JNJ-61186372), an EGFR– MET bispecific antibody,in diverse models of EGFR exon 20 insertion–drivenNSCLC.Cancer Discov 2020;10:1194-209.), the binding region of EGFR antibody is mainly located in the EGFR extracellular ligand domain, which can avoid the occurrence of drug-resistant mutations ;
  • EGFR antibodies can inhibit the growth of tumor cells by inhibiting the binding of EGFR and ligands, and can also use their own specific ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells. This can be achieved through a variety of methods. Mechanism of action, jointly exert anti-tumor killing effect.
  • B7-H3 (also known as CD276) is a type I transmembrane protein (Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: targeting B7–H3 (CD276) for human cancer immunotherapy. Clin Cancer Res. 2016; 22 :3425–31.and, Yang S, Wei W, Zhao Q. B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. Int J Biol Sci. 202016:1767–73), which has a very similar structure to PD-L1 and belongs to the B7/CD28 superfamily.
  • B7-H3 receptor has not yet been identified, but in tumor immunity, B7-H3 can participate in the regulation of immune function of toxic lymphocytes (Kraan J, van den BroekP, VerhoefC, Grunhagen DJ, Taal W, Gratama JW, et al. Endothelial CD276(B7-H3) expression is increased in human malignancies and distinguishes between normal and tumor-derived circulating endothelial cells. Br J Cancer.
  • B7-H3 expression may be related to the EGFR gene Expression status is related to anti-PD-1 therapeutic effect (Yonesaka K, Haratani K, Takamura S, Sakai H, Kato R, Takegawa N, et al. B7-H3 negatively modulates CTL-mediated cancer immunity. Clin Cancer Res. 2018; 24: 2653–64). Because B7-H3 has a good selective expression profile, some biological research and development companies have developed B7-H3 monoclonal monomers or B7-H3-ADC drugs for research and treatment in the field of related tumor diseases.
  • Antibody molecules can specifically bind to their corresponding antigens and are increasingly becoming important therapeutic, preventive and therapeutic agents for various diseases (such as cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.) /or diagnostic agents.
  • diseases such as cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.
  • monospecific antibodies targeting only one target have some limitations in clinical application. Patients may develop drug resistance or non-response after treatment with monospecific antibodies.
  • cancer and many other diseases it has been realized that multiple signal transduction pathways are often involved in the occurrence and development of the disease, and single-target immunotherapy is usually not enough to play a therapeutic role in many diseases.
  • multispecific antibodies eg., bispecific antibodies
  • they can be designed to simultaneously act on the signal transduction pathways of two or more different mediators. These advantageous properties open up broad application prospects for multispecific antibodies (e.g., bispecific antibodies).
  • bispecific antibodies with improved properties that are capable of binding different antigens simultaneously, in particular EGFR and B7-H3, while maintaining the corresponding distinct expression of each antigen binding site. binding activity, and other properties.
  • a first aspect of the invention relates to an antibody comprising an antigen-binding region that specifically recognizes EGFR (eg, human EGFR) and an antigen-binding region that specifically binds B7-H3.
  • the antibody is a multispecific antibody, such as a bispecific antibody.
  • the antibody or antigen-binding fragment thereof that binds EGFR and B7-H3 provided by the invention improves the efficacy and safety selectivity of the antibody molecule by reducing the affinity of EGFR and increasing the affinity of B7-H3; at the same time, Using GlymaxX low-fucose technology, antibody-dependent cell-mediated cytotoxicity (ADCC) is enhanced.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • B7-H3 Compared with Amivantamab (JNJ-372) for NSCLC-EGFR exon 20 insertion mutations (exon20ins), B7-H3 has a broader expression spectrum than cMET and has a wider range of tumor treatment applications; at the same time, The introduction of the B7-H3 parent not only improved the EGFR antibody blocking activity of the bispecific antibody, but also improved the overall ADCC effect of the bispecific antibody.
  • the bispecific antibody or antigen-binding fragment thereof that binds EGFR and B7-H3 provided by the invention has one or more of the following characteristics:
  • different antigen binding sites bind the same epitope on the same antigen, or different epitopes.
  • the first antigen binding region or the second antigen binding region is from a human, or humanized, or chimeric antibody.
  • the antibodies of the invention eg, bispecific antibodies, further comprise a heavy chain constant region.
  • the heavy chain constant domain is from IgG1. It will be appreciated that the Fc in the constant domain can be mutated to stabilize the antibody, or to enhance effector function.
  • the first antigen binding region is specific for the first antigen, and in one embodiment the first antigen is EGFR.
  • the second antigen binding region is specific for a second antigen, in one embodiment the second antigen is B7-H3.
  • the antibodies of the invention may also contain other antigen-binding regions that bind other antigens to constitute multispecific antibodies.
  • the types of other antigens specifically bound by the antibody molecules of the present invention are not particularly limited, and the antigens may be, for example, cytokines, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, cell surface receptors or fragments thereof.
  • the other antigens specifically bound by the antibody molecules of the invention are selected from the group consisting of tumor-associated antigens, immune checkpoint molecules, angiogenesis-inducing factors, tumor necrosis factor receptor superfamily members and costimulatory molecules in the immune system, and ligands and/or receptors for these molecules.
  • the invention provides a nucleic acid encoding any one or more polypeptide chains of the antibody molecule of the invention, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector.
  • the invention provides a vector, preferably an expression vector, such as pcDNA3.1, comprising a polynucleotide encoding any one or more polypeptide chains of the antibody molecule of the invention.
  • the invention provides methods for producing antibody molecules of the invention, or fragments thereof.
  • the invention provides immunoconjugates, pharmaceutical compositions, kits, combinations or articles of manufacture comprising an antibody of the invention.
  • the antibodies, pharmaceutical compositions or immunoconjugates or combination products or kits of the invention are used to prevent or treat diseases, such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. .
  • the disease is a tumor (such as cancer) or an infection.
  • the tumor is immune evasive.
  • the tumor is a gastrointestinal tract tumor or a lung tumor or a skin tumor.
  • the infection is a chronic infection.
  • the invention in another aspect, relates to a method of preventing or treating a disease in a subject or individual, said method comprising administering to said subject an effective amount of any antibody or fragment thereof, pharmaceutical composition or immunization agent described herein.
  • Conjugate or combination product or kit for example, the disease is a tumor (such as cancer) or an infection.
  • the tumor is immune evasive.
  • the tumor is a gastrointestinal tract tumor or a lung tumor or a skin tumor.
  • the infection is a chronic infection.
  • the invention also relates to any antibody or fragment thereof or immunoconjugate described herein for the preparation of a medicament or pharmaceutical composition or kit or combination for the treatment of tumors (e.g., cancer) or infections in a subject Product purpose.
  • tumors e.g., cancer
  • the tumor is immune evasive.
  • the tumor is a gastrointestinal tract tumor or a lung tumor or a skin tumor.
  • the infection is a chronic infection.
  • the invention also relates to methods of detecting antigens in samples.
  • the invention relates to the following specific embodiments:
  • the second antigen-binding region comprises the HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 3, 5 or 7, and as SEQ ID NO: 3, 5 or 7.
  • the HCDR1 adopts the Abm scheme
  • the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 adopt the Kabat scheme.
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:3; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in 4;
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:5; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in 6; or
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:7; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in 8;
  • the HCDR1 adopts the Abm scheme
  • the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 adopt the Kabat scheme.
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:15
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:16
  • HCDR3 includes SEQ ID NO: The amino acid sequence shown in 17 or consisting of said sequence
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:18
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:19
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence of or consisting of said sequence; or,
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:21
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:22
  • HCDR3 includes SEQ ID NO: The amino acid sequence shown in 23 or consisting of said sequence
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:24
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:25
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:26 The amino acid sequence of or consisting of said sequence; or,
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:27
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:28
  • HCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:28.
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:31
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:32 The amino acid sequence of or consisting of said sequence.
  • the second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises SEQ ID NO: 3, 5 or 7.
  • the amino acid sequence shown or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 3, 5 or 7 %, 98% or 99% sequence identity of an amino acid sequence or consisting of said amino acid sequence.
  • the second antigen-binding region comprises a light chain variable region VL, wherein the VL comprises SEQ ID NO: 4, 6 or 8
  • the VH contains the amino acid sequence shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence, and the VL includes the amino acid sequence shown in SEQ ID NO:4 or is identical to SEQ ID NO:4 An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or consisting of an amino acid sequence composition;
  • the VH includes the amino acid sequence shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence
  • the VL includes the amino acid sequence shown in SEQ ID NO: 6 or is identical to SEQ ID NO: 6
  • the VH includes the amino acid sequence shown in SEQ ID NO:7 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence
  • the VL includes the amino acid sequence shown in SEQ ID NO:8 or is identical to SEQ ID NO:8 An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or consisting of an amino acid sequence composition.
  • the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise the amino acid sequences shown below Or consisting of the amino acid sequence:
  • the first antigen-binding region comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2 and LCDR3 of the light chain variable region VL ,in
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:1; and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:2
  • the CDR is determined using the Kabat scheme.
  • HCDR1 of the first antigen-binding region comprises or consists of the amino acid sequence of SEQ ID NO: 9; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10 Composed; HCDR3 includes or consists of the amino acid sequence of SEQ ID NO:11; and LCDR1 of the first antigen-binding region includes or consists of the amino acid sequence of SEQ ID NO:12; LCDR2 includes SEQ ID NO:13 The amino acid sequence of or consisting of the sequence; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 14 or consists of the sequence.
  • the first antigen-binding region comprises a heavy chain variable Region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1 or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94% identical to the amino acid sequence of SEQ ID NO: 1 , 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences.
  • the first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 2 or is identical to SEQ ID NO.
  • the amino acid sequence of NO: 2 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the amino acid sequence or consists of The amino acid sequence consists of.
  • the VL includes the amino acid sequence shown in SEQ ID NO:2 or has at least 80%, 85%, 90%, 91% of the amino acid sequence with the amino acid sequence of SEQ ID NO:2 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences.
  • the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise the amino acid sequences shown below Or consisting of the amino acid sequences: SEQ ID NO: 1 and SEQ ID NO: 2.
  • HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:9;
  • HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 10;
  • HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 11;
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 12;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13;
  • LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 14;
  • the second antigen-binding region specifically binds B7H3, which includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL, where
  • HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 15;
  • HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 16;
  • HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 17;
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 18;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19;
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:20;
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 21;
  • HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 22;
  • HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 23;
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 24;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25;
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26; or
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 27;
  • HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 28;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 29;
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:30;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:31;
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:32.
  • the first antigen-binding region comprises a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 1 and a VH containing a VH shown in SEQ ID NO: 2
  • the amino acid sequence or VL consisting of the amino acid sequence, and the second antigen-binding region includes VH and VL respectively containing the amino acid sequence shown below or consisting of the amino acid sequence:
  • the bispecific antibody of embodiment 17, comprising a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.
  • first Fc region and the second Fc region are respectively human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc,
  • an amino acid sequence comprising or consisting of an amino acid sequence SEQ ID NO: 46 or 47 or having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity thereto.
  • One Fc region polypeptide includes or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide includes or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53;
  • An Fc region polypeptide contains an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 49 or 50, and Another Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 52 or 53 amino acid sequence; or
  • An Fc region polypeptide contains at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 49 or 50
  • the amino acid sequence of SEQ ID NO: 52 or 53 contains the mutations Y349T, K370S and K409D, and the other Fc region contains at least 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, 99% identity and containing mutations S364R and D399K.
  • One Fc region polypeptide contains the mutation T366W, and the other Fc region polypeptide contains T366S, L368A and Y407V (numbering follows the EU index), or
  • One Fc region contains the amino acid substitutions S354C and T366W, and the other Fc region contains the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index).
  • the bispecific antibody of any one of embodiments 1-25, wherein the first and/or second antigen-binding region (eg, the heavy chain variable region thereof) can also be combined with 1 or 2 heavy chains.
  • a constant region e.g., a heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4 comprising a CH1 and an Fc region, linked with or without a hinge region, e.g., a heavy chain variable region
  • the C-terminus is connected to the N-terminus of CH1 of the heavy chain constant region.
  • the bispecific antibody of embodiments 1-27, wherein the first and/or second antigen-binding region (e.g., the light chain variable region thereof) can also be linked to a light chain constant region, e.g., the light chain variable region
  • the C-terminus of the region is linked to the N-terminus of the light chain constant region.
  • the bispecific antibody of embodiment 20, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:54 or is at least 80%, 85%, or 90% identical to the amino acid sequence of SEQ ID NO:54. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to or consisting of an amino acid sequence.
  • bispecific antibody of embodiment 31 which comprises heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein heavy chain 1 and light chain 1 constitute the first half-antibody, and heavy chain 2 and light chain 2 constitute the second half-antibody; among which
  • Heavy chain 1 includes the heavy chain variable region of the first antigen binding region and the first heavy chain constant region; light chain 1 includes the light chain variable region of the first antigen binding region and the first light chain constant region; and heavy chain 2
  • the heavy chain variable region includes the second antigen-binding region and the second heavy chain constant region; the light chain 2 includes the light chain variable region and the second light chain constant region of the second antigen-binding region.
  • bispecific antibody of embodiment 32, wherein heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33 or has at least 80%, 85%, 90%, or 91% similarity with the amino acid sequence of SEQ ID NO:33. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences.
  • the bispecific antibody of embodiment 32 or 33, wherein the light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34 or has at least 80%, 85% or 90% of the amino acid sequence of SEQ ID NO: 34 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences.
  • heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33 or has at least 80% or 85% similarity with the amino acid sequence of SEQ ID NO:33. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to or consisting of an amino acid sequence
  • light chain 1 comprises SEQ The amino acid sequence shown in ID NO:34 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence of SEQ ID NO:34 , 98% or 99% identical amino acid sequence or consisting of said amino acid sequence.
  • the bispecific antibody of any one of embodiments 32-35, wherein the heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 35, 37 or 39 or is identical to the amino acid sequence shown in SEQ ID NO: 35, 37 or 39.
  • the bispecific antibody of any one of embodiments 32-36, wherein the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:36, 38 or 40 or is identical to the amino acid sequence shown in SEQ ID NO:36, 38 or 40.
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:35 or shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with the amino acid sequence shown in SEQ ID NO:35 %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of the amino acid sequence; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO: 36 or is identical to SEQ ID NO: The amino acid sequence shown in 36 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the amino acid sequence or is composed of The amino acid sequence consists of;
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:37 or is identical to the amino acid sequence shown in SEQ ID NO:37 An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence or consisting of said amino acid sequence Sequence composition; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO:38 or has at least 80%, 85%, 90%, 91%, 92%, 93%, An amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical or consists of said amino acid sequence;
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 39 or shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with the amino acid sequence shown in SEQ ID NO: 39 %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of the amino acid sequence; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO: 40 or is identical to SEQ ID NO: The amino acid sequence shown in 40 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the amino acid sequence or is composed of The amino acid sequence consists of.
  • Heavy chain 1 includes the amino acid sequence shown in SEQ ID NO:33 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence that is 96%, 97%, 98% or 99% identical or consists of said amino acid sequence, and the light chain 1 includes the amino acid sequence shown in SEQ ID NO: 34 or has the same amino acid sequence as SEQ ID NO: 34 or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; and Heavy chain 2 and light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise at least 85%, 90%, 95%, 96%, 97%, 98%, 99 with the amino acid sequence shown. % identity of the amino acid sequence, or consisting of the sequence shown in the following SEQ ID NO:
  • Heavy chain 1 includes or consists of a heavy chain with the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes or consists of the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:35, and light chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:36; or,
  • Heavy chain 1 includes or consists of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes or consists of the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:37, and light chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:38; or,
  • Heavy chain 1 includes or consists of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes or consists of the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:39, and light chain 2 includes or consists of the amino acid sequence shown in SEQ ID NO:40.
  • the antibody can block the binding of EGFR ligand to EGFR, inhibit biological signal transmission, and block the corresponding biological activity of tumors; on the other hand, it can stimulate EGFR endocytosis and eventually be degraded by intracellular lysosomes;
  • the antibody uses an EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the toxic side effects of a series of EGFR monoclonal antibodies on normal epithelial tissues such as skin;
  • the antibody quotes the high-affinity antibody parent sequence of B7-H3 on the basis of low affinity for EGER, which greatly improves the EGFR signal blocking activity and improves the pharmacological biological activity and efficacy of the bispecific antibody of the present invention. security window;
  • the antibody is a low-fucosylated antibody
  • the antibody has high pharmacodynamic biological activity and safety
  • the antibody has excellent tumor killing and inhibitory effects
  • the antibody has excellent ADCC pharmacodynamic activity in vivo and in vitro;
  • Vector comprising the nucleic acid of embodiment 42, preferably the vector is an expression vector, preferably the expression vector is pcDNA, such as pcDNA3.1.
  • Host cell comprising the nucleic acid of embodiment 42 or the vector of embodiment 43.
  • the host cell is prokaryotic or eukaryotic, more preferably yeast cells or mammalian cells (such as 293 cells or CHO cells , such as 293F cells or 293T cells or CHO-S cells).
  • the host cell of embodiment 44 which is glycoengineered to express an RMD enzyme.
  • the host cell is a CHO cell.
  • the host cell of embodiment 45 comprising a nucleic acid encoding an RMD enzyme.
  • RMD enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence with at least 90% identity thereto, preferably, the RMD
  • the enzyme is from Pseudomonas aeruginosa.
  • An immunoconjugate comprising the bispecific antibody of any one of the preceding embodiments 1-41 conjugated to a therapeutic or diagnostic agent.
  • composition comprising the bispecific antibody of any one of the aforementioned embodiments 1-41 or the immunoconjugate of embodiment 49, and optionally pharmaceutical excipients.
  • the pharmaceutical composition of embodiment 50 further comprising a second therapeutic agent; preferably, the second therapeutic agent is selected from the group consisting of anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, and anti-infectious active agents. , small molecule drugs or immune modulators (such as activators of costimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.
  • KRAS G12C inhibitors such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • a pharmaceutical combination product comprising the bispecific antibody of any one of embodiments 1-41 or the immunoconjugate of embodiment 49 or the pharmaceutical composition of embodiment 50, and one or more second therapeutic agents,
  • the second therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infectious active agents, small molecule drugs or immune modulators (such as activators of costimulatory molecules or immune modulators).
  • the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors agent.
  • KRAS G12C inhibitors such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitors agent KRAS small molecule inhibitors
  • a method of preventing or treating tumors or infectious diseases in a subject comprising administering to the subject an effective amount of the bispecific antibody of any one of embodiments 1-41, or The immunoconjugate of embodiment 49, or the pharmaceutical composition of embodiment 50.
  • the method of embodiment 53 further comprising co-administering to the subject one or more other therapies, for example, including a therapeutic modality and/or other therapeutic agents, preferably, the therapeutic modality Including surgical treatment and/or radiotherapy, or the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infectious agents, small molecule drugs or immunomodulators (such as costimulatory molecules activator or inhibitor of immune checkpoint molecules); preferably, the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133 ) or KRAS G12S inhibitor.
  • KRAS G12C inhibitors such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitor such as MRTX1133
  • a method of preventing or treating tumors or infectious diseases in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 51 or the pharmaceutical combination of embodiment 52.
  • the tumor is a cancer, such as a solid tumor or a hematological tumor, including cancers of epithelial origin, such as gastrointestinal tumors or lung tumors or skin tumors, e.g. Skin cancer (e.g. cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma), esophageal cancer (e.g. esophageal squamous cell carcinoma), bowel cancer (e.g. colon cancer, rectal cancer, colorectal cancer) or lung cancer (e.g. non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma).
  • Skin cancer e.g. cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma
  • esophageal cancer e.g. esophageal squamous cell carcinoma
  • bowel cancer e.g. colon cancer, rectal cancer, colorectal cancer
  • the mutated EGFR comprises one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (such as S768_D770dup), preferably, the The above-mentioned mutated EGFR includes R521K/Y1069C, R521K, L858R/T790M/C797S, Del19/T790M/C797S or S768_D770dup;
  • wild-type KRAS e.g., having elevated nucleic acid or protein levels of wild-type KRAS
  • a mutated KRAS preferably, the mutated KRAS comprises a G12 or G13 mutation, such as G12D or G12C;
  • B7-H3 with elevated nucleic acid levels or protein levels compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects;
  • the tumor cells are resistant to tyrosine kinase inhibitors, such as first generation (Erlotinib) and third generation (Osimertinib), such as resistance to osimertinib.
  • tyrosine kinase inhibitors such as first generation (Erlotinib) and third generation (Osimertinib), such as resistance to osimertinib.
  • a method for detecting the antigen EGFR and/or B7-H3 in a sample comprising
  • the antibody or antigen-binding fragment thereof according to any one of embodiments 1-41, and/or the isolated nucleic acid according to embodiment 42, and/or the vector according to embodiment 43, and/or embodiment 44
  • the invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any and all antibodies or fragments thereof or immunoconjugates or pharmaceutical compositions or combination products or kits, methods and uses of the invention described herein.
  • Figure 1 shows a schematic structural diagram of the anti-B7-H3/EGFR bispecific antibody of the present invention.
  • Figure 2 shows a graph of the screening proliferation inhibition experiment of the anti-B7-H3/EGFR bispecific antibody of the present invention.
  • Figure 3 shows the screening ADCC experiment diagram of the anti-B7-H3/EGFR bispecific antibody of the present invention.
  • Figure 4 shows the expression detection chart of B7H3 and EGFR in different cell lines of the Hz20G5.26/Zalu bsAb of the present invention.
  • Figure 5 shows the experimental results of the proliferation inhibition of Hz20G5.26/Zalu bsAb of the present invention in CRC tumor cell lines CCK-81, NCI-H508, HT-55 and LS180, in which the antibody is diluted as follows:
  • CCK-81, NCI-H508, HT-55 The final concentration of antibody dilution is up to 125nM, 4 times dilution;
  • LS180 The final concentration of antibody dilution is up to 300nM, 4 times dilution.
  • Figure 6 shows the experimental results of Hz20G5.26/Zalu bsAb of the present invention inhibiting proliferation in HNSCC tumor cell lines TE-1 and Colo680, in which the antibody is diluted as follows: TE-1, Colo680: the highest final concentration of antibody dilution is 300nM ,3.16 times dilution.
  • Figure 7.1 shows the experimental results of the proliferation inhibition of Hz20G5.26/Zalu bsAb of the present invention in different NSCLC-EGFR WT tumor cell lines;
  • Figure 7.2 shows the experimental results of the proliferation inhibition of Hz20G5.26/Zalu bsAb of the present invention in NSCLC-EGFR classic mutation tumor cell lines;
  • Figure 7.3 shows the experimental results of the proliferation inhibition of Hz20G5.26/Zalu bsAb of the present invention in NSCLC-EGFR abnormally amplified tumor cell lines;
  • Figure 7.4 shows the experimental results of the proliferation inhibition of Hz20G5.26/Zalu bsAb of the present invention in rare NSCLC-EGFR mutant tumor cell lines;
  • Figure 7.5 shows the comparative experimental results of the efficacy of the Hz20G5.26/Zalu bsAb of the present invention combined with gp120/Zalu and gp120/hz20G5.26;
  • the antibody or small molecule TKI (Osimertinim or Erlotinib) is diluted as follows:
  • Figure 7.1 NCI-292, NCI-H322: The highest final concentration of antibody dilution is 300nM, 3.16 times dilution;
  • Figure 7.3 NCI-H1703, SK-MES-1: Antibody molecules, the final dilution concentration is up to 300nM, 3.16-fold dilution; small molecule TKI (Osimertinim and Erlotinib), the final dilution concentration is up to 1000nM, 3.16-fold dilution;
  • Figure 7.5 LS180 and H292: final antibody dilution concentration up to 300nM, 4-fold dilution; SK-MES-1: final concentration up to 300nM, 3.16-fold dilution.
  • Figure 8 shows the comparative experimental results of the combined synergistic drug use of the Hz20G5.26/Zalu bsAb of the present invention and the AMG510 small molecule inhibitor in the H358 NSCLC cell line, wherein
  • AMG510 MedChem Expresss, HY-114277
  • AMG510+bsAb@10nm means adding fixed 10nM Hz20G5.26/Zalu bsAb to each well , and at the same time, AMG510 was serially diluted in each well (final concentration, 1000nM, 4-fold dilution); only AMG510: the final concentration was up to 1000nM, 4-fold dilution; only Hz20G5.26/Zalu bsAb: the final concentration was up to 10nM, 5-fold Double dilution; a total of 5 gradients were diluted.
  • Figure 9 shows the interaction between Hz20G5.26/Zalu bsAb of the invention and KRAS in the LS180 CRC cell line.
  • the G12D small molecule inhibitor MRTX1133 combined with synergistic medication comparative experimental results chart, among which
  • Figure 10 shows the experimental results of the EGFR phosphorylation signal blocking effect of the Hz20G5.26/Zalu bsAb of the present invention in the H358 cell line.
  • Figure 11 shows the experimental results of the blocking effect of Hz20G5.26/Zalu bsAb of the present invention on the EGFR phosphorylation signal induced by EGFR ligand (EGF or TGF- ⁇ ) in H358 cell line.
  • Figure 12.1 shows the results of the ADCC reporting experiment of Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-EGFR WT tumor cell line; in the three cell lines, the highest final dilution concentration of the antibody molecule was 6.25nM, 4-fold dilution;
  • Figure 12.2 shows the results of the ADCC reporting experiment of the Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-EGFR mutant tumor cell line; in the H1975 cell line, the final dilution concentration of the antibody molecule is up to 10 nM, 4-fold dilution; and in In the H1975 cell line, the final dilution concentration of the antibody molecule is up to 6.25nM, a 4-fold dilution;
  • Figure 12.3 shows the results of the ADCC reporting experiment of Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-EGFR abnormally amplified tumor cell line; in the SK-MES-1 cell line, the highest final concentration of antibody molecule dilution is 6.25nM , 4-fold dilution; and in the H1703 cell line, the final dilution concentration of the antibody molecule is up to 100nM, 4-fold dilution;
  • Figure 12.4 shows the results of the ADCC reporting experiment of Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-KRAS mutant tumor cell line; in the H358 cell line, the final dilution concentration of the antibody molecule is up to 25nM, 4-fold dilution ( Figure 12.4 (A)); and in the H358 cell line, the final dilution concentration of the antibody molecule is up to 1.56nM, 4-fold dilution ( Figure 12.4(B));
  • Figure 13.1 shows the results of the huPBMC ADCC reporting experiment of the Hz20G5.26/ZalubsAb of the present invention in the NSCLC-EGFR wild-type tumor cell line; wherein,
  • Figure 13.2 shows the results of the huPBMC ADCC reporting experiment of the Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-EGFR mutant tumor cell line; in the H1975 cell line, the highest final dilution concentration of the antibody molecule is 35nM, 4-fold dilution; In the H1650 cell line, the final concentration of antibody molecule dilution is up to 100nM, 4-fold dilution;
  • Figure 13.3 shows the Hz20G5.26/Zalu bsAb of the present invention in NSCLC-EGFR abnormal amplification type Picture of huPBMC ADCC reporting experiment results in tumor cell lines;
  • Figure 13.4 shows the results of the huPBMC ADCC reporting experiment of the Hz20G5.26/Zalu bsAb of the present invention in the NSCLC-KRAS mutant tumor cell line.
  • the final dilution concentration of the antibody molecule was up to 35nM, 4 times. dilution.
  • Figure 14 shows the results of in vitro research experiments of the Hz20G5.26/Zalu bsAb drug of the present invention to simulate skin toxicity, in which the antibody molecules were diluted using cell culture medium. In two human skin cell lines respectively, the final dilution concentration of the antibody molecules was up to 300nM. , 3.16 times dilution.
  • Figure 15A shows the experimental results of the anti-tumor effect of Hz20G5.26/Zalu bsAb of the present invention on NCI-H292 tumor-bearing mice
  • Figure 15B shows the effect of Hz20G5.26/Zalu bsAb of the present invention on NCI-H292 tumor-bearing mice.
  • Figure 15C shows the experimental result graph of the effect of Hz20G5.26/Zalu bsAb of the present invention on the body weight of NCI-H292 tumor-bearing mice.
  • Figure 16A shows the experimental results of the anti-tumor effect of Hz20G5.26/Zalu bsAb of the present invention on SK-MES-1 tumor-bearing mice
  • Figure 16B shows the effect of Hz20G5.26/Zalu bsAb of the present invention on SK-MES-1 tumor-bearing mice.
  • Experimental results showing the effect on body weight of MES-1 tumor-bearing mice.
  • Figure 17A shows the experimental results of the anti-tumor effect of Hz20G5.26/Zalu bsAb of the present invention on SK-MES-1 tumor-bearing mice
  • Figure 17B shows the effect of Hz20G5.26/Zalu bsAb of the present invention on SK-MES-1 tumor-bearing mice.
  • Experimental results showing the effect on body weight of MES-1 tumor-bearing mice.
  • Figure 18A shows the experimental results of the combination of Hz20G5.26/Zalu bsAb and AMG510 of the present invention on the anti-tumor effect of NCI-H358 tumor-bearing mice
  • Figure 18B shows the effect of the combination of Hz20G5.26/Zalu bsAb and AMG510 of the present invention on the anti-tumor effect of NCI-H358 tumor-bearing mice.
  • Experimental results showing the effect of NCI-H358 tumor-bearing mice on body weight.
  • the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
  • the term “comprises” or “includes” is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated.
  • reference is made to an antibody variable region that "comprises” a particular sequence it is also intended to encompass antibody variable regions that consist of that particular sequence.
  • amino acid positions of all variable regions of the heavy and light chains are as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) Kabat numbering system numbers and are referred to herein as "Kabat numbers”.
  • EU numbering when referring to an amino acid position in a domain of an antibody other than a variable region (e.g., a constant region, e.g., an Fc region), according to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed.
  • the EU numbering system is described in Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to herein as "EU numbering".
  • position numbers and/or amino acid residues are assigned to a particular antibody isotype, they are intended to apply to the corresponding positions and/or amino acid residues of any other antibody isotype, as is known to those skilled in the art.
  • antibody is used in the broadest sense herein to refer to proteins containing antigen-binding sites, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.
  • the terms “whole antibody”, “full-length antibody”, “complete antibody” and “intact antibody” are used interchangeably herein to refer to a naturally occurring antibody containing at least two heavy chains (H) interconnected by disulfide bonds and two light chains (L) glycoproteins.
  • Each heavy chain consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDR)), with more conservative regions (called framework regions (FR)) interposed.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FR composition, from amino terminal to carboxyl terminal as follows In the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • Hyf-antibody or “semipolymer” refers to a monovalent antigen-binding polypeptide.
  • a half-antibody or half-polymer comprises VH/VL units and optionally at least a portion of an immunoglobulin constant domain.
  • a half-antibody or half-polymer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain, or an antigen-binding fragment thereof.
  • a half-antibody or half-polymer is monospecific, ie, binds to a single antigen or epitope. In some specific embodiments, the half-antibody binds to EGFR and does not bind to B7-H3.
  • the half-antibody binds to B7-H3 and does not bind to EGFR.
  • a half-antibody may have an antigen-binding domain consisting of a single variable domain (eg, derived from the genus Camelidae).
  • antigen-binding fragment of an antibody is a molecule that is distinct from a full-length antibody and contains a portion of the full-length antibody, but which is capable of binding to the antigen of the full-length antibody or to the full-length antibody (i.e., to the full-length fragment from which the antigen-binding fragment is derived).
  • Antibodies compete for binding to antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single domain antibody (sdAb), and Nanobody.
  • Fab fragment or “Fab” is used interchangeably herein to refer to an immunoglobulin heavy chain variable region VH, a heavy chain constant domain CH1, and a light chain variable region consisting of two polypeptide chains.
  • An immunoglobulin fragment of region VL and light chain constant domain CL wherein one polypeptide chain from N-terminus to C-terminus contains VH and a constant region selected from CH1 and CL, and the other polypeptide chain contains from N-terminus to C-terminus VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site.
  • Fab' fragments differ from Fab fragments by the addition of some residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation for Fab' in which the cysteine residue of the constant domain carries a free thiol group.
  • F(ab') 2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.
  • the term "bispecific antibody” encompasses an antigen-binding domain that specifically binds two antigens or two epitopes. Unless otherwise stated, the order in which the bispecific antibody names are listed for the antigens bound by the bispecific antibody is arbitrary. That is, in some embodiments, the terms “anti-EGFR/B7-H3 bispecific antibody” and “anti-B7-H3/EGFR bispecific antibody” may be used interchangeably.
  • the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region and a single light chain variable region and optionally At least a portion of the light chain constant region.
  • the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than a single heavy chain variable region and does not comprise More than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to the first antigen/epitope and does not bind to the second antigen and the second half-antibody binds to the second antigen/epitope and does not bind to the first antigen.
  • Multispecific antibodies of the invention may contain linkers.
  • linker refers to any molecule that enables direct connection of different portions of a multispecific antibody. Examples of linkers that establish covalent connections between different portions of multispecific antibodies include peptide linkers and non-protein polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or polyethylene glycol, Copolymer of polypropylene glycol.
  • PEG polyethylene glycol
  • peptide linker refers to a sequence of amino acids, wherein said sequence will be multispecific The amino acid sequences of the various parts of the antibody are linked together.
  • the peptide linker is of a length sufficient to connect the two entities in a manner such that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the peptide linker may or may not consist primarily of the following amino acid residues: Gly, Ser, Ala or Thr.
  • Useful linkers include glycine-serine polymers including, for example, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n and (GGGGS)nG, where n is at least 1 (and preferably 2, 3, 4 , 5, 6, 7, 8, 9, 10) integers.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • Fc domain or "Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain.
  • the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of antibodies of the IgG, IgA, and IgD classes; or From the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the two heavy chains of IgM and IgE class antibodies.
  • CH2 domain and CH3 domain constant domains
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, e.g., Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, Numbering is carried out according to the EU numbering system described in 1991 (also called the EU Index). However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimeric Fc, and two different Fc regions heterodimerize to form a heterodimeric Fc.
  • the terms "Fc region”, “Fc portion” and “dimeric Fc (e.g. heterodimeric Fc)” exclude heavy chain variable regions VH and light chain variable regions VL as well as heavy chain constants of immunoglobulins. region CH1 and the light chain constant region CL, but in some cases may include a hinge region N-terminal to the heavy chain constant region.
  • the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxy terminus of the heavy chain.
  • the Fc region is a human Fc region.
  • the Fc region belongs to the human IgG4 subclass.
  • the Fc region belongs to the human IgG1 subclass.
  • Fc dimerization refers to the dimerization of two Fc regions to form a dimer.
  • Fc heterodimerization refers to the dimerization of two different Fc regions to form a dimer.
  • the heterodimerized Fc region constitutes the Fc scaffold in bispecific or multispecific antibodies. Therefore, a “heterodimeric Fc scaffold” refers to a scaffold that contains two different Fc regions or is formed by the dimerization of two different Fc regions, which can be connected to an antigen-binding domain at its N-terminus or C-terminus.
  • the heavy chain and/or light chain variable region of an antibody or an antigen-binding fragment of an antibody that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule used to constitute a multispecific Antibodies such as bispecific antibodies.
  • Amino acid mutations are represented by (original amino acid, amino acid position, mutated amino acid).
  • T366W means that the T at position 366 in EU numbering is replaced by W.
  • R521K/Y1069C means containing both mutations R521K and Y1069C. It should be noted that when describing a mutation, the specific position also encompasses its corresponding amino acid position on other polypeptide chains.
  • the original amino acid at a specific position may be the amino acid described, or it may be another amino acid at the corresponding position.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”) area.
  • CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are often referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable region of the antibody heavy chain are termed HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable region of the antibody light chain are termed LCDR1, LCDR2 and LCDR3.
  • each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any one or a combination of many well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al.
  • the residues of each CDR are as follows.
  • CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • residue positions in an antibody variable region refers to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDRs of the antibodies of the invention are bounded by Kabat's rules, or by AbM's rules, or by a combination thereof.
  • the HCDR1 of the VH in the antigen-binding region that binds to B7-H3 in the present invention is determined by the AbM rule, and HCDR2 and HCDR3 are determined by the Kabat rule, The CDR of VL is determined by Kabat rules.
  • the CDRs of VH and VL in the antigen-binding region that binds to EGFR in the present invention are determined by Kabat's rule.
  • hinge region refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, e.g., the IgG1 hinge region, e.g., the sequence D221 to P230 according to EU numbering.
  • the hinge regions of other IgG subclasses can be determined by alignment with hinge region cysteine residues of the IgG1 subclass sequence.
  • binding site or "antigen binding site” or “antigen binding region” as used herein refers to any part of an antibody molecule, such as a multispecific antibody, such as a bispecific antibody, that binds a specific target or antigen.
  • the antigen-binding region may be, for example, the antibody or immunoglobulin itself or an antibody fragment. This antigen-binding region may or may not have a tertiary structure independent of the remainder of the BsAB, and may or may not bind its antigen/epitope as a separate entity.
  • the antigen binding region includes a VH/VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), a heavy chain variable region derived from a camelid heavy chain antibody.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • v-NAR camelized human VH domain
  • humanized camelid antibody heavy chain variable region VH/VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), a heavy chain variable region derived from a camelid heavy chain antibody.
  • first antigen-binding region in a multispecific antibody or a bispecific antibody, it refers to the binding region that binds the first antigen and is not intended to limit the number of such antigen-binding regions contained in the antibody.
  • a multispecific antibody or a bispecific antibody may contain one or more first antigen-binding regions.
  • a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.
  • the antigen-binding region is derived from an antibody
  • the binding domain constituting the antigen-binding region is or is derived from the binding domain of the antibody that specifically binds the antigen, e.g.
  • a fragment such as a Fab is or is derived from a corresponding fragment of the antibody such as a Fab, or the heavy chain variable region and/or the light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and/or light chain variable region of the antibody.
  • the chain variable region, or 1, 2, 3, 4, 5 or 6 CDRs of the antigen-binding region are the CDRs of the antibody.
  • the antigen-binding region is an antigen-binding fragment of an antibody.
  • multispecific antibody refers to an antibody having at least two antigen-binding sites, each of the at least two antigen-binding sites being associated with a different epitope of the same antigen or with a different Different epitopes of the antigen bind.
  • Antibodies provided herein are typically multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies with binding specificities for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies having binding specificities for a first antigen and a second antigen.
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • IgG class immunoglobulins are approximately 150,000 dalton heterotetrameric glycoproteins composed of two disulfide-bonded light chains and two heavy chains. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL).
  • VH heavy chain variable region
  • CL light chain constant domain
  • the heavy chains of immunoglobulins can be assigned to one of five classes, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subdivisions. Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chains of immunoglobulins can be divided into two types based on the amino acid sequence of their constant domains. One of the types, called ⁇ and ⁇ . Immunoglobulins basically consist of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.
  • effector function refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with immunoglobulin isotype.
  • immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.
  • valency antibody refers to the number of antigen-binding sites present in the antibody molecule.
  • Bivalent, trivalent and tetravalent antibodies refer to the presence of 2, 3 and 4 antigen-binding sites respectively in the antibody molecule.
  • binding means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions.
  • the ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR) assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD assay biofilm thin layer interferometry
  • SPR surface plasmon resonance
  • Affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • knock-in-hole mutations or “knob-in-hole” mutations are used herein to refer to the introduction of mutations in the first Fc polypeptide and the second Fc polypeptide, respectively, using the "knob-in-hole” technique to create mutations in the first Fc polypeptide.
  • Protrusions (“knobs") and complementary holes (“holes") are formed at the interface of the polypeptide with the interface of the second Fc polypeptide.
  • "knot-in-button” technology can modify the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule.
  • the technique involves introducing "bumps/knots" at the interface of one chain and corresponding "cavities/buckles” at the interface of the other chain to be paired with, such that the bumps can be placed in the cavities.
  • One preferred interface contains the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to which it is intended to pair.
  • Bumps can be constructed by replacing small 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 tyrosine or tryptophan.
  • the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired is constructed to be identical or similar to the bulge by replacing the large amino acid side chain with a smaller one (such as alanine or threonine) Compensatory holes of different sizes.
  • Another optional interface is the Fab fragment described above, which contains the CL domain of the light chain and the CH1 domain of the heavy chain, which promotes correct interaction between the two chains of the Fab fragment by building a bulge-hole interaction. Heterodimerization.
  • the antibody constant region or antibody constant domain can be selected based on the expected function of the antibody molecule.
  • the constant region may be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of a human IgG, e.g. a constant domain of a human IgG1, IgG2, IgG3 or IgG4, preferably a constant structure of a human IgG1 area.
  • a Fab fragment of an antibody may comprise the CH and CL constant regions from IgGl.
  • the Fc region of an antibody may comprise CH2 and CH3 domains from IgGl. Immunoglobulin constant regions may have native or variant sequences.
  • antigen refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • the first antigen and the second antigen are two different antigens.
  • tumor-associated antigen or “cancer antigen” interchangeably refers to a molecule (usually a protein, carbohydrate or lipid) that is preferably expressed entirely or as fragments (e.g., MHC/peptides) on the surface of cancer cells compared to normal cells. substances), and the molecules can be used in the preferential targeting of cancer cells by pharmaceutical agents.
  • immune checkpoint molecule refers to a class of inhibitory signaling molecules present in the immune system that avoid tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens.
  • cytokine is a general term for proteins released by one cell population that act as intercellular mediators on another cell.
  • an “immunoconjugate” is an antibody conjugated to one or more other substances, including, but not limited to, cytotoxic agents or labels.
  • EGFR refers to epidermal growth factor receptor, a tyrosine kinase receptor, a giant transmembrane glycoprotein with a molecular weight of approximately 170 KDa and a member of the ErbB receptor family , is the most common cancer driver gene in NSCLC.
  • the EGFR activating mutation region mainly occurs in the tyrosine kinase domain of EGFR exons 18-21.
  • the binding region of EGFR antibodies is mainly located in the EGFR extracellular ligand domain, which can avoid resistance.
  • EGFR antibodies can inhibit the growth of tumor cells by inhibiting the binding of EGFR and ligands, and can also use their own specific ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells, so that It can jointly exert anti-tumor killing effects through multiple mechanisms of action.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • B7-H3 is a type I transmembrane glycoprotein that is a member of the B7/CD28 superfamily and is similar in sequence to the extracellular domain of PD-L1.
  • B7-H3 has 316 amino acids, including a putative signal peptide of 28 amino acids, an extracellular region of 217 amino acids, a transmembrane region and a cytoplasmic domain of 45 amino acids, with a molecular weight of approximately is 45-66kDa.
  • B7-H3 In humans, due to exon duplication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3), or an IgV-IgC-IgV-IgC-like domain (4Ig-B7- H3).
  • the sequence of cynomolgus B7-H3 is approximately 90% homologous to its human counterpart.
  • Antibody effector functions refer to those biological activities that are attributable to the Fc region of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g., B cell receptors) body) downregulation; and B cell activation.
  • GlymaxX technology is a technology platform developed from ProBioGen to increase the effect of dual-antibody ADCC. It is a CHO host cell that stably overexpresses bacterial RMD protein and ultimately blocks the Fc region by interfering with the formation of the substrate GDP-fucose. Fucose modification to enhance ADCC effect (WO2011035884A1).
  • the term "effective amount” refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention that produces the desired effect in a patient in need of treatment or prophylaxis when administered to the patient in single or multiple doses.
  • the effective amount can be easily determined by the attending physician, who is a person skilled in the art, by considering the following various factors: Determine: the species such as the mammal; its size, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; Selected dosing regimen; and use of any concomitant therapies.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • the therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof can vary depending on factors such as the disease state, the age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit the desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment, or conjugate or composition thereof, are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject.
  • % and still more preferably at least about 80%.
  • the ability of a compound to inhibit a measurable parameter can be evaluated in animal model systems that are predictive of efficacy in human tumors. Alternatively, this property of the composition may be assessed by testing the ability of the compound to inhibit in vitro by assays known to the skilled artisan.
  • prophylactically effective amount means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. p. 91 (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • VH or VL domains from antibodies that bind a specific antigen can be used to isolate antibodies that bind that antigen to screen libraries for complementary VL or VH domains, respectively.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cells.
  • a host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancers suitable for treatment by the antibodies of the invention include cancers of epithelial origin, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma), esophageal cancer (e.g.,: Esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma), including metastatic forms of those cancers.
  • skin cancer e.g., cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma
  • esophageal cancer e.g.,: Esophageal squamous cell carcinoma
  • intestinal cancer e.g., colon cancer, rectal cancer, colorectal cancer
  • lung cancer e.g., non-small cell lung cancer, lung s
  • tumor refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or Benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or Benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or Benign, and all pre-cancerous and cancerous cells and tissues.
  • infectious disease refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a fungal infection, or an infection by protozoa such as a parasite.
  • chronic infection refers to an infection in which an infectious agent (e.g., a pathogen such as a virus, a bacterium, a protozoa such as a parasite, a fungus, or the like) has induced an immune response in the infected host but has not yet induced an immune response in the infected host as in an acute infection. are removed or eliminated from the host during the same process.
  • infectious agent e.g., a pathogen such as a virus, a bacterium, a protozoa such as a parasite, a fungus, or the like
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent (such as a polynucleotide probe or antibody) and facilitates detection of the agent to which it is conjugated or fused.
  • the label itself may be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a detectable chemical change in the substrate compound or composition.
  • the term is intended to encompass direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody is one that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, e.g., electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC) confirmed.
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed phase HPLC
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but that are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
  • sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a first and a second amino acid sequence or nucleic acid sequence may be aligned for optimal alignment). Introducing gaps in one or both may allow non-homologous sequences to be discarded for comparison purposes).
  • the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • the GAP program from the GCG software package determine the percent identity between two nucleotide sequences using the NWSgapdna.CMP matrix with a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using the E. Meyers and W.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • composition refers to a composition that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain additional ingredients that would be unacceptable toxicities to the subject to whom the composition is administered. ingredients.
  • pharmaceutically acceptable excipient refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), carriers, excipients or stabilizers, etc., which are administered with the active substance.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.
  • the antibody molecules of the invention are used to delay disease progression or to slow the progression of disease.
  • prevention includes the inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventive regimen.
  • prevention refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • an effective amount refers to an amount or dose of an antibody or fragment or composition or combination of the invention that produces the desired effect in a patient in need of treatment or prophylaxis when administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject %, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • therapeutic agent encompasses any substance that is effective in preventing or treating disease, such as tumors (e.g., cancer) and infections (e.g., chronic infections), including anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, Other antibodies, anti-infectious agents, small molecule drugs or immunomodulators.
  • tumors e.g., cancer
  • infections e.g., chronic infections
  • anti-angiogenic agents e.g., chemotherapeutic agents, cytotoxic agents, vaccines, Other antibodies, anti-infectious agents, small molecule drugs or immunomodulators.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of cancer, including, but not limited to, antineoplastic agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; antiestrogens ; anti-androgens; and non-steroidal anti-androgens, etc.
  • immunomodulator refers to natural or synthetic agents or drugs that inhibit or modulate immune responses.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immune checkpoint molecule inhibitors and costimulatory molecule activators.
  • small molecule drugs refers to low molecular weight organic compounds capable of modulating biological processes.
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes; chemotherapeutics or drugs; growth inhibitors; enzymes and their fragments such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatic activities of bacterial, fungal, plant or animal origin. Toxins, including fragments and/or variants thereof.
  • Tumor immune escape refers to the process by which tumors escape immune recognition and clearance.
  • tumor immunity is “cured” when such evasion is attenuated, and tumors are recognized and attacked by the immune system.
  • Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • Immunogenicity refers to the ability of a specific substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps clear tumor cells through immune responses.
  • agonist activity of an antibody refers to the ability of an antibody to activate the biological activity of the antigen to which it binds.
  • Anti-angiogenic agent refers to a compound that blocks or interferes to some extent with the development of blood vessels.
  • Anti-angiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis.
  • drug combination or combination product refers to non-fixed combination products or fixed combination products, including but not limited to pharmaceutical kits and pharmaceutical compositions.
  • non-fixed combination means that the active ingredients (e.g., (i) the immunoconjugates of the invention, and (ii) other therapeutic agents) are administered in separate entities simultaneously, without specific time limits, or at the same or different times. Administration to the patient is spaced, sequentially, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient.
  • fixed combination means that two or more active agents are administered simultaneously to a patient in the form of a single entity.
  • the dosages and/or time intervals of two or more active agents are preferably selected so that the combined use of the components produces an effect in treating the disease or condition that is greater than that achieved by either component alone.
  • Each component may be in a separate formulation form, and the formulation forms may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents to treat cancer or infection as described in this disclosure.
  • Such administration involves co-administration of the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of the active ingredients.
  • such administration involves co-administration or separate or sequential administration of the individual active ingredients in several or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage prior to administration.
  • administering also includes administering each type of therapeutic agent at approximately the same time, or at different times in a sequential manner. In either case, the treatment regimen will provide for the beneficial effects of the drug combination in treating the disorder or condition described herein.
  • vector when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample may be solid tissue, like from fresh, frozen and/or preserved organ or tissue samples or biopsy or aspiration samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development.
  • Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.
  • the present invention provides a novel antibody molecule that can be used for immunotherapy, prevention and/or diagnosis of various diseases.
  • the antibody molecule of the present invention contains at least 2, 3 or 4 antigen-binding regions and can function as a bispecific antibody or a multispecific antibody. Preferably, it can function as a bispecific antibody.
  • the bispecific or multispecific antibodies of the invention comprise a first binding specificity for EGFR and a second binding specificity for B7H3, and optionally additional binding specificities.
  • one aspect of the invention relates to a bispecific antibody comprising
  • a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region specifically binds to EGFR, and/or the second antigen-binding region specifically binds to B7-H3.
  • Bispecific antibodies of the invention can be prepared using bispecific antibody formats or techniques known in the art. Specific exemplary bispecific forms that may be used in the context of the present invention are found, for example, Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8): 1-24.
  • the bispecific antibody format comprises an IgG-like antibody (Fan et al. (2015) Journal of Hematology & Oncology. 8:130).
  • the most common type of IgG-like antibody contains two Fab regions and two Fc regions, and the heavy and light chains of each Fab can be derived from separate monoclonal antibodies.
  • the bispecific antibodies of the invention are IgG-like bispecific antibodies that comprise a Fab fragment that specifically binds EGFR as one antigen-binding region and a Fab fragment that specifically binds B7H3 as the other antigen-binding region.
  • Antigen-binding region that specifically binds EGFR
  • the antigen-binding region that specifically binds EGFR is derived from an antibody that specifically binds EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.
  • the antigen-binding region that specifically binds to EGFR comprises 1, 2, 3, 4, 5, or 6 of known antibodies that specifically bind to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody. CDR.
  • the antigen-binding region that specifically binds EGFR includes 1, 2, and 3 heavy chain variable region CDRs of a known antibody that specifically binds EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody, Namely HCDR1, HCDR2 and HCDR3.
  • the antigen-binding region that specifically binds to EGFR includes the 1, 2, and 3 light chain variable region CDRs of a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody, Namely LCDR1, LCDR2 and LCDR3.
  • the antigen-binding region that specifically binds to EGFR includes a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as the 3 heavy chain variable region CDRs and 3 light chain of Zalutumumab monoclonal antibody. Variable region CDR.
  • the antigen-binding region that specifically binds to EGFR comprises a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as the heavy chain variable region and the light chain variable region of Zalutumumab monoclonal antibody.
  • the antigen-binding region that specifically binds to EGFR includes a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as the Fab of Zalutumumab monoclonal antibody.
  • the antigen-binding region that specifically binds EGFR includes three complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds EGFR includes three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds EGFR includes 3 complementarity-determining regions (HCDR) from the heavy chain variable region and 3 complementarity-determining regions (LCDR) from the light chain variable region.
  • the antigen-binding region that specifically binds EGFR comprises a heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds EGFR comprises a light chain variable region (VH). In some aspects, the antigen-binding region that specifically binds EGFR includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • the heavy chain variable region of the antigen-binding region that specifically binds EGFR that specifically binds EGFR
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:1 , more preferably amino acid conservative substitution) amino acid sequence consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.
  • the light chain variable region that specifically binds the antigen-binding region of EGFR is the light chain variable region that specifically binds the antigen-binding region of EGFR
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2 , more preferably amino acid conservative substitution) amino acid sequence consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.
  • three of the antigen-binding regions that specifically bind to EGFR are from the complementarity determining regions (HCDR) of the heavy chain variable region, HCDR1, HCDR2, and HCDR3 selected from
  • the three HCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the three complementarity determining regions (LCDRs) of the antigen-binding region that specifically bind to EGFR are from the light chain variable region, LCDR1, LCDR2, and LCDR3 are selected from
  • the three LCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the antigen-binding region that specifically binds EGFR comprises three complementarity determining regions (HCDRs) contained in the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 and three complementarity determining regions (HCDR) consisting of the amino acid sequence of SEQ ID NO:
  • HCDR complementarity determining regions
  • the light chain variable region consisting of 2 amino acid sequences contains a complementarity determining region (LCDR).
  • the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:9, or the HCDR1 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:9 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, or HCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 10 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11, or the HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 11 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:12, or LCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:12 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, or LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 13 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or LCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 14 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • the antigen-binding region that specifically binds EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
  • the antigen-binding region that specifically binds EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.
  • the antigen-binding region that specifically binds EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein
  • HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:9;
  • HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 10;
  • HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 11;
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 12;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13;
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:14.
  • the antigen-binding region that specifically binds to EGFR includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO:11 HCDR3; LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13 and LCDR3 as shown in SEQ ID NO:14.
  • the antigen-binding region that specifically binds EGFR comprises VH and VL, wherein
  • the VH contains the amino acid sequence shown in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of the amino acid sequence
  • the VL contains the amino acid sequence shown in SEQ ID NO: 2 or has at least 80%, 85%, 90%, 91%, 92%, or consist of an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the antigen-binding region that specifically binds to EGFR comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO:2.
  • Antigen-binding region that specifically binds B7-H3
  • the antigen-binding region that specifically binds B7-H3 is derived from an antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5 ( For example, Hz20G5.26) mentioned in the embodiment of the present invention.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. 1, 2, 3, 4, 5, or 6 CDRs.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. 1, 2 and 3 heavy chain variable region CDRs, namely HCDR1, HCDR2 and HCDR3.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. 1, 2 and 3 light chain variable region CDRs, namely LCDR1, LCDR2 and LCDR3.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. 3 heavy chain variable region CDRs and 3 light chain variable region CDRs.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. heavy chain variable region and light chain variable region.
  • the antigen-binding region that specifically binds B7-H3 includes a known antibody that specifically binds B7-H3, such as the B7-H3 antibody described in PCT/CN2021/140449, such as the monoclonal antibody numbered Hz20G5. Fab.
  • the antigen-binding region that specifically binds B7-H3 contains three complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds B7-H3 includes three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds B7-H3 includes 3 complementarity-determining regions (HCDR) from the heavy chain variable region and 3 complementarity-determining regions (LCDR) from the light chain variable region.
  • the antigen-binding region that specifically binds B7-H3 comprises a heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds B7-H3 comprises a light chain variable region (VH). In some aspects, the antigen-binding region that specifically binds B7-H3 includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 3, 5 or 7
  • the amino acid sequence preferably amino acid substitution, more preferably amino acid conservative substitution
  • the amino acid change does not occur in the CDR region.
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 4, 6 or 8
  • the amino acid sequence preferably amino acid substitution, more preferably amino acid conservative substitution
  • the amino acid change does not occur in the CDR region.
  • the three complementarity determining regions (HCDR) of the heavy chain variable region that specifically bind the antigen-binding region of B7-H3, HCDR1, HCDR2, and HCDR3 are selected from
  • the three HCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the HCDR1 adopts the Abm scheme
  • the HCDR2 and HCDR3 are determined by the Kabat scheme.
  • three complementarity determining regions (LCDRs) from the light chain variable region that specifically bind the antigen-binding region of B7-H3, LCDR1, LCDR2, and LCDR3 are selected from
  • the three LCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the antigen-binding region that specifically binds B7-H3 comprises three complementarity determining regions (HCDRs) contained in the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, 5 or 7 and 3 A complementarity determining region (LCDR) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, 6 or 8.
  • HCDRs complementarity determining regions
  • LCDR complementarity determining region
  • HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, 21 or 27, or HCDR1 comprises an amino acid sequence identical to SEQ ID NO: 15, 21 or 27.
  • the amino acid sequence has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence.
  • HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, 22, or 28, or HCDR2 comprises, compared to the amino acid sequence of SEQ ID NO: 16, 22, or 28, one, Amino acid sequence with two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, 23, or 29, or the HCDR3 comprises, compared to the amino acid sequence of SEQ ID NO: 17, 23, or 29, one, Amino acid sequence with two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, 24 or 30, or LCDR1 has an amino acid sequence of SEQ ID NO: 18, 24 or 30, Amino acid sequence with two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 19, 25, or 31, or LCDR2 comprises an amino acid sequence that has one, Amino acid sequence with two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, 26 or 32, or LCDR3 comprises an amino acid sequence compared to SEQ ID NO: 20, 26 or 32 that has one, Amino acid sequence with two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the antigen-binding region that specifically binds B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
  • the antigen-binding region that specifically binds B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.
  • the antigen-binding region that specifically binds B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:15
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:16
  • HCDR3 includes SEQ ID NO: The amino acid sequence shown in 17 or consisting of said sequence
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:18
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:19
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:20 The amino acid sequence of or consisting of said sequence; or,
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:21
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:22
  • HCDR3 includes SEQ ID NO: The amino acid sequence shown in 23 or consisting of said sequence
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:24
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:25
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:26 The amino acid sequence of or consisting of said sequence; or,
  • HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:27
  • HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:28
  • HCDR3 includes SEQ ID NO: The amino acid sequence shown in 29 or consisting of said sequence
  • LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO:31
  • LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO:32 The amino acid sequence of or consisting of said sequence.
  • the antigen-binding region that specifically binds B7-H3 comprises
  • HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, HCDR3 as shown in SEQ ID NO:17; LCDR1 as shown in SEQ ID NO:18, as shown in SEQ ID LCDR2 shown in NO:19 and LCDR3 shown in SEQ ID NO:20;
  • HCDR1 as shown in SEQ ID NO:21, HCDR2 as shown in SEQ ID NO:22, HCDR3 as shown in SEQ ID NO:23; LCDR1 as shown in SEQ ID NO:24, as shown in SEQ ID LCDR2 shown in NO:25 and LCDR3 shown in SEQ ID NO:26; or
  • HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29; LCDR1 as shown in SEQ ID NO:30, as shown in SEQ ID LCDR2 shown in NO:31 and LCDR3 shown in SEQ ID NO:32.
  • the antigen-binding region that specifically binds B7-H3 includes VH and VL, wherein
  • the VH contains the amino acid sequence shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence, and the VL includes the amino acid sequence shown in SEQ ID NO:4 or is identical to SEQ ID NO:4 An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or consisting of an amino acid sequence composition;
  • the VH includes the amino acid sequence shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence
  • the VL includes the amino acid sequence shown in SEQ ID NO: 6 or is identical to SEQ ID NO: 6
  • the VH comprises the amino acid sequence shown in SEQ ID NO:7 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical or consists of the amino acid sequence, and the VL includes the amino acid sequence shown in SEQ ID NO:8 or is identical to SEQ ID NO:8 Amino acid sequences having at least 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity Or consisting of the amino acid sequence.
  • the antigen-binding region that specifically binds B7-H3 includes VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequence shown below:
  • the present invention also relates to an antibody that specifically binds B7-H3 and includes the above-mentioned antigen-binding region that specifically binds to B7-H3, such as an antibody that includes HCDR1, HCDR2, and HCDR3 as defined in the present invention, as well as LCDR1, LCDR2, and LCDR3; or includes an antibody that specifically binds B7-H3.
  • the antibody molecules of the invention such as multispecific antibodies (eg, bispecific antibodies), further comprise an Fc region, wherein the included Fc regions may be the same or different.
  • the Fc region has hypofucosylation, such as that obtained after treatment with GlymaxX technology.
  • an antibody molecule of the invention comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.
  • the first Fc region and the second Fc region are different in that they are capable of dimerization to form a heterodimeric Fc scaffold.
  • an Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include native sequence Fc regions and variant Fc regions.
  • the natural sequence F region covers various naturally occurring immunoglobulin Fc sequences, such as various Ig subtypes and their allotype Fc regions (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389/fimmu.2014.00520).
  • the Fc region of the invention comprises antibodies CH2 and CH3.
  • the antibody Fc region may also have an IgG hinge region or part of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a part of an IgG1 hinge region, for example, the sequence from D221 to P230 according to EU numbering. Mutations may be contained in the hinge region.
  • the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.
  • the Fc region comprises the amino acid sequence SEQ ID NO: 46 or 47 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity thereto or consisting of The amino acid sequence composition.
  • the Fc region contained in the multispecific antibody of the present invention may contain a heterodimer that facilitates the heterodimerization of the first Fc region and the second Fc region.
  • the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) is no longer able to homodimerize with itself but is forced to Complementarily engineered other CH3 regions to heterodimerize (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimers are formed between the two first CH3 regions or the two second CH3 regions) ).
  • Knob-in-Hole technology corresponding Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region.
  • This technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); while in the CH3 region of the other Fc region
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation)
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the threonine residue at position 407 Tyrosine residues were replaced with valine residues (Y407V) (numbering according to EU index).
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue substitution (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue); while in another In the CH3 region of the Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) And the leucine residue at position 368 is replaced with an alanine residue (L368A) (the numbering method is in accordance with the EU index), and optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C
  • one Fc region contains the amino acid substitution T366W and the other Fc region contains the amino acid substitutions T366S, L368A and Y407V (numbering according to the EU index).
  • one Fc region contains the amino acid substitutions S354C and T366W, and the other Fc region contains the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index).
  • Corresponding mutations can also be introduced into the first Fc region and the second Fc region based on Innobody technology. See for example PCT/CN2021/143141 for this technology.
  • the first CH3 region contains the S364R/K mutation (preferably S364R), and optionally one or more other mutations.
  • the second CH3 region comprises the K370S/T/A/V mutation (preferably K370S), and optionally one or more other mutations.
  • the first CH3 region includes the S364R/K mutation and the second CH3 region includes the K370S/T/A/V mutation.
  • the first CH3 region includes the S364R mutation and the second CH3 region includes the K370S mutation.
  • the first CH3 region comprises S364R/K (preferably S364R) and D399K/R (preferably D399K) mutations.
  • the second CH3 region comprises K370S/T/A/V (preferably K370S) mutations and K409D/E (preferably K409D) mutations.
  • the first CH3 region includes S364R/K+D399K/R and the second CH3 region includes K370S/T/A/V+Y349T/S/A/V.
  • the first CH3 region comprises S364R +D399K, and the second CH3 region contains K370S+Y349T.
  • the first CH3 region further comprises E375N/Q (preferably E375N) and/or T350V/A (preferably T350V).
  • the second CH3 region further comprises K409D/E (preferably K409D), Q347D/E (preferably Q347D), and/or T350V/A (preferably T350V).
  • the first CH3 region includes S364R+D399K and the second CH3 region includes K370S+Y349T+K409D.
  • the first CH3 region further comprises E357N.
  • the second CH3 region further comprises Q347D.
  • the first CH3 region further comprises E357N and the second CH3 region further comprises Q347D.
  • the first CH3 region and the second CH3 region each further comprise T350V, or both comprise T350V.
  • the first CH3 region includes S364R+D399K and the second CH3 region includes K370S+Y349T+K409D+Q347D.
  • the first CH3 region includes S364R+D399K+E357N and the second CH3 region includes K370S+Y349T+K409D+Q347D.
  • the first CH3 region includes S364R+D399K+E357N+T350V and the second CH3 region includes K370S+Y349T+K409D+Q347D+T350V.
  • the first CH3 region comprises K409E/D (preferably K409E).
  • the second CH3 region comprises D399K/R (preferably D399K) or K370T/S/A/V (preferably K370T).
  • the first CH3 region comprises K409E/D (preferably K409E) and the second CH3 region comprises D399K/R (preferably D399K).
  • the first CH3 region further comprises T411R/K (preferably T411R).
  • the second CH3 region further comprises K370T/S/A/V (preferably K370T).
  • the CH3 region includes K409E/D+T411R/K and the second CH3 region includes D399K/R+K370T/S/A/V. In some embodiments, the CH3 region includes K409E+T411R and the second CH3 region includes D399K+K370T.
  • the first and second CH3 regions have the following combination of mutations:
  • the CH3 mutations of one Fc region comprise the S364R and D399K mutations and the CH3 mutations of the other Fc region comprise the Y349T, K370S and K409D mutations.
  • the multispecific antibodies of the invention comprise two Fc regions that heterodimerize, wherein
  • One Fc region polypeptide contains mutation T366W and the other Fc region polypeptide contains T366S, L368A and Y407V, or
  • Fc-region polypeptide contains mutations S354C and T366W, and the other Fc-region polypeptide contains mutations Y349C, T366S, L368A and Y407V, or
  • Fc-region polypeptide contains mutations S364R and D399K, while the other Fc-region polypeptide contains mutations Y349T, K370S and K409D.
  • the multispecific antibody of the invention comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50 , and another Fc region polypeptide includes or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53.
  • the multispecific antibody of the invention comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 49 or 50. %, 90%, 95%, 96%, 97%, 98%, 99% identity, and another Fc region polypeptide contains at least 85% of the amino acid sequence shown in SEQ ID NO: 52 or 53 , 90%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the multispecific antibody of the invention comprises two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 49 or 50. %, 90%, 95%, 96%, 97%, 98%, 99% identical amino acid sequence and contains mutations Y349T, K370S and K409D, while another Fc region contains the same amino acid sequence as SEQ ID NO: 52 or 53
  • the amino acid sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence and contains the mutations S364R and D399K.
  • the Fc region also contains other mutations that facilitate heterodimer purification.
  • the amino acid changes described herein include substitutions, insertions or deletions of amino acids.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described in the present invention occur in regions outside the CDRs (eg in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • substitutions are conservative substitutions.
  • a conservative substitution is when one amino acid is replaced by another amino acid within the same class, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid Displacement. Exemplary substitutions are shown in Table A below:
  • the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation.
  • Antibodies can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structure.
  • fucosylation in the constant region eg, Fc region
  • the constant region eg, Fc region
  • Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies.
  • carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation system.
  • afucosylated antibodies are expressed in ⁇ -1,6-fucosyltransferase 8 (Fut8) knockout host cells (WO2000061739).
  • Fut8 ⁇ -1,6-fucosyltransferase 8
  • low or salt-free fucosylated antibodies are obtained by introducing host cells encoding enzymes RMD related to sugar chain modification (GlymaxX technology, ProBioGen AG, see patent publication number: WO2011035884A1).
  • fucosidase enzymes can be used to cleave fucosyl residues from antibodies; for example, fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. 1975) Biochem. 14:5516-23).
  • the anti-B7-H3/EGFR bispecific antibodies of the invention have one or more of the following properties:
  • the bispecific antibody of the present invention can block the binding of EGFR ligands to EGFR, inhibit biological signal transmission, and block the corresponding biological activity of tumors; on the other hand, it can stimulate EGFR endocytosis and eventually be lysed by intracellular enzymes. Degradation in the body;
  • the bispecific antibody of the present invention uses an EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the toxic side effects of a series of EGFR monoclonal antibodies on normal epithelial tissues such as skin;
  • the bispecific antibody of the present invention uses the high-affinity antibody parent sequence of B7-H3 on the basis of low affinity for EGER, which greatly improves the EGFR signal blocking activity and improves the efficacy of the bispecific antibody of the present invention.
  • the bispecific antibody of the present invention is a low-fucosylated antibody
  • the bispecific antibody of the present invention has high pharmacodynamic biological activity and safety
  • the bispecific antibody of the present invention has excellent tumor killing and inhibitory effects
  • the bispecific antibody of the present invention has excellent ADCC pharmacodynamic activity in vivo and in vitro;
  • the combination of the bispecific antibody of the present invention and a KRAS small molecule inhibitor has excellent anti-tumor effect, especially a synergistic effect.
  • the antigen-binding region that specifically binds EGFR is linked to the heavy chain constant region CH, for example, the heavy chain variable region thereof is linked to the heavy chain constant region CH, for example, the heavy chain constant region CH
  • the C-terminus of the chain variable region is linked to the N-terminus of the CH constant region of the heavy chain.
  • the antigen-binding region that specifically binds EGFR is linked to the light chain constant region, for example, the light chain variable region thereof is linked to the light chain constant region CL, for example, the light chain wherein The C-terminus of the variable region is linked to the N-terminus of the light chain constant region CL.
  • the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL.
  • the antigen-binding region that specifically binds B7-H3 is linked to the heavy chain constant region CH, for example, where the heavy chain variable region is linked to the heavy chain constant region CH, for example, where The C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region CH.
  • the antigen-binding region that specifically binds B7-H3 is connected to the light chain constant region, for example, the light chain variable region thereof is connected to the light chain constant region CL, such as where The C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL.
  • the heavy chain variable region in the antigen-binding region that specifically binds B7-H3, is connected to the heavy chain constant region CH and the light chain variable region is connected to the light chain constant region CL. connect.
  • the heavy chain constant region comprises a CH1 and an Fc region, linked with or without a hinge region.
  • the heavy chain constant region is an IgGl, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgGl, human IgG2, human IgG3, or human IgG4 heavy chain constant region.
  • the CH1 comprises the amino acid sequence shown in SEQ ID NO:42 or is identical to the amino acid sequence of SEQ ID NO:42. A sequence having or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. .
  • the light chain constant region is a kappa light chain constant region or a lambda light chain constant region, such as a human kappa or human lambda light chain constant region.
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 54 or is at least 80%, 85%, 90%, 91%, 92%, or identical to the amino acid sequence of SEQ ID NO: 54. or consist of an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the invention provides a bispecific antibody molecule comprising a Fab fragment that specifically binds EGFR, a Fab fragment that specifically binds B7-H3, and an Fc dimer, wherein specifically The Fab fragment that specifically binds to EGFR and an Fc form a half-antibody that specifically binds to EGFR, and the Fab fragment that specifically binds to B7-H3 and an Fc form a half-antibody that specifically binds to B7-H3.
  • the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.
  • the bispecific antibody comprises heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein heavy chain 1 and light chain 1 constitute the first half-antibody, and heavy chain 2 and Light chain 2 constitutes the second half-antibody; wherein heavy chain 1 includes the heavy chain variable region of the first antigen-binding region and the first heavy chain constant region; light chain 1 includes the light chain variable region of the first antigen-binding region and the first heavy chain constant region. a light chain constant region; and heavy chain 2 includes a heavy chain variable region of a second antigen-binding region and a second heavy chain constant region; light chain 2 includes a light chain variable region of a second antigen-binding region and a second light chain constant region.
  • heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33 or has at least 80% or 85% of the amino acid sequence of SEQ ID NO: 33. or consist of an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34 or is at least 90%, 91%, 92%, or identical to the amino acid sequence of SEQ ID NO: 34. or consist of an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33 or is at least 90%, 91%, 92%, or identical to the amino acid sequence of SEQ ID NO: 33.
  • the light chain 1 includes the amino acid sequence shown in SEQ ID NO: 34 or is identical to
  • the amino acid sequence of SEQ ID NO:34 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence or consists of said amino acid sequence composition.
  • heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 35, 37 or 39 or has the same amino acid sequence as SEQ ID NO: 35, 37 or 39. or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 36, 38 or 40 or is identical to the amino acid sequence shown in SEQ ID NO: 36, 38 or 40.
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 35 or shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence shown in SEQ ID NO: 35 %, 97%, 98% or 99% identity of the amino acid sequence or consisting of the amino acid sequence; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO: 36 or is identical to the amino acid sequence shown in SEQ ID NO: 36 having or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical;
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:37 or shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence shown in SEQ ID NO:37 %, 97%, 98% or 99% identity of the amino acid sequence or consisting of the amino acid sequence; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO: 38 or is identical to the amino acid sequence shown in SEQ ID NO: 38 having or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical;
  • Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 39 or shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence shown in SEQ ID NO: 39 %, 97%, 98% or 99% identical amino acid sequence or consisting of the amino acid sequence; and the light chain 2 includes the amino acid sequence shown in SEQ ID NO: 40 or is identical to the amino acid sequence shown in SEQ ID NO: 40 Be or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • Heavy chain 1 includes the amino acid sequence shown in SEQ ID NO:33 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence that is 98% or 99% identical or consists of the amino acid sequence
  • the light chain 1 includes the amino acid sequence shown in SEQ ID NO: 34 or has at least 90% or 91% of the amino acid sequence of SEQ ID NO: 34 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences; and heavy chain 2 and light chain 2 respectively comprise the following SEQ ID NO:
  • the sequence shown in ID NO consists of:
  • Heavy chain 1 includes a heavy chain with the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes the amino acid sequence shown in SEQ ID NO:35, and light chain 2 includes the amino acid sequence shown in SEQ ID NO:36; or,
  • Heavy chain 1 includes the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes the amino acid sequence shown in SEQ ID NO:37, and light chain 2 includes the amino acid sequence shown in SEQ ID NO:38; or,
  • Heavy chain 1 includes the amino acid sequence shown in SEQ ID NO:33, and light chain 1 includes the amino acid sequence shown in SEQ ID NO:34,
  • Heavy chain 2 includes the amino acid sequence shown in SEQ ID NO:39, and light chain 2 includes the amino acid sequence shown in SEQ ID NO:40.
  • the invention also encompasses antibodies conjugated to other substances. Accordingly, the present invention relates to immunoconjugates comprising antibodies conjugated to other substances.
  • immunoconjugate comprising antibodies conjugated to other substances.
  • other substances such as therapeutic agents or labels, such as cytotoxic agents, immunomodulatory agents (eg, immune agonists), or chemotherapeutic agents.
  • Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (eg, chemotherapeutic agents) suitable for forming immunoconjugates are known in the art.
  • cytotoxic agents include, but are not limited to: radioactive isotopes; growth inhibitors; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including their Fragments and/or variants; and various known anti-tumor or anti-cancer agents.
  • the antibody molecules of the invention can be conjugated to labeling sequences (eg, peptides) to facilitate purification.
  • the antibody molecules of the invention are conjugated to a diagnostic or detectable agent.
  • a diagnostic or detectable agent may be used to monitor or predict the onset, development, progression and/or severity of a disease or condition as part of a clinical assay (eg, to determine the efficacy of a particular therapy).
  • diagnosis and detection can be accomplished by conjugating antibodies to detectable substances, including but not limited to various enzymes; prosthetic groups; fluorescent substances; luminescent substances; radioactive substances; and for various positron emission Positron-emitting metal and nonradioactive paramagnetic metal ions in imaging.
  • the antibody molecules of the invention can be conjugated to therapeutic or drug moieties that modulate a given biological response.
  • the therapeutic or drug portion includes, but is not limited to, classic chemotherapeutic drugs.
  • the drug moiety may be a protein, peptide or polypeptide possessing the desired biological activity.
  • antibody molecules of the invention can be conjugated to therapeutic moieties such as radioactive metal ions.
  • Antibodies can also be linked to solid supports which are particularly useful for immunoassays or purification of target antigens.
  • the immunoconjugates are used to prevent or treat diseases, such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • diseases such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • the disease is a tumor (such as cancer) or an infection.
  • the tumor is immune evasive.
  • the tumor is a cancer of epithelial origin, such as a gastrointestinal tract tumor or a lung tumor or a skin tumor, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal cancer) squamous carcinoma), intestinal cancer (eg colon cancer, rectal cancer, colorectal cancer) or lung cancer (eg non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma).
  • the infection is a chronic infection.
  • the infection is, e.g., bacterial infection, viral infection infections, fungal infections, protozoal infections, etc.
  • the invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any chain thereof.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising said nucleic acid or said vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for preparation of antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 3-8 and 33-40, or encoding an amino acid sequence selected from any one of SEQ ID NO: 3-8 and 33-40.
  • each antibody or polypeptide amino acid sequence may be encoded by multiple nucleic acid sequences because of codon degeneracy.
  • Nucleic acid sequences encoding molecules of the invention can be generated using methods well known in the art, such as by de novo solid-phase DNA synthesis, or by PCR amplification.
  • the invention provides nucleic acids encoding any of the above antibodies or any antibody chain.
  • the polypeptide encoded by the nucleic acid is capable of displaying human EGFR/or B7-H3 antigen binding ability.
  • the invention provides nucleic acids encoding any of the above bispecific antibodies.
  • the polypeptide encoded by the nucleic acid is capable of displaying human EGFR/or B7-H3 antigen binding ability.
  • the nucleic acids encoding each chain of the bispecific antibody can be in the same vector or in different vectors.
  • nucleic acids encoding each chain of a bispecific antibody can be introduced into the same or different host cells for expression.
  • a method for producing a bispecific antibody of the invention includes the steps of culturing a host cell comprising a nucleic acid encoding each chain of the molecule under conditions suitable for expression of each chain of the molecule, Bispecific antibodies of the invention are produced.
  • one or more vectors comprising the nucleic acids are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • the vector is, for example, a pcDNA vector, such as pcDNA3.1.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion cells are grown in culture medium and screened for appropriate activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present description and methods known from the prior art, depending on the specific expression vector used and Mammalian host cell changes or optimizations.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.
  • a host cell comprising one or more polynucleotides of the invention.
  • host cells comprising expression vectors of the invention are provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells such as CHO cells (eg CHO-S, such as ExpiCHO-S) or 293 cells (eg 293F or HEK293 cells)) or is suitable for the preparation of antibodies or fragments thereof of other cells.
  • the host cell is prokaryotic, such as a bacterium, such as E. coli.
  • Suitable host cells include prokaryotic microorganisms such as E. coli.
  • the host cell can also be a eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells. Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered to be suitable for suspension growth may be used.
  • Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOK1SV cells, CHOK1SV GS-KO cells, CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc.
  • the host cell is a CHO cell, such as a CHOS cell CHOK1SV cell or CHOK1SV GS-KO, or the host cell is a 293 cell, such as a HEK293 cell.
  • the host cell is a CHO cell.
  • the host cell of the invention is a glycosylated host cell, preferably a glycosylated CHO cell.
  • the host cell is engineered to express an RMD enzyme.
  • the host cell comprises a nucleic acid encoding an RMD enzyme.
  • the RMD enzyme comprises the amino acid sequence shown in SEQ ID NO: 41 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical thereto. % or 99% identity of or consisting of the amino acid sequence.
  • the RMD enzyme is from Pseudomonas aeruginosa.
  • a host cell of the invention comprises a nucleic acid encoding one or more or all chains of an antibody molecule of the invention and a nucleic acid encoding an RMD enzyme.
  • the invention provides a method for preparing an antibody molecule of the invention, wherein said method comprises culturing said host cell under conditions suitable for expression of a nucleic acid encoding an antibody molecule of the invention, and optionally isolating said antibody .
  • the method further includes recovering the antibody molecule of the invention from the host cell.
  • a method for preparing an antibody molecule of the present invention includes culturing a compound encoding the antibody (eg, any one polypeptide chain and/or multiple polypeptide chains) under conditions suitable for antibody expression. a host cell of a nucleic acid or an expression vector comprising said nucleic acid, as provided above, and optionally recovering said antibody from said host cell (or host cell culture medium).
  • a compound encoding the antibody eg, any one polypeptide chain and/or multiple polypeptide chains
  • the nucleic acid encoding the antibody (such as the antibody described above, such as any one polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for use in a host. cells for further cloning and/or expression.
  • Such nucleic acids are easy to isolate and sequence using routine protocols (For example, by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).
  • Antibody molecules prepared as described herein can be purified by known state-of-the-art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the antibody molecules provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the antibodies of the invention are tested for their antigen-binding activity, for example by known methods such as ELISA, Western blotting, etc. Binding to the bound antigen can be determined using methods known in the art, exemplary methods are disclosed herein, such as biofilm layer interference techniques and SPR.
  • the invention also provides assays for identifying biologically active antibodies.
  • Biological activities may include, for example, binding to antigens, binding to cell surface antigens, inhibition or activation of antigens, etc.
  • Antibodies having such biological activity in vivo and/or in vitro are also provided.
  • the antibodies of the invention are tested for such biological activity.
  • the invention also provides methods for identifying properties of antibodies, such as properties relevant to druggability.
  • druggability-related properties include, for example, thermal stability, such as long-term thermal stability.
  • Cells for use in any of the above in vitro assays include cell lines that naturally express the antigen or that are engineered to express the antigen. Such cells also include cell lines that express the antigen and cell lines that are transfected with DNA encoding the antigen that does not normally express the antigen.
  • the antigen is EGFR (eg, human EGFR) and/or B7-H3 (eg, human B7-H3).
  • the invention provides a composition comprising any antibody molecule described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises an antibody molecule of the invention, or a fragment thereof, or an immunoconjugate thereof, in combination with one or more other therapeutic agents.
  • the other therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cells Cytotoxic agents, vaccines, anti-infectious agents, small molecule drugs or immune modulators (such as activators of costimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug,
  • the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (eg AMG510 (Sotorasib) or GFH925), KRAS G12D (eg MRTX1133) or KRAS G12S inhibitors.
  • the compositions are used to prevent or treat diseases, such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • diseases such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • the disease is a tumor (such as cancer) or an infection.
  • the tumor is immune evasive.
  • the tumor is a cancer of epithelial origin, such as a gastrointestinal tract tumor or a lung tumor or a skin tumor, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal cancer) squamous carcinoma), intestinal cancer (eg colon cancer, rectal cancer, colorectal cancer) or lung cancer (eg non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma).
  • the infection is a chronic infection.
  • the infection is, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, or the like.
  • compositions comprising the antibodies of the invention or immunoconjugates thereof and/or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding the antibodies of the invention.
  • compositions comprise one or more antibodies of the invention, or fragments thereof, or one or more polynucleotides encoding one or more antibodies of the invention, or fragments thereof.
  • compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Pharmaceutical carriers suitable for use in the present invention 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, etc. When administering pharmaceutical compositions intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerin , propylene, glycol, water, ethanol, etc.
  • excipients see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.
  • Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • compositions of the present invention may be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • dispersions or suspensions e.g., liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.
  • the preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (ip), intramuscular) injection.
  • the antibody molecule is administered by intravenous infusion or injection.
  • the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • Antibodies containing the compounds described herein can be prepared by mixing the antibodies of the invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)).
  • the pharmaceutical preparation of the antibody is preferably in the form of a freeze-dried preparation or an aqueous solution.
  • compositions or preparations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • the other therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infectious agents, small molecule drugs, or immunomodulators (e.g., activators of costimulatory molecules or immunomodulators). Inhibitors of checkpoint molecules);
  • the second therapeutic agent is a small molecule drug.
  • the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.
  • KRAS G12C inhibitors such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitors such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitors such as MRTX113
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include a semipermeable matrix of a solid hydrophobic polymer containing the antibody in the form of a shaped article, such as a film or microcapsules.
  • compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the antibody molecule) in the required amount in an appropriate solvent, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other ingredients. Coating agents such as lecithin can be used.
  • proper fluidity of the solution can be maintained by using surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • Kits containing the antibody molecules described herein are also within the scope of the invention.
  • a kit may contain one or more other elements, including, for example, a packaging insert; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject.
  • the invention also provides a combination product comprising an antibody of the invention, or an antigen-binding fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents.
  • the other therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infectious agents, small molecule drugs, or immunomodulators (e.g., activators of costimulatory molecules or immunomodulators). Inhibitors of checkpoint molecules);
  • the second therapeutic agent is a small molecule drug.
  • the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.
  • KRAS G12C inhibitors such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitors such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925
  • KRAS G12D such as MRTX1133
  • KRAS G12S inhibitors such as MRTX113
  • the combination product is used to prevent or treat diseases, such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • diseases such as acute and chronic inflammatory diseases, infections (eg, chronic infections), tumors, and the like.
  • the disease is a tumor (such as cancer) or an infection.
  • the tumor is immune evasive.
  • the tumor is a cancer of epithelial origin, such as a gastrointestinal tract tumor or a lung tumor or a skin tumor, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancer such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal cancer) squamous carcinoma), intestinal cancer (eg colon cancer, rectal cancer, colorectal cancer) or lung cancer (eg non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma).
  • the infection is chronic.
  • the infection is, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, or the like.
  • two or more components of the combination may be administered to the subject sequentially, separately, or simultaneously.
  • kits comprising the antibodies, pharmaceutical compositions, immunoconjugates or combinations of the invention, and optionally a package insert to guide administration.
  • the invention also provides pharmaceutical products comprising the antibodies, pharmaceutical compositions, immunoconjugates, combinations of the invention, optionally further comprising a package insert to guide administration.
  • the invention in another aspect, relates to a method of preventing or treating tumors (e.g., cancer) in a subject, comprising administering to said subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein. compound or combination product or kit.
  • tumors in subjects of the invention include solid tumors and hematological tumors.
  • the tumor is immune evasive.
  • the tumor is cancer.
  • the tumor cells of the tumor have one of the following characteristics compared to normal cells of the same tissue adjacent to the same subject or normal cells of adjacent normal tissue, or compared to normal cells of the same tissue of a healthy subject.
  • One or more characteristics compared to normal cells of the same tissue adjacent to the same subject or normal cells of adjacent normal tissue, or compared to normal cells of the same tissue of a healthy subject.
  • the mutated EGFR comprises a mutation selected from the group consisting of R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion ( Del19), one or more mutations in Exon20ins (such as S768_D770dup), preferably, the mutated EGFR includes R521K/Y1069C, R521K, L858R/T790M/C797S, Del19/T790M/C797S or S768_D770dup;
  • wild-type KRAS e.g., wild-type KRAS with elevated nucleic acid or protein levels
  • the mutated KRAS includes a G12 or G13 mutation, such as G12D or G12C;
  • B7-H3 with elevated nucleic acid levels or protein levels compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects;
  • the tumor cells are resistant to tyrosine kinase inhibitors, such as first generation (Erlotinib) and third generation (Osimertinib), such as resistance to osimertinib.
  • tyrosine kinase inhibitors such as first generation (Erlotinib) and third generation (Osimertinib), such as resistance to osimertinib.
  • the invention in another aspect, relates to a method of preventing or treating an infectious disease in a subject, said method comprising administering to said subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein or combination products or kits.
  • the infectious disease is a chronic infection.
  • the subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., patients who have or are at risk of suffering from a disease described herein).
  • a mammal such as a primate, preferably a higher primate, such as a human
  • the subject suffers from or is at risk of suffering from a disease described herein (eg, a tumor or an infection or an autoimmune disease as described herein).
  • the subject receives or has received other treatments, such as chemotherapy treatments and/or radiation therapy.
  • the present invention provides the use of an antibody molecule or fragment thereof or an immunoconjugate or composition or combination product or kit thereof in the production or preparation of a medicament for the treatment of the relevant diseases or conditions mentioned herein .
  • an antibody or antibody fragment or immunoconjugate or composition or combination product or kit of the invention delays the onset of a disorder and/or symptoms associated with the disorder.
  • the antibodies or pharmaceutical compositions or immunoconjugates or combinations or kits of the invention can also be administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents, for use herein. prevention and/or treatment.
  • treatment modalities include surgery (eg, tumor resection) or radiation therapy.
  • the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infectious agents, small molecule drugs, or immunomodulatory agents.
  • the small molecule drug is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133), or a KRAS G12S inhibitor.
  • a KRAS G12C inhibitor e.g., AMG510 (Sotorasib) or GFH925
  • KRAS G12D e.g., MRTX1133
  • KRAS G12S inhibitor e.g., MRTX113
  • Immunomodulators include immune checkpoint molecule inhibitors and costimulatory molecule activators.
  • the antibodies of the invention or fragments thereof are used in combination with KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133 ) or KRAS G12S inhibitor.
  • KRAS G12C inhibitors e.g., AMG510 (Sotorasib) or GFH925
  • KRAS G12D e.g., MRTX1133
  • KRAS G12S inhibitor e.g., MRTX1133
  • the antibodies of the invention, or fragments thereof may be administered in conjunction with a treatment comprising the adoptive transfer of T cells (eg, cytotoxic T cells or CTLs) expressing chimeric antigen receptors (CARs).
  • T cells eg, cytotoxic T cells or CTLs
  • CARs chimeric antigen receptors
  • the antibodies of the invention, or fragments thereof can be administered in combination with an anti-tumor agent.
  • the antibodies of the invention or fragments thereof can be administered in combination with cytokines.
  • Cytokines can be administered as fusion molecules with the antibody molecules of the invention, or as separate compositions.
  • an antibody of the invention is administered in combination with one, two, three or more cytokines (eg, as a fusion molecule or as separate compositions).
  • the antibodies of the invention or fragments thereof can be combined with conventional cancer therapies in the art, including but not limited to: (i) radiotherapy; (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapies, or agents that specifically affect the deregulation of cancer cell proteins; (iv) immunotherapy, or enhancement of the host immune response (e.g., vaccines); (v) hormonal therapies, or block hormones (eg, when the tumor is hormone sensitive), (vi) angiogenesis inhibitors, or block blood vessel formation and growth, and (vii) palliative care.
  • conventional cancer therapies including but not limited to: (i) radiotherapy; (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapies, or agents that specifically affect the deregulation of cancer cell proteins; (iv) immunotherapy, or enhancement of the host immune response (e.g., vaccines); (v) hormonal therapies, or block hormones (eg, when the tumor is hormone sensitive), (vi
  • the antibodies or fragments thereof of the invention can be combined with conventional methods of enhancing host immune function.
  • Such combination therapy encompasses both combined administration (in which two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the other therapy, e.g., treatment modality and Administration of an antibody of the invention occurs before, simultaneously with, and/or after the therapeutic agent.
  • Antibody molecules and/or other therapies such as therapeutic agents or treatment modalities, may be administered during active disease or during remission or less active disease.
  • Antibody molecules can be administered before, concurrently with other treatments, after treatment, or during disease remission.
  • the antibodies of the invention may be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the administration may be by any suitable route, such as by injection, for example intravenously or subcutaneously.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease , whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • Dosages and treatment regimens for the antibody molecules of the invention can be determined by the skilled artisan. In some embodiments, dosage regimens are adjusted to provide optimal desired response (eg, therapeutic response).
  • the antibodies (and pharmaceutical compositions or immunoconjugates comprising the same) of the invention can be administered twice a week, or once a week, or once every two weeks.
  • immunoconjugates or compositions or combinations or kits of the invention can be used in place of or in addition to the antibodies of the invention for any treatment.
  • the anti-B7-H3/EGFR bispecific antibody molecule of the present invention is developed through the protein science Innobody technology platform of Innovent Biopharmaceutical (Suzhou) Co., Ltd. (Application No.: PCT/CN2021/143141, invention name: heterodimer-containing antibody Fc protein and its preparation method) or use conventional methods in the art to assemble the anti-B7-H3 antibody parent and the anti-EGFR antibody parent into the IgG1 antibody form (as shown in Figure 1).
  • This bispecific antibody format contains four polypeptide chains and can bind to two antigens, antigen A is EGFR and antigen B is B7-H3.
  • the parent antibodies used to construct bispecific antibodies are anti-EGFR monoclonal antibodies anti-Zalutumumab (hereinafter referred to as Zalu, publication number: WO02100348A2, invention name: Human monoclonal antibodies to epidermal growth factor receptor (EGFR)) and anti-B7- H3 monoclonal antibody, which is derived from the hybridoma technology screening platform of Innovent Biologics and humanized to obtain the antigen-binding region sequences of anti-B7-H3 monoclonal antibodies Hz20G5.26 and Hz19A2.25 (Application No.: PCT/CN2021/ 140449, invention name: anti-B7-H3 antibodies and Hz20G5, Hz19A2 in their uses)
  • the antigen-binding region sequence of the anti-B7-H3 monoclonal antibody Hz5C2.9 was obtained from Innobody's hybridoma technology screening platform and antibody humanization; at the same time, Innobody technology was used to
  • the heavy chain sequence of the anti-EGFR antibody, the light chain sequence of the anti-EGFR antibody, the heavy chain sequence of the anti-B7-H3 antibody, and the light chain sequence of the anti-B7-H3 antibody listed in Table 1 were inserted into the vector pcDNA3.1 (Invitrogen , V790-20), the heavy chain plasmid and light chain plasmid anti-EGFR end, and the heavy chain plasmid and light chain plasmid anti-B7-H3 end were obtained respectively.
  • the RMD enzyme GDP-6-deoxy-D -lyxo-4-hexylose reductase, sequence: SEQ ID NO: 41
  • plasmid was transiently transfected with the heavy chain plasmid and light chain plasmid of the anti-EGFR parental antibody and the heavy chain plasmid and light chain plasmid of the anti-B7-H3 parental antibody, respectively.
  • ExpiCHO Invitrogen, A29133
  • the low fucose content anti-EGFR and anti-B7-H3 end antibody parent was expressed. After 7 days, the cell fermentation broth was harvested, filtered and clarified, and captured using Hitrap Mabselect Sure chromatography columns (GE Healthcare, 11-0034-95) to obtain antibodies against the EGFR parent and the B7H3 parent.
  • Biofilm thin layer interferometry is used to determine the affinity (KD) of the bispecific antibody of the present invention for binding to B7H3 and EGFR.
  • the anti-B7-H3 and anti-EGFR antibody parent sequences are expressed and assembled into Hz5C2.9/Zalu bsAb, Hz19A2.25/Zalu bsAb and Hz20G5.26/Zalu bsAb bispecific antibody molecules.
  • the B7H3/Zalu bsAb bispecific antibody molecule has been evaluated in non-small cell lung cancer (NSCLC) and head and neck.
  • NSCLC non-small cell lung cancer
  • In vitro activity testing was conducted on squamous cell carcinoma (HNSCC), and safety verification was conducted on human skin squamous cell carcinoma cell line (A431).
  • PC9 (NSCLC): Shanghai Yubo Biotech, YB-H3210D; culture medium: MEM+10% FBS+1% Pen/strep
  • TE-1 HNSCC: CoBioer, CBP60655; culture medium: RPMI 1640+10% FBS+1% Pen/strep
  • SK-MES-1 (NSCLC): CoBioer, CBP60152; culture medium: MEM+1% NEAA+1mM Sodium Pyruvate+10% FBS+1% Pen/strep
  • A431 Cutaneous squamous cell carcinoma cell, ATCC, CRL-1555; culture medium: DMEM+10%FBS+1%Pen/strep
  • NSCLC Plate 96-well low-adsorption plate (Corning, CLS7007-24EA) with 1500-2500cells/100ul for 3D cell culture.
  • HNSCC Plate 1500-2000cells/100ul on a 96-well white bottom plate (NUNC, 136101) for 2D cell culture.
  • NSCLC Transfer the Cell-Titer and cell mixture into a 96-well white bottom plate (NUNC, 136101), and detect with a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).
  • the percentage of surviving cells (% of surviving cells) ( Figure 2A, B, C, D and F) is the relative survival rate of the cells detected.
  • PC9 is a type of NSCLC.
  • TE-1 is a type of HNSCC.
  • A431 is a skin squamous cell carcinoma cell that does not respond to antibody treatment and is used to measure drug side effects.
  • the growth inhibition percentage (Growth inhibition%) in Figure 2E is obtained after analysis and statistics based on the results (% of surviving cells) in Figure 2. The details are as follows: At the highest concentration of antibody 300nM, the maximum concentration of the drug in each cell Inhibition rate, calculation method: 300nM, (1-% of surviving cells) ⁇ 100%.
  • the bisAb-specific molecule Hz20G5.26/Zalu bsAb not only has excellent efficacy, but also has poor efficacy in vitro against A431 ( Figure 2D), indicating that Hz20G5 .26/Zalu bsAb can reduce or reduce the skin toxicity and other side effects of the EGFR antibody series clinical drugs.
  • the bisAb of the present invention is less toxic to human skin and has a larger effective and safe drug dose when it does not cause or weakly causes toxic side effects, that is, it has a wider range of The drug effect selection window makes the drug effect safer.
  • EGFR After EGFR ligand binds to EGFR on the surface of tumor cells, EGFR will form homodimers and transmit signals into cells. Through various cascade reactions, tumor cells will proliferate, invade, metastasize, and resist apoptosis. biological activity.
  • EGFR is a broad-spectrum tyrosine kinase receptor of epithelial origin.
  • Hz20G5.26/Zalu bs Ab uses the EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the effects of EGFR monoclonal antibodies on normal epithelial tissues such as skin. toxic side effect.
  • B7-H3 is highly expressed in a variety of tumor cell lines and low in normal tissues and organs. It is a highly selective TAA with a wide expression spectrum.
  • Hz20G5.26/Zalu bs Ab quotes B7-H3 on the basis of low affinity for EGER.
  • the high-affinity Hz20G5.26 parent greatly improves the EGFR signal blocking activity and improves the efficacy, biological activity and efficacy safety window of Hz20G5.26/Zalu bs Ab.
  • the drug sensitivity of Hz20G5.26/Zalu bsAb to tumor cell lines has a certain positive correlation with the receptor expression abundance on the surface of tumor cells.
  • Select some cell lines from NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colon cancer) and normal cell lines to detect the relative expression of B7-H3 and EGFR.
  • NSCLC non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • CRC colon cancer
  • B7-H3 and EGFR The expression abundance on the cell surface was tested and verified in vitro for the efficacy and activity of Hz20G5.26/Zalu bsAb. The experimental results are shown in Figure 4.
  • Figure 4 shows the expression of B7H3 and EGFR in different cell lines, among which cell lines with higher B7H3 expression and dependence on the EGFR signaling pathway (such as cell lines containing EGFR mutations obtained through subsequent sequencing analysis (results not shown) ) or EGFR overexpressing cell lines) can be selected for activity detection of bispecific antibodies.
  • EGFR is highly expressed on the cell surface of a variety of solid tumors, such as lung cancer (30%-80%), head and neck squamous cell carcinoma (36%-100%), colorectal cancer (25%-77%), and esophageal cancer (43%-89 %), etc., is a broad-spectrum anti-tumor tyrosine kinase target protein.
  • EGFR also has the highest mutation rate among mutation types in patients with non-small cell lung cancer (the global average mutation rate is about 35%, reaching 40% in China).
  • H1975-EGFR L858R/T790M/C797S PC9+B7H3-EGFR Del19/T790M/C797S and H322-EGFR S768_D770dup cell lines: Construct and package Lentvirus+EGFR L858R/T790M/C797S , Lentvirus+EGFR Del19/T790M/C797 respectively.
  • Lentvirus+EGFR S768_D770dup virus respectively use Lentvirus+EGFR L858R/T790M/C797S , Lentvirus+EGFR Del19/T790M/C797S and Lentvirus+EGFR S768_D770du virus to infect NCI-H1975 (ATCC, CRL-5908), PC9+hB7H3 (as described above) Construct) and H322, through pressure screening, H1975-EGFR L858R/T790M/C797S , PC9+B7H3-EGFR Del19/T790M/C797S and H322-EGFR S768_D770dup stably transfected cell lines were obtained.
  • NSCLC 1500-2500cells/100ul was spread on a 96-well low-adsorption plate (Corning, CLS7007-24EA) for 3D cell culture. The following cell lines and specific amounts were used:
  • NSCLC 2000cells/100 ⁇ l/well:
  • CRC and HNSCC 1500-2000cells/100ul were spread on a 96-well white bottom plate (NUNC, 136101) for 2D cell culture. The following cell lines and specific amounts were used:
  • HNSCC 1500cells/100ul/well
  • Colo680 CoBioer,CBP60452.
  • NSCLC Transfer the Cell-Titer and cell mixture into a 96-well white bottom plate (NUNC, 136101), and detect with a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).
  • EGFR is a broad-spectrum anti-tumor target protein. From the in vitro proliferation inhibition anti-tumor effect, it was found that Hz20G5.26/Zalu bsAb has anti-tumor effect in esophageal cancer tumor cell lines (as shown in Figure 6), among which TE- 1 Esophageal cancer tumor cell line, Hz20G5.26/Zalu bsAb has better anti-tumor effect than JNJ372. In colon cancer and other tumor cell lines (as shown in Figure 5), Hz20G5.26/Zalu bsAb can achieve the anti-tumor effect of EGFR monoclonal antibody.
  • EGFR-TKI small molecules such as Erlotinib ( Selleck Chemicals, cat: S7786) or Osimertinib (Selleck Chemicals, cat: S7297)
  • Erlotinib Selleck Chemicals, cat: S7786
  • Osimertinib Selleck Chemicals, cat: S7297
  • New targets need to be continuously developed for new mutation sites. drug.
  • Hz20G5.26/Zalu bsAb is not only effective against EGFR-mutated NSCLC tumor cell lines, but also has anti-tumor in vitro effects against wild-type EGFR and abnormally amplified EGFR non-small cell lung cancer tumor cell lines.
  • the in vitro anti-tumor effect of Hz20G5.26/Zalu bsAb is much better than that of JNJ-372.
  • the maximum anti-tumor killing rate of NCI-H292 in vitro was 74.2 ⁇ 2.8%; in the NSCLC-EGFR classic mutation (L858R, T790M or Exon19 deletion) tumor cell line ( Figure 7.2), against NCI-H1975 (EGFR L858R/T790M ) ( Figure 7.2B), the maximum anti-tumor inhibition killing rate in vitro is 51.2 ⁇ 1.4%, and the IC50 is 0.99nM.
  • the in vitro efficacy is superior to JNJ-372 and EGFR monoclonal antibodies; in NSCLC-EGFR Abnormally amplified tumor cell lines ( Figure 7.3), the maximum anti-tumor killing rate against SK-MES-1 in vitro is 60.7 ⁇ 15.9%, the IC50 is 0.40nM, the overall in vitro efficacy is better than JNJ-372 and EGFR monoclonal antibodies, and SK - MES-1 and NCI-H1703, tumor cell lines with abnormally amplified NSCLC-EGFR, are insensitive and resistant to first-generation (Erlotinib) and third-generation (Osimertinib).
  • Osimertinib a third-generation EGFR-TKI small molecule inhibitor that has been marketed and used for the treatment of T790M mutations, has poor efficacy against rare EGFR exon20ins mutations.
  • H322-EGFR S768_D770dup stably transfected cell line based on NCI-H322-EGFR WT cells.
  • Figure 7.4 In vitro proliferation inhibition and anti-tumor efficacy were shown ( Figure 7.4).
  • Hz20G5.26/Zalu bsAb has in vitro pharmacodynamic activity against EGFR exon20ins . of.
  • Hz20G5.26/Zalu bsAb was combined with gp120/Zalu and gp120/Hz20G5.26 for comparative analysis of in vitro proliferation inhibition efficacy (Figure 7.5).
  • KRAS gene mutation activation is also a common driver of cancer tumors.
  • About 25% of human cancers are KRAS mutations, and KRAS mutations are closely related to cancer prognosis and treatment.
  • KRAS-G12D and KRAS-G12C are common abnormal mutations in CRC and NSCLC tumors.
  • KRAS small molecule inhibitors it is expected that KRAS acquired resistance will develop later.
  • KRAS small molecule inhibitors are used in combination with other drugs (such as immunotherapy drugs or targeted therapy drugs) or are the direction of new drug research and development.
  • NSCLC H358 (Cobioer, CBP60136) and CRC (LS180) (Cobioer, CBP60034): 1500-2500cells/100ul are spread on a 96-well low-adsorption plate (Corning, CLS7007-24EA) for 3D cell culture.
  • Example 5.4 Study on the in vitro pharmacodynamic mechanism of Hz20G5.26/Zalu bsAb bispecific antibody molecule proliferation inhibition
  • Hz20G5.26/Zalu bsAb In the proliferation inhibition experiment, Hz20G5.26/Zalu bsAb, with the help of B7H3 antibody, further improved EGFR signal blocking to achieve stronger anti-tumor killing effect.
  • NCI-H358 cells we conducted studies in two directions: EGFR signal inhibition and EGFR ligand blocking through Western blotting to explore the in vitro anti-tumor effect of Hz20G5.26/Zalu bsAb, which is superior to EGFR monoclonal antibodies and Gp120/Zalu and Reasons for combined use of Gp120/Hz20G5.26.
  • ⁇ 1> Prepare cell lysis solution in advance.
  • the composition of cell lysis solution is: 100-200ul/tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001 ).
  • Zalu group 200nM Zalu mAb
  • Gp120/Zalu+Gp120/B7H3 group 200nM Gp120/Zalu+200nM Gp120/Hz20G5.26)
  • 30nM EGF ACRO, EGF-H52b
  • 30 nM TGF- ⁇ R&D, 239-A-100
  • ⁇ 1> Prepare cell lysis solution in advance.
  • the composition of cell lysis solution is: 100-200ul/tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001 ).
  • Antibody-dependent cell-mediated cytotoxicity is an important mechanism for anti-tumor antibodies to exert anti-tumor effects. Its principle is to use the Fab segment of the antibody to bind to the antigenic epitope on the surface of tumor cells, and its Fc segment binds to the killing immune system.
  • the FcR on the surface of (NK cells, macrophages, neutrophils, etc.) binds to directly kill tumor cells through immune cell mediation.
  • the ADCC effect is mainly achieved through the antibody Fc and the FcRIIIa receptor on the surface of NK.
  • Hz20G5.26/Zalu bsAb Due to the introduction of Hz20G5.26 parent, Hz20G5.26/Zalu bsAb not only improved the EGFR antibody blocking activity in Hz20G5.26/Zalu bsAb, but also improved the overall ADCC effect of Hz20G5.26/Zalu bsAb (presumably due to the combination of Hz20G5.26 The specific epitope on the proximal membrane end of B7H3 triggers a strong ADCC function); at the same time, the use of GlymaxX low fucose technology further enhances the ADCC effect of Hz20G5.26/Zalu bsAb.
  • the Hz20G5.26/Zalu bsAb dual-characteristic antibody molecule can jointly exert a variety of anti-tumor effects through two mechanisms of EGFR signal blocking and ADCC.
  • the specific experimental steps are as follows:
  • ADCC effector cells ((Promega, G7102)) are engineered to overexpress FcRIIIa (V158) receptors into Jurkat T cells.
  • FcRIIIa V158 receptors into Jurkat T cells.
  • the NFAT-RE driving element in Jurkat T cells can quickly respond to the strength of ADCC. of engineered cells.
  • ADCC activity of Hz20G5.26/Zalu bsAb is stronger than the combination of JNJ372, EGFR monoclonal antibody Zalu, Gp120/Zalu and Gp120/Hz20G5.26; in order to more truly and effectively reflect the ADCC activity of Hz20G5.26/Zalu bsAb , we designed the huPBMC ADCC experiment and used normal human PBMC to verify ADCC activity.
  • Hz20G5.26/Zalu bsAb huPBMC ADCC activity is stronger than JNJ373 and EGFR monoclonal antibodies, and stronger than Gp120/Zalu and Gp120/Hz20G5.26 combined ADCC activity; through the huPBMCADCC effect, although Hz20G5.26/Zalu bsAb has the lowest anti-tumor killing rate in H1975, the killing rate is also 36.53% ; Hz20G5.26/Zalu bsAb has the highest anti-tumor killing rate in H322, with a killing rate as high as 100%.
  • Hz20G5.26/Zalu bsAb on the basis of enhancing EGFR signal blockade, and participating in ADCC through immune cells, can further exert the anti-tumor activity of Hz20G5.26/Zalu bsAb in many cancer tumors.
  • EGFR is an epithelial-derived epidermal growth factor. In addition to overexpression or abnormal activation leading to cancer tumorigenesis, EGFR is also expressed in some epithelial cells and keratinocytes. Compared with the adverse reactions of EGFR-TKI small molecule inhibitors, such as rash, diarrhea, paronychia, oral mucositis, liver damage, and interstitial lung disease, EGFR monoclonal antibodies also have certain adverse reactions, mainly on the skin. Toxicity, such as pustular rash, paronychia, dry and itchy skin, etc., which greatly affects the quality of life and treatment compliance of cancer patients.
  • Hz20G5.26/Zalu bs Ab uses the B7H3 high-affinity Hz20G5.26 parent, which improves the pharmacological biological activity and pharmacological safety window of Hz20G5.26/Zalu bs Ab.
  • Example 7 In vivo pharmacodynamic activity of Hz20G5.26/Zalu bsAb bispecific antibody molecules
  • the NSCLC-EGFR WT tumor cell line NCI-H292 cells (ATCC) were used to inoculate Balb/c Nude mice to determine the Hz20G5.26/Zalu bispecific antibody molecule of the present invention.
  • Antitumor efficacy of specific antibodies SPF grade female Balb/c Nude mice (purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.) were used in the experiment, and the certificate number is NO.110011211108430747.
  • NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. Collect the cells by centrifugation, mix PBS (1 ⁇ ) and Matrigel Matix (Corning) in equal proportions and resuspend the NCI-H292 cells to prepare a cell suspension with a cell concentration of 20 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of Balb/c Nude mice to establish the NCI-H292 tumor-bearing mouse model.
  • mice Four days after tumor cell inoculation, the tumor volume of each mouse was measured and divided into groups (5 mice per group). The dosage and method of administration are shown in Table 6.
  • h-IgG is the isotype control antibody, purchased from Equitech-Bio, lot number 210112-0356.
  • h-IgG, JNJ372, Gp120/Zalu, Hz20G5.26/Zalu and Zalu mAb are all used at a concentration of 0.5 mg/ml, and are administered once every 3-4 days for a total of 4 times (Q3-4D x4).
  • Body weight was measured using an electronic balance. Mice with tumor volume exceeding 2000 mm3 or body weight loss exceeding 20% were euthanized.
  • the tumor inhibition rate results are shown in Table 7: On the 22nd day after vaccination, compared with the h-IgG, 5mg/kg group, the tumor inhibition rates of JNJ372, Gp120/Zalu, Hz20G5.26/Zalu and Zalu mAb were 100% respectively. , 72%, 108% and 107%. There was 1 mouse with complete tumor remission in each of the Hz20G5.26/Zalu and Zalu mAb groups. The mouse survival curve is shown in Figure 15B. Hz20G5.26/Zalu can significantly extend the survival period of mice.
  • the anti-tumor efficacy of Hz20G5.26/Zalu on NCI-H292 tumor-bearing mice is similar to that of the parent Zalu
  • the mAb was comparable and better than JNJ372 and the untargeted GP120/Zalu.
  • SK-MES-1 cells Najing Kebai
  • Balb/c Nude mice mice to measure the Hz20G5.26 of the present invention.
  • Zalu bispecific antibody molecule anti-tumor efficacy SPF-grade female Balb/c Nude mice (purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.) were used in the experiment, and the certificate number is NO.110011211108966881.
  • SK-MES-1 cells were routinely subcultured for subsequent in vivo experiments. Collect cells by centrifugation, mix SK-MES-1 cells with PBS (1 ⁇ ) and Matrigel Matix (Corning) in equal proportions, and prepare a cell suspension with a cell concentration of 20 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of Balb/cNude mice to establish the SK-MES-1 tumor-bearing mouse model.
  • mice Six days after tumor cell inoculation, the tumor volume of each mouse was measured and divided into groups (7 mice per group). The dosage and method of administration are shown in Table 8.
  • h-IgG is the isotype control antibody, purchased from Equitech-Bio, lot number 210112-0356.
  • the usage concentrations of h-IgG, Hz20G5.26/Zalu, JNJ372, Zalu mAb and Gp120/Zalu are all 0.1mg/ml.
  • the SK-MES-1 cells were administered once on the 6th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 16A, and the monitoring ended after 24 days.
  • TGI% 100% * (tumor volume in the control group – tumor volume in the treatment group) / (tumor volume in the control group – tumor volume in the control group before administration) ).
  • the tumor inhibition rate results are shown in Table 9: On the 24th day after vaccination, compared with the h-IgG, 1mg/kg group, the tumor inhibition rates of Hz20G5.26/Zalu, JNJ372, Zalu mAb and Gp120/Zalu were 143% respectively. , 122%, 145% and 113%. The results of monitoring the body weight of the mice at the same time (Figure 16B) showed that there was no significant difference in the body weight of the mice on the 24th day after vaccination.
  • SK-MES-1 cells Najing Kebai
  • Balb/c Nude mice were used to inoculate Balb/c Nude mice to measure Hz20G5.26/Zalu of the present invention.
  • Antitumor efficacy of bispecific antibody molecules The experiment used SPF grade female Balb/c Nude mice (purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.), the certificate number is NO.110011221103705231.
  • SK-MES-1 cells were routinely subcultured for subsequent in vivo experiments. Collect cells by centrifugation, mix SK-MES-1 cells with PBS (1 ⁇ ) and Matrigel Matix (Corning) in equal proportions, and prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of Balb/c Nude mice to establish the SK-MES-1 tumor-bearing mouse model.
  • mice Fourteen days after tumor cell inoculation, the tumor volume of each mouse was measured and divided into groups (6 mice per group). The dosage and method of administration are shown in Table 10.
  • h-IgG is the isotype control antibody, purchased from Equitech-Bio, lot number 210112-0356.
  • the usage concentration of h-IgG is 0.06mg/ml
  • the usage concentration of Hz20G5.26/Zalu, Gp120/Zalu and Gp120/Hz20G5.26 is 0.03mg/ml.
  • the SK-MES-1 cells were administered once on the 14th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 17A, and the monitoring ended after 35 days.
  • TGI% 100% * (tumor volume in the control group – tumor volume in the treatment group) / (tumor volume in the control group – tumor volume in the control group before administration) ).
  • the tumor inhibition rate results are shown in Table 11: on the 35th day after vaccination, compared with the h-IgG, 0.6mg/kg group, Hz20G5.26/Zalu, Gp120/Zalu, Gp120/Hz20G5.26 and Gp120/Zalu+Gp120
  • the tumor inhibition rates of /Hz20G5.26 were 121%, 31%, 1% and 16% respectively.
  • the anti-tumor efficacy of Hz20G5.26/Zalu is better than that of the control non-targeting monoclonal antibody Gp120/Zalu, Gp120/Hz20G5.26 or a combination of the two, proving the unique mechanism of Hz20G5.26/Zalu as a bispecific antibody, namely Hz20G5.26 has a pulling effect on zalu blocking EGFR signal.
  • the results of monitoring the body weight of the mice at the same time showed that there was no significant difference in the body weight of the mice on the 24th day after vaccination.
  • Example 8 In vivo pharmacodynamic activity of Hz20G5.26/Zalu bsAb bispecific antibody molecule combined with KRAS small molecule inhibitor
  • the KRAS G12C mutated NSCLC tumor cell line NCI-H358 (Nanjing Kebai) was used to inoculate NOG mice to measure Hz20G5 of the present invention.
  • the anti-tumor efficacy of 26/Zalu bispecific antibody combined with the KRAS small molecule inhibitor AMG510 SPF grade female NOG mice (purchased from Beijing Vitong Lever Laboratory Animal Technology Co., Ltd.) were used in the experiment, and the certificate number is NO.1100112211001153625.
  • NCI-H358 cells were routinely subcultured for subsequent in vivo experiments. Collect the cells by centrifugation, mix PBS (1 ⁇ ) and Matrigel Matix (Corning) in equal proportions, and resuspend the NCI-H358 cells to prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/ml. On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of NOG mice to establish the NCI-H358 tumor-bearing mouse model.
  • mice Six days after tumor cell inoculation, the tumor volume of each mouse was measured and divided into groups (7 mice per group). The dosage and method of administration are shown in Table 12.
  • h-IgG is the isotype control antibody, purchased from Equitech-Bio, lot number 210112-0356.
  • the concentration of h-IgG used is 2mg/ml, and the concentration of Hz20G5.26/Zalu, JNJ372 and Zalu mAb is 1mg/ml. It is administered once every 3-4 days for a total of 4 times (Q3-4D x4).
  • the NCI-H358 cells were administered on the 6th, 9th, 12th, and 15th days after inoculation, and the tumor volume and body weight of the mice were monitored twice a week.
  • the concentration of AMG510 used is 1 mg/ml.
  • Administration begins on the 6th day after NCI-H358 cell inoculation. Administration is once a day for a total of 14 times (QD x14). The mouse tumor volume is monitored twice a week.
  • TGI% 100% * (tumor volume in the control group – tumor volume in the treatment group) / (tumor volume in the control group – tumor in the control group before administration) volume).
  • the results of tumor inhibition rate are shown in Table 13: on the 33rd day after vaccination, compared with h-IgG, 20mg/kg group, AMG510, Hz20G5.26/Zalu, AMG510+Hz20G5.26/Zalu, JNJ372 and Zalu mAb
  • the tumor inhibition rates were 89%, 110%, 130%, 82% and 111% respectively.
  • Hz20G5.26/Zalu combined with AMG510 had the best anti-tumor effect on NCI-H358 tumor-bearing mice and had a synergistic effect.

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Abstract

提供了一种经人工设计的双特异性抗体分子，特别是抗B7-H3/EGFR双特异性抗体分子，其能同时结合B7-H3和EGFR。

Description

结合EGFR和B7-H3的双特异性抗体 发明领域
本发明总体上涉及免疫学和抗体工程领域。具体而言，本发明涉及新型的经人工设计的双特异性抗体分子，特别是同时结合EGFR和B7-H3的双特异性抗体、编码所述抗体分子或其各条链的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、包含所述抗体分子的免疫缀合物和药物组合物、以及所述抗体分子在疾病的免疫治疗、预防和/或诊断上的用途。
发明背景
在全球范围内，肺癌是最常见的癌症肿瘤类型之一，在肺癌中约有80％-85％是非小细胞肺癌(NSCLC)患者，NSCLC可分为肺腺癌、肺鳞状细胞癌和大细胞肺癌等常见类型。表皮生长因子受体(EGFR)是一种酪氨酸激酶受体，是一个巨大的跨膜糖蛋白，分子量约为170KDa，属于ErbB受体家族的一个成员，是NSCLC最常见的癌变驱动基因，在亚洲NSCLC患者中约有40％是EGFR突变导致的，在高加索人群中约有15％是EGFR突变导致的(Gower A,Wang Y,Giaccone G.Oncogenic drivers,targeted therapies,and acquired resistance in non-small-cell lung cancer.J.Mol.Med.2014；92:697-707.)，在其他癌症肿瘤患者，如肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌和头颈癌等上皮源癌症，都存在着EGFR过度表达和/或异常突变激活现象。
针对NSCLC-EGFR异常激活和扩增，现已有EGFR-TKI小分子抑制剂(如吉非替尼、厄洛替尼(Erlotinib)，阿法替尼、达克替尼和奥希替尼(Osimertinib)、阿美替尼等)和生物单抗大分子(西妥昔单抗、帕尼单抗、耐昔妥珠单抗和尼妥珠单抗)靶向治疗方法。虽EGFR-TKI小分子抑制剂仍是现在非小细胞肺癌(NSCLC)治疗的标准方法，但EGFR-TKI小分子主要针对酪氨酸激酶活性结构突变患者，常因靶点基因突变而造成耐药失效，因此需要不断针对新突变位点进行新的靶向药物的研发，极大限制了该类药物的临床运用，这也是EGFR-TKI小分子整个行业所面临的严峻挑战。
EGFR激活突变区主要发生在EGFR外显子18-21酪氨酸激酶结构域(Jiyeon Yun,Soo-Hwan Lee,Seok-Young Kim,et al.Antitumor activity of Amivantamab(JNJ-61186372),an EGFR–MET bispecific antibody,in diverse models of EGFR exon 20 insertion–drivenNSCLC.Cancer Discov 2020；10:1194-209.)，EGFR抗体的结合区主要位于EGFR胞外配体结构域区，可避免耐药突变的发生；同时，EGFR抗体可通过抑制EGFR与配体的结合而抑制肿瘤细胞的生长，也可利用自身特定ADCC(抗体依赖细胞介导的细胞毒性作用)与免疫细胞共同杀伤肿瘤，这样可通过多种作用机制，共同发挥抗肿瘤杀伤作用。
B7-H3(又称之为CD276)是一种I型跨膜蛋白(Picarda E,Ohaegbulam KC,Zang X.Molecular pathways:targeting B7–H3(CD276)for human cancer immunotherapy.Clin Cancer Res.2016；22:3425–31.and，Yang S,Wei W,Zhao Q.B7-H3,a checkpoint molecule,as a target for cancer immunotherapy.Int J Biol Sci. 202016:1767–73)，和PD-L1结构非常类似，同属于B7/CD28超家族。在绝大多数正常人体组织低水平表达，在肺癌、结肠癌、头颈癌、乳腺癌、卵巢癌、胰腺癌等肿瘤细胞中却异常高表达(Lee YH,Martin-OrozcoN,Zheng P,Li J,Zhang P,Tan H,et al.Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function.Cell Res.2017；27:1034-45.and，Kontos F,Michelakos T,Kurokawa T,Sadagopan A,Schwab JH,Ferrone CR,et al.B7-H3:an attractive target for antibody-based immunotherapy.Clin Cancer Res.2020.https://doi.org/10.1158/1078-0432.CCR-20-2584.and，Seaman S,Zhu Z,Saha S,Zhang XM,Yang MY,Hilton MB,et al.Eradication of tumors through simultaneous ablation of CD276/B7-H3-positive tumor cells and tumor vasculature.Cancer Cell.2017；31:501–15)。B7-H3受体仍未被确认，但在肿瘤免疫中，B7-H3可参与毒性淋巴细胞的免疫功能调节(Kraan J,van den BroekP,VerhoefC,Grunhagen DJ,Taal W,Gratama JW,et al.Endothelial CD276(B7-H3)expression is increased in human malignancies and distinguishes between normal and tumour-derived circulating endothelial cells.Br J Cancer.2014；111:149–56)；有证据显示，B7-H3表达可能与EGFR基因表达状况及抗PD-1治疗效果相关(Yonesaka K,Haratani K,Takamura S,Sakai H,Kato R,Takegawa N,et al.B7-H3negatively modulates CTL-mediated cancer immunity.Clin Cancer Res.2018；24:2653–64)。由于B7-H3有着良好的选择表达谱，现已有生物研发公司研发出B7-H3单克隆单体或B7-H3-ADC药物，用于相关肿瘤疾病领域的研究和治疗。
抗体分子能够与其相应的抗原发生靶向性的特异性结合，正日益成为针对各种疾病(例如，癌症、自身免疫病、炎性疾病、感染性疾病等)的重要的治疗剂、预防剂和/或诊断剂。但是，仅针对一种靶点的单特异性抗体在临床应用上存在一些局限性。患者在接受单特异性抗体治疗后可能产生耐药性或无应答。随着对癌症和其他多种疾病的研究，认识到了往往有多种信号转导通路参与疾病的发生和发展，单一靶点的免疫疗法在许多疾病中通常并不足以发挥对疾病的治疗作用。
由于多特异性抗体(例如，双特异性抗体)能够特异性结合不同抗原，能够设计为同时作用于两种或多种不同介质的信号转导通路。这些优势特性为多特异性抗体(例如，双特异性抗体)开辟了广阔的应用前景。
本领域仍然需要可供选择的具有改善性能的双特异性抗体，所述的双特异性抗体能够同时结合不同的抗原，特别是EGFR和B7-H3，保持各抗原结合位点与相应的不同表位结合的结合活性，以及其他性质。同时，需要获得具有一定稳定性的，且具有更好的生产性和可发展性的双特异性抗体样式是物理稳定的和生物学稳定的，这允许该抗体具有更好的生产性和可发展性。
发明概述
本发明的第一方面涉及一种抗体，其包含特异性识别EGFR(例如人EGFR)的抗原结合区和特异性结合B7-H3的抗原结合区。在一些实施方案中，所述抗体为多特异性抗体，例如双特异性抗体。
本发明提供的结合EGFR和B7-H3的抗体或其抗原结合片段，通过降低EGFR亲和力，提高B7-H3亲和力，提高了抗体分子的药效和安全选择性；同时， 采用GlymaxX低岩藻糖技术，增强了抗体依赖的细胞介导的细胞毒性作用(ADCC)。相比用于NSCLC-EGFR 20外显子插入突变(exon20ins)的埃万妥希单抗(Amivantamab，JNJ-372)，B7-H3比cMET表达谱更广泛，肿瘤治疗适用范围更广；同时，在B7-H3亲本的引入下，不仅提高了双特异性抗体中EGFR抗体阻断活性，而且提高了双特异性抗体整体的ADCC作用。
本发明提供的结合EGFR和B7-H3的双特异性抗体或其抗原结合片段具有以下一个或多个特性：
(a)以高亲和力与一种或两种抗原特异性结合；
(b)易于在体外的培养细胞中表达，且抗体分子的各条链之间能够正确偶合或配对；
(c)具有良好的物理稳定性，特别地，具有良好的长期热稳定性；且能长时间保持生物学活性；
(d)在与一种或两种抗原特异性结合后，通过调节(例如，抑制或者激活)各抗原所参与的信号传导通路来发挥生物学功能；
(e)发挥效应子功能；
(f)具有更好的抗肿瘤活性。
在一个实施方案中，不同抗原结合位点结合相同的抗原上的相同表位，或不同表位。
在一个实施方案中，第一抗原结合区或第二抗原结合区来自人的，或人源化的，或嵌合的抗体。
在一些实施方案中，本发明的抗体，例如双特异性抗体还包含重链恒定区。在一个实施方案中，所述重链恒定结构域来自IgG1。应当理解的是，可以对恒定结构域中的Fc进行突变，以实现稳定抗体的作用，或增强效应子功能的作用。
在一个实施方案中，第一抗原结合区对第一抗原是特异性的，在一个实施方案中，第一抗原是EGFR。
在一个实施方案中，第二抗原结合区对第二抗原是特异性的，在一个实施方案中，第二抗原是B7-H3。
本发明的抗体还可以包含结合其他抗原的其他抗原结合区以构成多特异性抗体。不特别地限制本发明的抗体分子特异性结合的其他抗原类型，抗原可以是例如细胞因子、生长因子、激素、信号传导蛋白、炎性介质、配体、细胞表面受体或其片段。在一个实施方案中，本发明的抗体分子特异性结合的其他抗原选自肿瘤相关抗原、免疫检查点分子、血管新生诱导因子、肿瘤坏死因子受体超家族成员和免疫系统中的共刺激分子，以及这些分子的配体和/或受体。
在一个方面，本发明提供了编码本发明抗体分子中的任意一条或者多条多肽链的核酸，包含所述核酸的载体，包含所述核酸或载体的宿主细胞。
在一个方面，本发明提供了包含编码本发明抗体分子中的任意一条或者多条多肽链的多核苷酸的载体，优选地表达载体，例如pcDNA3.1。
在一个方面，本发明提供了用于产生本发明抗体分子或其片段的方法。
在一些实施方案中，本发明提供了包含本发明抗体的免疫缀合物、药物组合物、试剂盒、组合产品或制品。
在一些实施方案中，本发明的抗体、药物组合物或免疫缀合物或组合产品或试剂盒用于预防或治疗疾病，如急性和慢性炎性疾病、感染(例如，慢性感染)、肿瘤等。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤是胃肠道肿瘤或肺部肿瘤或皮肤肿瘤。在一些实施方案中，感染是慢性感染。
在另一方面中，本发明涉及预防或治疗受试者或个体疾病的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗体或其片段、药物组合物或免疫缀合物或组合产品或试剂盒。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。在一个实施方案中，肿瘤是胃肠道肿瘤或肺部肿瘤或皮肤肿瘤。在一个实施方案中，感染是慢性感染。
在另一方面，本发明还涉及本文所述的任何抗体或其片段或免疫缀合物制备用于在受试者中治疗肿瘤(例如癌症)或感染的药物或药物组合物或试剂盒或组合产品的用途。在一些实施方案中，肿瘤是肿瘤免疫逃逸。在一个实施方案中，肿瘤是胃肠道肿瘤或肺部肿瘤或皮肤肿瘤。在一个实施方案中，感染是慢性感染。
本发明还涉及在样品中检测抗原的方法。
在另一个方面，本发明涉及以下的具体的实施方案：
1、结合EGFR和B7-H3的双特异性抗体，其包含第一抗原结合区和第二抗原结合区，所述第一抗原结合区特异性结合EGFR，且所述第二抗原结合区特异性结合B7H3。
2、实施方案1的双特异性抗体，其中所述第二抗原结合区包含如SEQ ID NO:3、5或7所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:4、6或8所示的轻链可变区的LCDR1、2和3序列，优选地，所述HCDR1采用Abm方案，所述HCDR2、HCDR3、LCDR1、LCDR2和LCDR3采用Kabat方案。
3、实施方案1所述的双特异性抗体，其中所述第二抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
(i)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:3所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:4所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(ii)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:5所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:6所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；或
(iii)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:7所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:8所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
优选地，所述HCDR1采用Abm方案，所述HCDR2、HCDR3、LCDR1、LCDR2和LCDR3采用Kabat方案。
4、实施方案1所述的双特异性抗体，其中所述第二抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
(i)HCDR1包含如SEQ ID NO:15所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:16所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:17所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:18所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:19所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:20所示的氨基酸序列或由所述序列组成；或，
(ii)HCDR1包含如SEQ ID NO:21所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:22所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:23所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:24所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:25所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:26所示的氨基酸序列或由所述序列组成；或，
(iii)HCDR1包含如SEQ ID NO:27所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:28所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:29所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:30所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:31所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:32所示的氨基酸序列或由所述序列组成。
5、实施方案1-4任一项所述的双特异性抗体，其中，所述第二抗原结合区包含重链可变区VH，其中所述VH包含SEQ ID NO:3、5或7所示的氨基酸序列或与SEQ ID NO:3、5或7所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列或由所述氨基酸序列组成。
6、实施方案1-5任一项所述的双特异性抗体，其中，所述第二抗原结合区包含轻链可变区VL，其中所述VL包含SEQ ID NO:4、6或8所示的氨基酸序列或与SEQ ID NO:4、6或8所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列或由所述氨基酸序列组成。
7、实施方案1-6中任一项的双特异性抗体，其中第二抗原结合区包含重链可变区VH和轻链可变区VL，其中
(i)所述VH包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)所述VH包含SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(iii)所述VH包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:8所示的氨基酸序列或与SEQ ID NO:8的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
8、实施方案1-7中任一项的双特异性抗体，其中第二抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
SEQ ID NO:3和SEQ ID NO:4；
SEQ ID NO:5和SEQ ID NO:6；或
SEQ ID NO:7和SEQ ID NO:8。
9、实施方案1-7中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:2所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
优选地，所述CDR采用Kabat方案确定。
10、实施方案9的双特异性抗体，其中第一抗原结合区的HCDR1包含SEQ ID NO:9的氨基酸序列或由所述序列组成；HCDR2包含SEQ ID NO:10的氨基酸序列或由所述序列组成；HCDR3包含SEQ ID NO:11的氨基酸序列或由所述序列组成；且第一抗原结合区的LCDR1包含SEQ ID NO:12的氨基酸序列或由所述序列组成；LCDR2包含SEQ ID NO:13的氨基酸序列或由所述序列组成；且LCDR3包含SEQ ID NO:14的氨基酸序列或由所述序列组成。
11、实施方案9或10的双特异性抗体，其中第一抗原结合区包含重链可变 区VH，其中所述VH包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
12、实施方案9-11中任一项的双特异性抗体，其中第一抗原结合区包含轻链可变区VL，其中所述VL包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
13、实施方案9-12中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH和轻链可变区VL，其中所述VH包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
14、实施方案9-13中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:2。
15、实施方案1-14中任一项的双特异性抗体，其中第一抗原结合区特异性结合EGFR，其包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
HCDR1包含SEQ ID NO:9的氨基酸序列或由所述序列组成；
HCDR2包含SEQ ID NO:10的氨基酸序列或由所述序列组成；
HCDR3包含SEQ ID NO:11的氨基酸序列或由所述序列组成；
LCDR1包含SEQ ID NO:12的氨基酸序列或由所述序列组成；
LCDR2包含SEQ ID NO:13的氨基酸序列或由所述序列组成；且
LCDR3包含SEQ ID NO:14的氨基酸序列或由所述序列组成；
且
第二抗原结合区特异性结合B7H3，其包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
(i)HCDR1包含SEQ ID NO:15的氨基酸序列或由所述序列组成；
HCDR2包含SEQ ID NO:16的氨基酸序列或由所述序列组成；
HCDR3包含SEQ ID NO:17的氨基酸序列或由所述序列组成；
LCDR1包含SEQ ID NO:18的氨基酸序列或由所述序列组成；
LCDR2包含SEQ ID NO:19的氨基酸序列或由所述序列组成；且
LCDR3包含SEQ ID NO:20的氨基酸序列或由所述序列组成；
(ii)HCDR1包含SEQ ID NO:21的氨基酸序列或由所述序列组成；
HCDR2包含SEQ ID NO:22的氨基酸序列或由所述序列组成；
HCDR3包含SEQ ID NO:23的氨基酸序列或由所述序列组成；
LCDR1包含SEQ ID NO:24的氨基酸序列或由所述序列组成；
LCDR2包含SEQ ID NO:25的氨基酸序列或由所述序列组成；且
LCDR3包含SEQ ID NO:26的氨基酸序列或由所述序列组成；或者
(iii)HCDR1包含SEQ ID NO:27的氨基酸序列或由所述序列组成；
HCDR2包含SEQ ID NO:28的氨基酸序列或由所述序列组成；
HCDR3包含SEQ ID NO:29的氨基酸序列或由所述序列组成；
LCDR1包含SEQ ID NO:30的氨基酸序列或由所述序列组成；
LCDR2包含SEQ ID NO:31的氨基酸序列或由所述序列组成；且
LCDR3包含SEQ ID NO:32的氨基酸序列或由所述序列组成。
16、实施方案15的双特异性抗体，其中第一抗原结合区包含含有如SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成的VH和含有如SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成的VL，且第二抗原结合区包含分别含有如下所示的氨基酸序列或由所述氨基酸序列组成的VH和VL:
SEQ ID NO:3和SEQ ID NO:4；
SEQ ID NO:5和SEQ ID NO:6；或
SEQ ID NO:7和SEQ ID NO:8。
17、实施方案1-16中任一项的双特异性抗体，其包含Fc区，优选地，所述Fc区具有低岩藻糖基化，例如通过GlymaxX技术处理后获得的低岩藻糖基化。
18、实施方案17的双特异性抗体，其包含第一Fc区和第二Fc区，其中第一Fc区和第二Fc区相同或不同。
19、实施方案17或18所述的双特异性抗体，其中第一Fc区和第二Fc区分别为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc，或人IgG4 Fc，例如包含氨基酸序列SEQ ID NO:46或47或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。
20、实施方案18或19的双特异性抗体，其中在第一Fc区和第二Fc区中引入促进第一Fc区和第二Fc区的异二聚化的突变。
21、实施方案20的双特异性抗体，其中所述突变是基于Innobody技术引入的。
22、实施方案21的多特异性抗体，其中一个Fc区的CH3包含S364R和D399K突变，且另一个Fc区的CH3突变包含Y349T、K370S和K409D突变。
23、实施方案22的双特异性抗体，其中
a)一个Fc区多肽包含SEQ ID NO:49或50所示的氨基酸序列或由其组成，而另一个Fc区多肽包含SEQ ID NO:52或53所示的氨基酸序列或由其组成；
b)一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，而另一个Fc区多肽包含与SEQ ID NO:52或53所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列；或
c)一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变Y349T、K370S和K409D，而另一个Fc区包含与SEQ ID NO:52或53所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变S364R和D399K。
24、实施方案20的双特异性抗体，其中所述突变是基于Knob-into-Hole技术引入的，其中在第一Fc区和第二Fc区中引入相应的Knob突变和Hole突变。
25、实施方案24的双特异性抗体，其中
a)一个Fc区多肽包含突变T366W，而另一个Fc区多肽包含T366S,L368A和Y407V(编号方式依照EU索引)，或
b)一个Fc区包含氨基酸替代S354C和T366W,且另一个Fc区包含氨基酸替代Y349C,T366S,L368A和Y407V(编号方式依照EU索引)。
26、实施方案1-25中任一项所述的双特异性抗体，其中第一和/或第二抗原结合区(例如其中的重链可变区)还可以与1个或2个重链恒定区(例如人IgG1,人IgG2,人IgG3，或人IgG4的重链恒定区)连接，所述重链恒定区包含CH1和Fc区，经由或不经由铰链区连接，例如重链可变区的C末端与重链恒定区的CH1的N末端连接。
27、实施方案26所述的双特异性抗体，其中所述CH1包含SEQ ID NO:42所示的氨基酸序列或与SEQ ID NO:42的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
28、实施方案1-27所述的双特异性抗体，其中第一和/或第二抗原结合区(例如其中的轻链可变区)还可以与轻链恒定区连接，例如轻链可变区的C末端与轻链恒定区的N末端连接。
29、实施方案28所述的双特异性抗体，其中轻链恒定区为kappa轻链恒定区或lambda轻链恒定区。
30、实施方案20所述的双特异性抗体，其中所述轻链恒定区包含SEQ ID NO:54所示的氨基酸序列或与SEQ ID NO:54的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
31、实施方案1-30中任一项所述的双特异性抗体，其中所述双特异性抗体为 IgG样抗体，其具有如图1所示的构型。
32、实施方案31所述的双特异性抗体，其中包含重链1和轻链1，以及重链2和轻链2，其中重链1和轻链1构成第一半抗体，且重链2和轻链2构成第二半抗体；其中
重链1包含第一抗原结合区的重链可变区和第一重链恒定区；轻链1包含第一抗原结合区的轻链可变区和第一轻链恒定区；且重链2包含第二抗原结合区的重链可变区和第二重链恒定区；轻链2包含第二抗原结合区的轻链可变区和第二轻链恒定区。
33、实施方案32所述的双特异性抗体，其中重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
34、实施方案32或33所述的双特异性抗体，其中轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
35、实施方案32-34中任一项所述的双特异性抗体，其中重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
36、实施方案32-35中任一项所述的双特异性抗体，其中重链2包含SEQ ID NO:35、37或39所示的氨基酸序列或与SEQ ID NO:35、37或39所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
37、实施方案32-36中任一项所述的双特异性抗体，其中轻链2包含SEQ ID NO:36、38或40所示的氨基酸序列或与SEQ ID NO:36、38或40所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
38、实施方案32-37中任一项所述的双特异性抗体，其中
(1)重链2包含SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:36所示的氨基酸序列或与SEQ ID NO:36所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(2)重链2包含SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37所示 的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:38所示的氨基酸序列或与SEQ ID NO:38所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(3)重链2包含SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:40所示的氨基酸序列或与SEQ ID NO:40所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
39、实施方案32-38中任一项所述的双特异性抗体，其中
重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且重链2和轻链2分别包含如下的SEQ ID NO:所示的氨基酸序列，或包含与所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，或由如下SEQ ID NO所示的序列组成：
i)SEQ ID NO:35和SEQ ID NO:36；
ii)SEQ ID NO:37和SEQ ID NO:38；
iii)SEQ ID NO:39和SEQ ID NO:40。
40、实施方案32-39任一项所述的双特异性抗体，其中，
(i)重链1包含SEQ ID NO:33所示的氨基酸序列的重链或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
重链2包含SEQ ID NO:35所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:36所示的氨基酸序列或由其组成；或，
(ii)重链1包含SEQ ID NO:33所示的氨基酸序列或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
重链2包含SEQ ID NO:37所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:38所示的氨基酸序列或由其组成；或，
(iii)重链1包含SEQ ID NO:33所示的氨基酸序列或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
重链2包含SEQ ID NO:39所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:40所示的氨基酸序列或由其组成。
41、实施方案1-40任一项所述的结合EGFR和B7-H3的双特异性抗体或其 抗原结合片段，其中，所述抗体或其抗原结合片段具有以下一个或多个特性：
(i)所述抗体一方面可以阻断EGFR配体与EGFR的结合，抑制生物信号传递，阻断肿瘤相应生物活性；另一方面会刺激EGFR内吞而最终被细胞内溶酶体等降解；
(ii)所述抗体采用对EGFR低亲和力的EGFR抗体亲本序列，极大降低了系列EGFR单克隆抗体对皮肤等正常上皮组织带来的毒副作用；
(iii)所述抗体在EGER低亲和力的基础上引用B7-H3高亲和力的抗体亲本序列，极大提高了EGFR信号阻断活性，提高了本发明双特异性抗体的药效生物活性和药效安全窗口；
(iv)所述抗体为低岩藻糖基化的抗体；
(ii)所述抗体具有较高的药效生物活性和安全性；
(v)所述抗体具有优良的肿瘤杀伤和抑制效果；
(vi)所述抗体具有优良的ADCC体内外药效活性；
(vii)所述抗体与KRAS小分子抑制剂联用具有优良的抗肿瘤协同效果。
42、分离的核酸，其编码实施方案1-41任一项的所述的结合EGFR和B7-H3的双特异性抗体中的任一条链。
43、载体，其包含实施方案42的核酸，优选地所述载体是表达载体，优选地，所述表达载体为pcDNA，例如pcDNA3.1。
44、宿主细胞，其包含实施方案42的核酸或实施方案43的载体，优选地，所述宿主细胞是原核的或真核的，更优选的酵母细胞或哺乳动物细胞(例如293细胞或CHO细胞，例如293F细胞或293T细胞或CHO-S细胞)。
45、实施方案44的宿主细胞，其被糖工程化而表达RMD酶，优选地，所述宿主细胞是CHO细胞。
46、实施方案45的宿主细胞，其包含编码RMD酶的核酸。
47、实施方案46的宿主细胞，其中所述RMD酶包含SEQ ID NO:41所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成，优选地，所述RMD酶来自铜绿假单胞菌。
48、制备结合EGFR和B7-H3的双特异性抗体的方法，所述方法包括在适于表达编码前述实施方案1-41任一项的双特异性抗体的核酸的条件下培养实施方案44-47中任一项所述的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收所述抗体或其抗原结合片段。
49、免疫缀合物，其包含与治疗剂或诊断剂缀合的前述实施方案1-41中任一项的双特异性抗体。
50、药物组合物，其包含前述实施方案1-41任一项的双特异性抗体或实施方案49的免疫缀合物，以及任选地药用辅料。
51、实施方案50的药物组合物，其还包含第二治疗剂；优选地，所述第二治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
52、药物组合产品，其包含实施方案1-41任一项的双特异性抗体或实施方案49的免疫缀合物或实施方案50的药物组合物，以及一种或多种第二治疗剂，优选地，所述第二治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
53、在受试者中预防或治疗肿瘤或感染性疾病的方法，所述方法包括向所述受试者施用有效量的实施方案1-41中任一项所述的双特异性抗体、或实施方案49所述的免疫缀合物、或实施方案50的药物组合物。
54、实施方案53所述的方法，其还包括向所述受试者联合施用一种或多种其它疗法，所述疗法例如包括治疗方式和/或其它治疗剂，优选地，所述治疗方式包括手术治疗和/或放射疗法，或者所述治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
55、在受试者中预防或治疗肿瘤或感染性疾病的方法，所述方法包括向所述受试者施用有效量的实施方案51的药物组合物或实施方案52的药物组合产品。
56、实施方案53-55中任一项所述的方法，其中所述肿瘤为癌症，例如实体肿瘤或血液肿瘤，包括上皮来源的癌症，例如胃肠道肿瘤或肺部肿瘤或皮肤肿瘤，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)。
57、实施方案53-56中任一项的方法，其中所述肿瘤的肿瘤细胞中
(i)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型EGFR(例如具有升高的核酸或蛋白质水平的野生型EGFR)，和/或表达突变的EGFR，优选地，所述突变的EGFR包含选自R521K、L858R、T790M、G719X、C797S、Y1069C、Exon19缺失(Del19)、Exon20ins(例如S768_D770dup)中的一个或多个突变，优选地，所述突变的EGFR包含R521K/Y1069C、R521K、L858R/T790M/C797S、Del19/T790M/C797S或S768_D770dup；
(ii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型KRAS(例如具有升高的核酸或蛋白质水平的野生型KRAS)， 或表达突变的KRAS，优选地，所述突变的KRAS包含G12位或G13位突变，例如G12D或G12C；
(iii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，具有升高的核酸水平或蛋白水平的B7-H3；和/或
(iv)所述肿瘤细胞对于酪氨酸激酶抑制剂，例如对一代(厄洛替尼Erlotinib)和三代(奥希替尼Osimertinib)耐药，例如对于奥希替尼耐药。
58、实施方案57的方法，其中所述肿瘤的肿瘤细胞中表达突变的EGFR和突变的KRAS，例如包含具有R521K的突变的EGFR和具有G120D的突变的KRAS。
59、检测样品中抗原EGFR和/或B7-H3的方法，所述方法包括
(a)将样品与实施方案1-41任一项所述的双特异性抗体接触；和
(b)检测抗体或其抗原结合片段和EGFR和/或B7-H3间的复合物的形成，任选地，所述抗体被可检测的标记。
60、实施方案1-41任一项所述的抗体或其抗原结合片段，和/或实施方案42所述的分离的核酸，和/或实施方案43所述的载体，和/或实施方案44-47中任一项所述的宿主细胞，和/或实施方案49所述的免疫缀合物，和/或实施方案50或51所述的药物组合物或实施方案52的药物组合产品，在制备用于预防和/或治疗受试者疾病的药物中的用途。
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述的发明的任何和所有抗体或其片段或免疫缀合物或药物组合物或组合产品或试剂盒、方法和用途。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案。
图1显示了本发明的抗B7-H3/EGFR双特异性抗体的结构示意图。
图2显示了本发明的抗B7-H3/EGFR双特异性抗体的筛选增殖抑制实验图。
图3显示了本发明的抗B7-H3/EGFR双特异性抗体的筛选ADCC实验图。
图4显示了本发明的Hz20G5.26/Zalu bsAb的B7H3和EGFR在不同细胞系表达检测图。
图5显示了本发明的Hz20G5.26/Zalu bsAb在CRC肿瘤细胞系CCK-81、NCI-H508、HT-55和LS180中增殖抑制的实验结果图，其中抗体稀释如下：
CCK-81,NCI-H508,HT-55:抗体稀释终浓度中最高为125nM,4倍稀释；
LS180:抗体稀释终浓度中最高为300nM,4倍稀释。
图6显示了本发明的Hz20G5.26/Zalu bsAb在HNSCC肿瘤细胞系TE-1和Colo680中增殖抑制的实验结果图，其中抗体稀释如下：TE-1,Colo680:抗体稀释终浓度中最高为300nM,3.16倍稀释。
图7.1显示了本发明的Hz20G5.26/Zalu bsAb在不同的NSCLC-EGFRWT肿瘤细胞系中增殖抑制的实验结果图；
图7.2显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR经典突变肿瘤细胞系中增殖抑制的实验结果图；
图7.3显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR异常扩增肿瘤细胞系中增殖抑制的实验结果图；
图7.4显示了本发明的Hz20G5.26/Zalu bsAb在罕见NSCLC-EGFR突变型肿瘤细胞系中增殖抑制的实验结果图；
图7.5显示了本发明的Hz20G5.26/Zalu bsAb与gp120/Zalu和gp120/hz20G5.26联合的药效对比实验结果图；
其中抗体或小分子TKI(Osimertinim或Erlotinib)稀释如下：
图7.1：NCI-292，NCI-H322：抗体稀释终浓度中最高为300nM,3.16倍稀释；
图7.2：NCI-H1650，NCI-H1975：抗体稀释终浓度中最高为300nM,3.16倍稀释；
图7.3：NCI-H1703，SK-MES-1：抗体分子，稀释终浓度中最高为300nM,3.16倍稀释；小分子TKI(Osimertinim和Erlotinib)，稀释终浓度中最高为1000nM,3.16倍稀释；
图7.4：H1975(EGFRL858R/T790M/C797S):各种抗体分子和Osimertinib的稀释终浓度中最高为300nM,3.16倍稀释；H322(EGFRS768-D77dup)：各种抗体分子和Osimertinib的稀释终浓度中最高为300nM,4倍稀释；PC9+B7H3(EGFRDel19/T790M/C79S):各种抗体分子和Osimertinib的稀释终浓度中最高为100nM,4倍稀释；
图7.5：LS180和H292:抗体稀释终浓度中最高为300nM,4倍稀释；SK-MES-1:终浓度,最高300nM,3.16倍稀释。
图8显示了在H358 NSCLC细胞系中，本发明的Hz20G5.26/Zalu bsAb与AMG510小分子抑制剂联合协同用药对比实验结果图，其中
图8(A)：H358体外增殖抑制实验：各个抗体分子的最终浓度中，最高浓度为300nM，4倍稀释；
图8(B)：Hz20G5.26/Zalu bsAb与KRAS G12C抑制剂AMG510(MedChem Expresss,HY-114277)联合用药实验：AMG510+bsAb@10nm表示每个孔中加入固定的10nM Hz20G5.26/Zalu bsAb，同时又在每个孔中梯度稀释AMG510(终浓度，1000nM,4倍稀释)；仅有AMG510：终浓度最高1000nM,4倍稀释；仅有Hz20G5.26/Zalu bsAb:终浓度最高10nM,5倍稀释；共稀释5个梯度。
图9显示了在LS180 CRC细胞系，本发明的Hz20G5.26/Zalu bsAb与KRAS  G12D小分子抑制剂MRTX1133联联合协同用药对比实验结果图，其中
图9(A)：Hz20G5.26/Zalu bsAb与G12D抑制剂MRTX1133(MCE,cat:HY-134813A-10mg)联合用药实验：MRTX1133+bsAb@100nm表示每个孔中加入固定的100nM Hz20G5.26/Zalu bsAb，同时又在每个孔中梯度稀释MRTX1133(终浓度，1000nM,4倍稀释)；仅有MRTX1133：终浓度最高1000nM,4倍稀释；仅有Hz20G5.26/Zalu bsAb:终浓度最高100nM,4倍稀释；共稀释6个梯度。
图10显示了在H358细胞系，本发明的Hz20G5.26/Zalu bsAb针对EGFR磷酸化信号阻断作用的实验结果图。
图11显示了在H358细胞系，本发明的Hz20G5.26/Zalu bsAb针对EGFR配体(EGF或者TGF-α)诱导的EGFR磷酸化信号阻断作用的实验结果图。
图12.1显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFRWT肿瘤细胞系中ADCC报告实验结果图；在3个细胞系中，抗体分子稀释终浓度最高为6.25nM，4倍稀释；
图12.2显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR突变型肿瘤细胞系中ADCC报告实验结果图；在H1975细胞系中，抗体分子稀释终浓度最高为10nM，4倍稀释；且在H1975细胞系中，抗体分子稀释终浓度最高为6.25nM，4倍稀释；
图12.3显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR异常扩增型肿瘤细胞系中ADCC报告实验结果图；在SK-MES-1细胞系中，抗体分子稀释终浓度最高为6.25nM，4倍稀释；且在H1703细胞系中，抗体分子稀释终浓度最高为100nM，4倍稀释；
图12.4显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-KRAS突变型肿瘤细胞系中ADCC报告实验结果图；在H358细胞系中，抗体分子稀释终浓度最高为25nM，4倍稀释(图12.4(A))；且在H358细胞系中，抗体分子稀释终浓度最高为1.56nM，4倍稀释(图12.4(B))；
图13.1显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR野生型肿瘤细胞系中huPBMC ADCC报告实验结果图；其中，
图13.1(A)，在H292细胞系中，抗体分子稀释终浓度最高为2.5nM，4倍稀释；
图13.1(B)：在H322细胞系中，抗体分子稀释终浓度最高为100nM，4倍稀释；
图13.1(C)：在H292细胞系中，抗体分子稀释终浓度最高为100nM，4倍稀释；
图13.2显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR突变型肿瘤细胞系中huPBMC ADCC报告实验结果图；其中在H1975细胞系中，抗体分子稀释终浓度最高为35nM，4倍稀释；在H1650细胞系中，抗体分子稀释终浓度最高为100nM，4倍稀释；
图13.3显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-EGFR异常扩增型 肿瘤细胞系中huPBMC ADCC报告实验结果图；
图13.3(A)，在SK-MES-1细胞系中，抗体分子稀释终浓度最高为35nM，4倍稀释；
图13.3(B)：在H1703细胞系中，抗体分子稀释终浓度最高为25nM，4倍稀释；
图13.3(C)：在SK-MES-1细胞系中，抗体分子稀释终浓度最高为25nM，4倍稀释；
图13.4显示了本发明的Hz20G5.26/Zalu bsAb在NSCLC-KRAS突变型肿瘤细胞系中huPBMC ADCC报告实验结果图，其中分别在两个细胞系中，抗体分子稀释终浓度最高为35nM，4倍稀释。
图14显示了本发明的Hz20G5.26/Zalu bsAb药物模拟皮肤毒性的体外研究实验结果图，其中应用细胞培养基稀释抗体分子，分别在两个人皮肤细胞系中，抗体分子稀释终浓度最高为300nM，3.16倍稀释。
图15A显示了本发明的Hz20G5.26/Zalu bsAb对NCl-H292荷瘤小鼠抗肿瘤作用影响的实验结果图；图15B显示了本发明的Hz20G5.26/Zalu bsAb对NCI-H292荷瘤小鼠抗肿瘤作用的影响-生存曲线的实验结果图；图15C显示了本发明的Hz20G5.26/Zalu bsAb对NCI-H292荷瘤小鼠体重的影响的实验结果图。
图16A显示了本发明的Hz20G5.26/Zalu bsAb对SK-MES-1荷瘤小鼠的抗肿瘤作用的影响的实验结果图；图16B显示了本发明的Hz20G5.26/Zalu bsAb对SK-MES-1荷瘤小鼠体重的影响的实验结果图。
图17A显示了本发明的Hz20G5.26/Zalu bsAb对SK-MES-1荷瘤小鼠的抗肿瘤作用的影响的实验结果图；图17B显示了本发明的Hz20G5.26/Zalu bsAb对SK-MES-1荷瘤小鼠体重的影响的实验结果图。
图18A显示了本发明的Hz20G5.26/Zalu bsAb与AMG510联合对NCI-H358荷瘤小鼠抗肿瘤作用影响的实验结果图；图18B显示了本发明的Hz20G5.26/Zalu bsAb与AMG510联合对NCI-H358荷瘤小鼠体重影响的实验结果图。
发明详述
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本 发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本文中当提及“第一”“第二”时，仅为了区分两个结构域或两条链，而不以任何方式表明两个结构域的位置。
如本文所用，重链和轻链的全部可变区的氨基酸位置根据Kabat等人,Sequences of Proteins of Immunological Interest,第5版.Public Health Service,National Institutes of Health,Bethesda,MD(1991)中描述的Kabat编号系统编号并且在本文中称作“Kabat编号”。
如本文所用，用于提及抗体除可变区之外的结构域(例如恒定区，例如Fc区)中的氨基酸位置时，根据Kabat,E.A.等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)中描述的EU编号系统编号并且在本文中称作“EU编号”。当将位置编号和/或氨基酸残基赋予特定抗体同种型时，其旨在适用于任何其它抗体同种型的相应的位置和/或氨基酸残基，这是本领域技术人员己知的。
关于人免疫球蛋白轻链和重链的核苷酸序列的一般信息在Kabat,E.A.等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)中给出。
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指天然存在的包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如 下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
“半抗体”或“半聚物”指单价抗原结合多肽。在一些实施方案中，半抗体或半聚物包含VH/VL单元和任选地免疫球蛋白恒定结构域的至少一部分。在一些实施方案中，半抗体或半聚物包含与一条免疫球蛋白轻链缔合的一条免疫球蛋白重链，或其抗原结合片段。在一些实施方案中，半抗体或半聚物是单特异的，即，与单个抗原或表位结合。在一些具体的实施方案中，半抗体与EGFR结合并且不与B7-H3结合。在一些具体的实施方案中，半抗体与B7-H3结合并且不与EGFR结合。本领域技术人员将会轻易地理解，半抗体可以具有由单一可变结构域组成(例如，源自骆驼属(Camelidae))的抗原结合结构域。
术语抗体的“抗原结合片段”是与全长抗体不同的分子，其包含全长抗体的一部分，但其能结合全长抗体的抗原或与全长抗体(即与抗原结合片段所来源的全长抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、双体抗体(diabody)、单域抗体(sdAb)、纳米抗体。
“Fab片段”或“Fab”在本文中可互换使用，用于指，由两条多肽链组成的、包含免疫球蛋白重链可变区VH、重链恒定结构域CH1、轻链可变区VL和轻链恒定结构域CL的免疫球蛋白片段，其中，一条多肽链从N端到C端包含VH和选自CH1和CL的一个恒定区，另一条多肽链从N端到C端包含VL和选自CL和CH1的另一恒定区，其中所述VH结构域和VL结构域配对形成抗原结合位点。Fab’片段因在重链CH1结构域的羧基末端增加了一些残基(包括来自抗体铰链区的一个或多个半胱氨酸)而与Fab片段不同。Fab’-SH是对其中恒定结构域的半胱氨酸残基携带一个游离硫醇基的Fab’的称谓。F(ab’)2抗体片段最初是作为成对Fab’片段生成的，在Fab’片段之间具有铰链半胱氨酸。抗体片段的其它化学偶联也是已知的。
在本文中，术语“双特异性抗体”包含与两种抗原或两种表位特异性结合的抗原结合结构域。除非另外说明，否则列出的双特异性抗体名称中双特异性抗体结合的抗原的顺序是任意的。即，在一些实施方案中，术语“抗EGFR/B7-H3双特异性抗体”和“抗B7-H3/EGFR双特异性抗体”可以互换使用。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和任选地重链恒定区的至少一部分以及单个轻链可变区和任选地轻链恒定区的至少一部分。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区并且不包含多于一个单个重链可变区且不包含多于一个单个轻链可变区。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区，并且其中第一半抗体与第一抗原/表位结合且不与第二抗原结合并且第二半抗体与第二抗原/表位结合且不与第一抗原结合。
本发明的多特异性抗体可以包含接头。如本文所用的术语“接头”是指使得能够直接连接多特异性抗体的不同部分的任何分子。在多特异性抗体不同部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。在一些实施方案中，根据本发明的术语“肽接头”是指氨基酸的序列，其中所述序列将多特异性 抗体的各个部分的氨基酸序列连接在一起。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。肽接头可以主要包括或可以不主要包括以下氨基酸残基：Gly、Ser、Ala或Thr。有用的接头包括甘氨酸-丝氨酸聚合物，包括例如(GS)n、(GSGGS)n、(GGGGS)n、(GGGS)n和(GGGGS)nG，其中n是至少1(且优选2、3、4、5、6、7、8、9、10)的整数。有用的接头还包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。
术语“Fc结构域”或“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如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(也称作EU索引)进行编号。然而，Fc区的C端赖氨酸(Lys447)可以存在或可以不存在。两个Fc区可以实现二聚化以构成二聚Fc，两个不同的Fc异二聚化形成异二聚Fc。在本文中，术语“Fc区”、“Fc部分”和“二聚Fc(例如异二聚Fc)”不包括免疫球蛋白的重链可变区VH和轻链可变区VL以及重链恒定区CH1和轻链恒定区CL，但在一些情况下可以包括在重链恒定区N端的铰链区。在一个实施方案中，人IgG重链Fc区从Asp221或从Cys226或从Asp231延伸至重链的羧基端。在一个实施方案中，Fc区是人Fc区。在一个实施方案中，Fc区属于人IgG4亚类。在一个实施方案中，Fc区属于人IgG1亚类。
在本文中，“Fc二聚化”是指两个Fc区二聚化后形成二聚体。“Fc异二聚化”是指两个不同的Fc区二聚化后形成二聚体。异二聚化的Fc区在双特异性抗体或多特异性抗体中构成Fc支架。因此，“异二聚体Fc支架”是指包含两个不同的Fc区或由两个不同的Fc区二聚化后形成的支架，其可以在其N末端或C末端连接结合抗原的结构域(例如可以与靶分子结合的抗体的重链和/或轻链可变区或抗体的抗原结合片段，或可以与靶分子结合的配体或受体的可溶性部分)，用于构成多特异性抗体例如双特异性抗体。
在氨基酸突变用(原始氨基酸,氨基酸位置,突变氨基酸)来表示。例如，当突变位点位于Fc区时，“T366W”是指，位于EU编号位置366的T被W取代。当述及突变组合时，组合的突变之间用“和”或“/”连接。“R521K/Y1069C”表示同时包含突变R521K和Y1069C。应当注意，在描述突变时，具体的位置同时涵盖其在其他多肽链上所对应的氨基酸位置。例如，如果提及C220，其涵盖IgG1重链通过EU编号下的第220位氨基酸，以及在其他重链中对应的氨基酸，例如在IgG2、IgG3或IgG4中的第131位氨基酸。在描述突变时，在一个具体位置上的原始氨基酸可以是描述的氨基酸，或者也可以是在对应位置上的其他氨基酸。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的 区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变区内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变区内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的CDR通过Kabat规则确定边界，或通过AbM规则确定，或通过其组合确定规则。
在本发明的一个实施方案中，本发明中与B7-H3结合的抗原结合区中的VH的HCDR1是通过AbM规则确定，HCDR2和HCDR3是通过Kabat规则确定， VL的CDR是通过Kabat规则确定。
在本发明的一个实施方案中，本发明中与EGFR结合的抗原结合区中的VH和VL的CDR是通过Kabat规则确定。
术语“铰链区”指抗体重链多肽的部分，所述部分在野生型抗体重链中连接CH1区和CH2区，例如，IgG1铰链区，例如根据EU编号，D221到P230的序列。可以通过与IgG1亚类序列的铰链区半胱氨酸残基对齐，确定其他IgG亚类的铰链区。
如本文所用的术语“结合位点”或“抗原结合位点”或“抗原结合区”表示抗体分子，例如多特异性抗体，例如双特异性抗体的结合特定靶标或抗原的任何部分。抗原结合区可以是例如抗体或免疫球蛋白本身或抗体片段。这种抗原结合区可以具有或可以不具有独立于BsAB的剩余部分的三级结构，并且可以作为单独实体结合或不结合其抗原/表位。在一些实施方案中，抗原结合区包括由抗体轻链可变区(VL)和抗体重链可变区(VH)组成的VH/VL对、衍生自骆驼科重链抗体的重链可变区、来自鲨鱼科动物的IgNAR的VH样单结构域(v-NAR)、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变区。
当在多特异性抗体或双特异性抗体中提及“第一抗原结合区”时，是指结合第一抗原的结合区，而不意图限制抗体中所含的该抗原结合区的个数，例如多特异性抗体或双特异性抗体中可以包含1个或1个以上的第一抗原结合区。例如，双特异性抗体包含第一抗原结合区和第二抗原结合区，但是可以包含1个或1个以上的第一抗原结合区和1个或1个以上的第二抗原结合区。
当提及“抗原结合区来源于抗体”时，是指构成该抗原结合区的结合结构域为或衍生自该抗体的特异性结合抗原的结合结构域，例如，抗原结合区的特异性结合抗原的片段例如Fab为或衍生自该抗体的相应片段例如Fab，或抗原结合区的重链可变区和/或轻链可变区为或衍生自该抗体的重链可变区和/或轻链可变区，或者该抗原结合区的1个、2个、3个、4个、5个或6个CDR为该抗体的CDR。在一些实施方案中所述抗原结合区是抗体的抗原结合片段。
如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。本文提供的抗体通常是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原和第二抗原的结合特异性。
术语“免疫球蛋白分子”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键键合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个重链可变区(VH)，也称作重链可变结构域，随后是三个重链恒定结构域(CH1、CH2和CH3)。类似地，从N端至C端，每条免疫球蛋白轻链具有一个轻链可变区(VL)，也称作轻链可变结构域，随后是一个轻链恒定结构域(CL)。免疫球蛋白的重链可以归属5个类别之一，称作α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，其中某些类别可以进一步划分成亚类，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。免疫球蛋白的轻链可以基于其恒定结构域的氨基酸序列而划分成两种 类型之一，称作κ和λ。免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和一个Fc结构域组成。
术语“效应子功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白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)、Fc受体结合作用、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、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。
术语“…价”抗体指抗体分子中存在的抗原结合位点的数目。“二价”、“三价”和“四价”抗体指抗体分子中分别存在2个抗原结合位点、3个抗原结合位点和4个抗原结合位点。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗原结合位点与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法测定如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由解离常数(KD)表示，解离常数是解离速率常数和缔合速率常数(分别是Kdis和Kon)的比例。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
“knob-in-hole”突变或“结入扣”突变在本文中用于指，利用“结入扣”技术，在第一Fc多肽和第二Fc多肽中分别引入突变，以在第一Fc多肽的界面上与在第二Fc多肽的界面上形成凸起(“结(knob)”)和互补的空穴(“扣(hole)”)。本领域已知，“结入扣”技术可在抗体分子的不同链之间改造界面，以促进抗体分子的各链正确缔合。通常，该技术涉及在一条链的界面引入“凸起/结”，在欲与之配对的另一条链的界面引入相应的“空穴/扣”，使得凸起可置于空穴中。一个优选的界面包含一条链的重链恒定结构域的CH3结构域和欲与之配对的另一条链的重链恒定结构域的CH3结构域。可通过将来自一条链的重链恒定结构域的CH3结构域的界面的小氨基酸侧链替换为较大的侧链(例如酪氨酸或色氨酸)来构建凸起。通过将大氨基酸侧链替换为较小的侧链(例如丙氨酸或苏氨酸)，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。另一可选的界面是上文所述Fab片段的包含轻链的CL结构域和重链的CH1结构域，通过构建凸起-空穴相互作用促进Fab片段的两条链之间发生正确的异二聚化。
在本文中，抗体恒定区或抗体恒定结构域，包括CH1，CL和Fc结构域以及构成Fc结构域的CH2,CH3和可选的CH4结构域，可以根据抗体分子的预期功能进行选择。例如，恒定区可以是IgA,IgD,IgE,IgG或IgM区，尤其是人IgG的免疫球蛋白恒定结构域，例如，人IgG1，IgG2,IgG3或IgG4的恒定结构域，优选人IgG1的恒定结构域。再例如，抗体的Fab片段可以包含来自IgG1的CH和CL恒定区。再例如，抗体的Fc区可以包含来自IgG1的CH2和CH3结构域。免疫球蛋白恒定区可以具有天然序列或变体序列。
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。在本文的一些实施方案中，第一抗原、第二抗原是两种不同的抗原。
术语“肿瘤相关抗原”或“癌症抗原”可互换地指与正常细胞相比，优选在癌细胞表面完全或作为片段(例如，MHC/肽)表达的分子(通常为蛋白质、碳水化合物或脂质)，并且所述分子可用在药剂对癌细胞的优先靶向中。
术语“免疫检查点分子”意指免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。
“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
如本文中使用的，术语“EGFR”是指表皮生长因子受体，是一种酪氨酸激酶受体，是一个巨大的跨膜糖蛋白，分子量约为170KDa，属于ErbB受体家族的一个成员，是NSCLC最常见的癌变驱动基因，EGFR激活突变区主要发生在EGFR外显子18-21酪氨酸激酶结构域，EGFR抗体的结合区主要位于EGFR胞外配体结构域区，可避免耐药突变的发生；同时，EGFR抗体可通过抑制EGFR与配体的结合而抑制肿瘤细胞的生长，也可利用自身特定ADCC(抗体依赖细胞介导的细胞毒性作用)与免疫细胞共同杀伤肿瘤，这样可通过多种作用机制，共同发挥抗肿瘤杀伤作用。
术语“B7-H3”、“B7H3”和“CD276”在本文中可互换使用。B7-H3是属于B7/CD28超家族成员的I型跨膜糖蛋白，与PD-L1的胞外结构域在序列上相似。B7-H3具有316个氨基酸，其中包含一个推定的由28个氨基酸组成的信号肽，一个217个氨基酸组成的胞外区以及一个跨膜区和一个45个氨基酸组成的胞质结构域，分子量约为45-66kDa。在人体中，由于外显子复制，B7-H3的胞外结构可以为IgV-IgC样结构域(2Ig-B7-H3)，或者为IgV-IgC-IgV-IgC样结构域(4Ig-B7-H3)。食蟹猴B7-H3的序列与其人类对应物具有约90％的同源性。
“效应子功能”指那些可归于抗体Fc区且随抗体同种型而变化的生物学活性。抗体效应子功能的实例包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“GlymaxX技术”是从ProBioGen公司开发的增加双抗ADCC效应的技术平台，其是一个稳定过表达细菌RMD蛋白的CHO宿主细胞，通过干扰底物GDP-fucose的形成，最终阻断Fc区域的岩藻糖修饰，以此增强ADCC效应(WO2011035884A1)。
术语“有效量”指本发明的抗体或片段或缀合物或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确 定：诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。可选地，可以通过检验化合物抑制的能力评价组合物的这种特性，所述抑制在体外通过熟练技术人员已知的测定法。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括上皮来源的癌症，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)，包括那些癌症的转移性形式。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是 良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
术语“感染性疾病”是指病原体引发的疾病，包括例如病毒感染、细菌感染、真菌感染或者原生动物例如寄生虫感染。
术语“慢性感染”是指这样的感染，其中传染原(例如，病原体如病毒、细菌、原生动物例如寄生虫、真菌或诸如此类)已经在感染的宿主中诱导了免疫应答，但尚未如在急性感染过程中一样被从宿主中清除或消除。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体，以及通过与直接被标记的另一种试剂反应来间接标记探针或抗体。间接标记的实例包括使用荧光标记的二级抗体进行的一级抗体的检测和具有生物素的DNA探针的末端标记，使得其可以用荧光标记的链霉抗生素蛋白来检测。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。
如下进行序列之间序列同一性的计算。为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com 可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、载体、赋形剂或稳定剂等。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数或改善可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
本文所述的术语“治疗剂”涵盖在预防或治疗疾病，例如肿瘤(例如癌症)和感染(例如慢性感染)中有效的任何物质，包括抗血管发生剂、化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
“化疗剂”包括在治疗癌症中有用的化学化合物，包括但不限于抗肿瘤剂，包括烷化剂；抗代谢物；天然产物；抗生素；酶；杂类试剂；激素和拮抗剂；抗雌激素；抗雄激素；以及非类固醇抗雄激素等。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包括免疫检查点分子抑制剂和共刺激性分子激活剂。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。
如本文所用，术语“细胞毒性剂”指抑制或阻止细胞功能和/或造成细胞死亡或破坏的物质。细胞毒性剂包括但不限于放射性同位素；化疗药或药物；生长抑制剂；酶及其片段如溶核酶；抗生素；毒素如细菌源、真菌源、植物源或动物源的小分子毒素或酶活性毒素，包括其片段和/或变体。
“肿瘤免疫逃逸”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。
“免疫原性”指特定物质引发免疫应答的能力。肿瘤是免疫原性的，并且增强肿瘤免疫原性有助于通过免疫应答清除肿瘤细胞。
如本文中使用的，“抗体的激动剂活性”指抗体能活化它所结合的抗原的生物学活性。
“抗血管发生剂”指阻断或在某种程度上干扰血管发育的化合物。抗血管发生剂可以是例如结合涉及促进血管发生的生长因子或生长因子受体的小分子或抗体。
术语“药物组合或组合产品”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的免疫缀合物、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”或“联合疗法”是指施用两种或更多种治疗剂以治疗如本公开所述的癌症或感染。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用或分开施用或依次施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。在一些实施方案中，施用还包括以大致相同的时间，或在不同的时间以顺序的方式，使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
II.本发明的抗体分子
本发明提供了一种新型的抗体分子，其能够用于多种疾病的免疫治疗、预防和/或诊断。本发明的抗体分子包含至少2个、3个或4个抗原结合区，其能够作为双特异性抗体或多特异性抗体发挥作用，优选地，其能够作为双特异性抗体发挥作用。
在一些实施方案中，本发明的双特异性抗体或多特异性抗体包含针对EGFR的第一结合特异性和针对B7H3的第二结合特异性，以及任选地其他结合特异性。
因此，本发明的一个方面涉及一种双特异性抗体，其包含
第一抗原结合区和第二抗原结合区，其中第一抗原结合区特异性结合EGFR，和/或第二抗原结合区特异性结合B7-H3。
可以使用本领域已知的双特异性抗体形式或技术来制备本发明的双特异性抗体。可以在本发明的背景下使用的具体示例性双特异性形式参见例如，Labrijn,et al.Bispecific antibodies:a mechanistic review of the pipeline.Nature Reviews Drug Discovery,2019,18(8):1-24。在一个实施方案中，双特异性抗体形式包含IgG样抗体(Fan等人(2015)Journal of Hematology&Oncology.8:130)。最常见的IgG样抗体类型包含两个Fab区域和两个Fc区域,每个Fab的重链和轻链可以来自单独的单克隆抗体。在一些实施方案中，本发明的双特异性抗体是IgG样双特异性抗体，其包含特异性结合EGFR的Fab片段作为一个抗原结合区和特异性结合B7H3的Fab片段作为另一个抗原结合区。
特异性结合EGFR的抗原结合区
在一些实施方案中，特异性结合EGFR的抗原结合区来源于特异性结合EGFR的抗体，例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的1、2、3、4、5、或6个CDR。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的1、2和3个重链可变区CDR，即HCDR1、HCDR2和HCDR3。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的1、2和3个轻链可变区CDR，即LCDR1、LCDR2和LCDR3。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的3个重链可变区CDR和3个轻链可变区CDR。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的重链可变区和轻链可变区。
在一些实施方案中，特异性结合EGFR的抗原结合区包含已知的特异性结合EGFR的抗体例如WO02100348A2中公开的EGFR抗体，例如Zalutumumab单抗的Fab。
在一些实施方案中，特异性结合EGFR的抗原结合区包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，特异性结合EGFR的抗原结合区包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。在一些实施方案中，特异性结合EGFR的抗原结合区包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。
在一些方面中，特异性结合EGFR的抗原结合区包含重链可变区(VH)。在一些方面中，特异性结合EGFR的抗原结合区包含轻链可变区(VH)。在一些方面中，特异性结合EGFR的抗原结合区包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，特异性结合EGFR的抗原结合区的重链可变区
(i)包含与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:1的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:1的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，特异性结合EGFR的抗原结合区的轻链可变区
(i)包含与SEQ ID NO:2的氨基酸序列具有至少80％、85％、90％、91％、92％、 93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:2的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:2的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，特异性结合EGFR的抗原结合区的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3选自
(i)如SEQ ID NO:1中所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，特异性结合EGFR的抗原结合区的3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3选自
(i)如SEQ ID NO:2所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，特异性结合EGFR的抗原结合区包含3个由SEQ ID NO:1的氨基酸序列组成的重链可变区所含的互补决定区(HCDR)以及3个由SEQ ID NO:2的氨基酸序列组成的轻链可变区所含的互补决定区(LCDR)。
在一些实施方案中，HCDR1包含SEQ ID NO:9的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO:9的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO:10的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO:10的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR3包含SEQ ID NO:11的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO:11的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO:12的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO:12的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR2包含SEQ ID NO:13的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO:13的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO:14的氨基酸序列，或由所述氨基酸序列组成，或者LCDR3包含与SEQ ID NO:14的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含如上所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、和/或LCDR3。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含如上所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、和LCDR3。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中
HCDR1包含SEQ ID NO:9的氨基酸序列或由所述序列组成；
HCDR2包含SEQ ID NO:10的氨基酸序列或由所述序列组成；
HCDR3包含SEQ ID NO:11的氨基酸序列或由所述序列组成；
LCDR1包含SEQ ID NO:12的氨基酸序列或由所述序列组成；
LCDR2包含SEQ ID NO:13的氨基酸序列或由所述序列组成；且
LCDR3包含SEQ ID NO:14的氨基酸序列或由所述序列组成。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含如SEQ ID NO:9所示的HCDR1、如SEQ ID NO:10所示的HCDR2、如SEQ ID NO:11所示的HCDR3；如SEQ ID NO:12所示的LCDR1、如SEQ ID NO:13所示的LCDR2和如SEQ ID NO:14所示的LCDR3。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含VH和VL，其中
所述VH含有SEQ ID NO:1所示的氨基酸序列或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，和所述VL含有SEQ ID NO:2所示的氨基酸序列或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，特异性结合EGFR的抗原结合区包含VH和VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:2。
特异性结合B7-H3的抗原结合区
在一些实施方案中，特异性结合B7-H3的抗原结合区来源于特异性结合B7-H3的抗体，例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗(例如本发明实施例中提及的Hz20G5.26)。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的1、2、3、4、5、或6个CDR。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的1、2和3个重链可变区CDR，即HCDR1、HCDR2和HCDR3。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的1、2和3个轻链可变区CDR，即LCDR1、LCDR2和LCDR3。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的3个重链可变区CDR和3个轻链可变区CDR。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的重链可变区和轻链可变区。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含已知的特异性结合B7-H3的抗体例如PCT/CN2021/140449中描述的B7-H3抗体，例如其中编号为Hz20G5的单抗的Fab。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，特异性结合B7-H3的抗原结合区包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。在一些实施方案中，特异性结合B7-H3的抗原结合区包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。
在一些方面中，特异性结合B7-H3的抗原结合区包含重链可变区(VH)。在一些方面中，特异性结合B7-H3的抗原结合区包含轻链可变区(VH)。在一些方面中，特异性结合B7-H3的抗原结合区包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，特异性结合B7-H3的抗原结合区的重链可变区
(i)包含与SEQ ID NO:3、5或7的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:3、5或7的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:3、5或7的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，特异性结合B7-H3的抗原结合区的轻链可变区
(i)包含与SEQ ID NO:4、6或8的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:4、6或8的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:4、6或8的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，特异性结合B7-H3的抗原结合区的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3选自
(i)如SEQ ID NO:3、5或7中所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述HCDR1采用Abm方案，所述HCDR2、HCDR3通过Kabat方案确定。
在一些实施方案中，特异性结合B7-H3的抗原结合区的3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3选自
(i)如SEQ ID NO:4、6或8所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，特异性结合B7-H3的抗原结合区包含3个由SEQ ID NO:3、5或7的氨基酸序列组成的重链可变区所含的互补决定区(HCDR)以及3个由SEQ ID NO:4、6或8的氨基酸序列组成的轻链可变区所含的互补决定区(LCDR)。
在一些实施方案中，HCDR1包含SEQ ID NO:15、21或27的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO:15、21或27的氨 基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO:16、22或28的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO:16、22或28的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR3包含SEQ ID NO:17、23或29的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO:17、23或29的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO:18、24或30的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO:18、24或30的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR2包含SEQ ID NO:19、25或31的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO:19、25或31的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO:20、26或32的氨基酸序列，或由所述氨基酸序列组成，或者LCDR3包含与SEQ ID NO:20、26或32的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含如上所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、和/或LCDR3。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含如上所述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、和LCDR3。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中
(i)HCDR1包含如SEQ ID NO:15所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:16所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:17所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:18所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:19所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:20所示的氨基酸序列或由所述序列组成；或，
(ii)HCDR1包含如SEQ ID NO:21所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:22所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:23所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:24所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:25所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:26所示的氨基酸序列或由所述序列组成；或，
(iii)HCDR1包含如SEQ ID NO:27所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:28所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:29所示的氨基酸序列或由所述序列组成，
LCDR1包含如SEQ ID NO:30所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:31所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:32所示的氨基酸序列或由所述序列组成。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含
(i)如SEQ ID NO:15所示的HCDR1、如SEQ ID NO:16所示的HCDR2、如SEQ ID NO:17所示的HCDR3；如SEQ ID NO:18所示的LCDR1、如SEQ ID NO:19所示的LCDR2和如SEQ ID NO:20所示的LCDR3；
(ii)如SEQ ID NO:21所示的HCDR1、如SEQ ID NO:22所示的HCDR2、如SEQ ID NO:23所示的HCDR3；如SEQ ID NO:24所示的LCDR1、如SEQ ID NO:25所示的LCDR2和如SEQ ID NO:26所示的LCDR3；或
(iii)如SEQ ID NO:27所示的HCDR1、如SEQ ID NO:28所示的HCDR2、如SEQ ID NO:29所示的HCDR3；如SEQ ID NO:30所示的LCDR1、如SEQ ID NO:31所示的LCDR2和如SEQ ID NO:32所示的LCDR3。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含VH和VL，其中
(i)所述VH包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)所述VH包含SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(iii)所述VH包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:8所示的氨基酸序列或与SEQ ID NO:8的氨基酸序列具有至少80％、85％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，特异性结合B7-H3的抗原结合区包含VH和VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
SEQ ID NO:3和SEQ ID NO:4；
SEQ ID NO:5和SEQ ID NO:6；或
SEQ ID NO:7和SEQ ID NO:8。
本发明还涉及包含上述特异性结合B7-H3的抗原结合区的特异性结合B7-H3的抗体，例如包含本发明所定义的HCDR1、HCDR2和HCDR3，以及LCDR1、LCDR2和LCDR3的抗体；或包含本发明所定义的VH或VL的抗体，或包含本发明所定义的VH和VL的抗体。
Fc区
在一些实施方案中，本发明的抗体分子，例如多特异性抗体(例如双特异性抗体)还包含Fc区，其中所包含的Fc区可以相同或不同。
在一些实施方案中，所述Fc区具有低岩藻糖基化，例如通过GlymaxX技术处理后获得的低岩藻糖基化。
在一些实施方案中，本发明的抗体分子包含第一Fc区和第二Fc区，其中第一Fc区和第二Fc区相同或不同。
在一些实施方案中，第一Fc区和第二Fc区不同，其能够二聚化形成异二聚体Fc支架。
在本文中，Fc区是指含有至少一部分的恒定区的免疫球蛋白重链的C-端区域，并且可以包括天然序列Fc区和变体Fc区。天然序列F区涵盖天然存在的各种免疫球蛋白Fc序列，例如各种Ig亚型以及其同种异型的Fc区(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions，20 October 2014,doi:10.3389/fimmu.2014.00520)。在一些实施方案中，本发明的Fc区包含抗体CH2和CH3。在一些实施方案中，抗体Fc区还可以在N端带有IgG铰链区或部分IgG铰链区，例如，IgG1铰链区或部分IgG1铰链区，例如根据EU编号，D221到P230的序列。在所述铰链区中可以含有突变。
除非本文中另外指出，Fc区中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。
在一些实施方案中，Fc区为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc或人IgG4 Fc。在一个实施方案中，Fc区包含氨基酸序列SEQ ID NO:46或47或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由所述的氨基酸序列组成。
如本领域技术人员理解的，为了促进本发明的多特异性抗体作为异二聚体的形成，本发明多特异性抗体所包含的Fc区可以包含利于第一Fc区与第二Fc区异二聚化的突变。在一个实施方案中，在两个Fc区的CH3区中引入突变。
本领域已知促进Fc区异二聚化的方法。例如，第一Fc区的CH3区和第二Fc区的CH3区以互补方式进行工程化改造使得每个CH3区(或包含它的重链)不再能与其自身同二聚化但被迫与互补工程化改造的其它CH3区异二聚化(使得第一和第二Fc区的CH3区异二聚化且两个第一CH3区或两个第二CH3区之间不形成同二聚体)。
优选地，基于Knob-in-Hole技术，在第一Fc区和第二Fc区中引入相应的Knob突变和Hole突变。此技术参见例如US 5,731,168；US 7,695,936；Ridgway等,Prot Eng 9,617-621(1996)和Carter,J Immunol Meth 248,7-15(2001)。
在一个特定的实施方案中,在一个Fc区的CH3区中,第366位的苏氨酸残基用色氨酸残基替换(T366W)(knob突变)；而在另一个Fc区的CH3区中,第407位的酪氨酸残基用缬氨酸残基替换(Y407V)(hole突变)，任选地366位的苏氨酸残基用丝氨酸残基替换(T366S)且第407位的酪氨酸残基用缬氨酸残基替换(Y407V)(编号方式依照EU索引)。
在又一个实施方案中,在一个Fc区的CH3区中，第366位的苏氨酸残基用色氨酸残基替换(T366W)且第354位的丝氨酸残基用半胱氨酸残基替换(S354C)或第356位的谷氨酸残基用半胱氨酸残基替换(E356C)(特别是,第354位的丝氨酸残基用半胱氨酸残基替换)；而在另一个Fc区的CH3区中，第407位的酪氨酸残基用缬氨酸残基替换(Y407V)(hole突变)，任选地366位的苏氨酸残基用丝氨酸残基替换(T366S)且第368位的亮氨酸残基用丙氨酸残基替换(L368A)(编号方式依照EU索引)，任选地第349位的酪氨酸残基用半胱氨酸残基替换(Y349C)(编号方式依照EU索引)。
在一个具体的实施方案中，一个Fc区包含氨基酸替代T366W,且另一个Fc区包含氨基酸替代T366S,L368A和Y407V(编号方式依照EU索引)。
在一个具体的实施方案中，一个Fc区包含氨基酸替代S354C和T366W,且另一个Fc区包含氨基酸替代Y349C,T366S,L368A和Y407V(编号方式依照EU索引)。
还可以基于Innobody技术，在第一Fc区和第二Fc区中引入相应的突变。此技术参见例如PCT/CN2021/143141。
在一个特定的实施方案中，
所述第一CH3区包含S364R/K突变(优选地S364R)，以及任选的一个或多个其他突变。在一些实施方案中，所述第二CH3区包含K370S/T/A/V突变(优选地K370S)，以及任选的一个或多个其他突变。在一些实施方案中，所述第一CH3区包含S364R/K突变，且所述第二CH3区包含K370S/T/A/V突变。在一些实施方案中，所述第一CH3区包含S364R突变，且所述第二CH3区包含K370S突变。
在一些实施方案中，所述第一CH3区包含S364R/K(优选地S364R)和D399K/R(优选地D399K)突变。在一些实施方案中，所述第二CH3区包含K370S/T/A/V(优选地K370S)突变和K409D/E(优选地K409D)突变。在一些实施方案中，所述第一CH3区包含S364R/K+D399K/R，且所述第二CH3区包含K370S/T/A/V+Y349T/S/A/V。在一些实施方案中，所述第一CH3区包含S364R +D399K，且所述第二CH3区包含K370S+Y349T。在一些实施方案中，所述第一CH3区还包含E375N/Q(优选地E375N)和/或T350V/A(优选地T350V)。在一些实施方案中，所述第二CH3区还包含K409D/E(优选地K409D)、Q347D/E(优选地Q347D)和/或T350V/A(优选地T350V)。
在一些实施方案中，所述第一CH3区包含S364R+D399K，且所述第二CH3区包含K370S+Y349T+K409D。在一些实施方案中，所述第一CH3区还包含E357N。在一些实施方案中，所述第二CH3区还包含Q347D。在一些实施方案中，所述第一CH3区还包含E357N，且所述第二CH3区还包含Q347D。在一些实施方案中，所述第一CH3区和所述第二CH3区还分别包含T350V，或都包含T350V。
因此，在一些实施方案中，所述第一CH3区包含S364R+D399K，且所述第二CH3区包含K370S+Y349T+K409D+Q347D。在一些实施方案中，所述第一CH3区包含S364R+D399K+E357N，且所述第二CH3区包含K370S+Y349T+K409D+Q347D。在一些实施方案中，所述第一CH3区包含S364R+D399K+E357N+T350V，且所述第二CH3区包含K370S+Y349T+K409D+Q347D+T350V。
在一些实施方案中，所述第一CH3区包含K409E/D(优选地K409E)。在一些实施方案中，所述第二CH3区包含D399K/R(优选地D399K)或K370T/S/A/V(优选地K370T)。在一些实施方案中，所述第一CH3区包含K409E/D(优选地K409E)，且所述第二CH3区包含D399K/R(优选地D399K)。在一些实施方案中，所述第一CH3区还包含T411R/K(优选地T411R)。在一些实施方案中，所述第二CH3区还包含K370T/S/A/V(优选地K370T)。在一些实施方案中，所述CH3区包含K409E/D+T411R/K，且所述第二CH3区包含D399K/R+K370T/S/A/V。在一些实施方案中，所述CH3区包含K409E+T411R，且所述第二CH3区包含D399K+K370T。
在一些具体的实施方案中，所述第一和第二CH3区具有以下突变组合：
在一个实施方案中，一个Fc区的CH3包含S364R和D399K突变，且另一个Fc区的CH3突变包含Y349T、K370S和K409D突变。
因此，在一个具体的实施方案中，本发明的多特异性抗体包含的两个Fc区异二聚化，其中
a)一个Fc区多肽包含突变T366W，而另一个Fc区多肽包含T366S,L368A和Y407V，或
b)一个Fc-区多肽包含突变S354C和T366W，而另一个Fc-区多肽包含突变Y349C,T366S,L368A和Y407V，或
c)一个Fc-区多肽包含突变S364R和D399K，而另一个Fc-区多肽包含突变Y349T、K370S和K409D。
因此，在一个具体的实施方案中，本发明的多特异性抗体包含的两个Fc区异二聚化，其中一个Fc区多肽包含SEQ ID NO:49或50所示的氨基酸序列或由其组成，而另一个Fc区多肽包含SEQ ID NO:52或53所示的氨基酸序列或由其组成。
因此，在一个具体的实施方案中，本发明的多特异性抗体包含的两个Fc区异二聚化，其中一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，而另一个Fc区多肽包含与SEQ ID NO:52或53所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列。
因此，在一个具体的实施方案中，本发明的多特异性抗体包含的两个Fc区异二聚化，其中一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变Y349T、K370S和K409D，而另一个Fc区包含与SEQ ID NO:52或53所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变S364R和D399K。
在一些实施方案中,Fc区还包含其他利于异二聚体纯化的突变。
抗体修饰
在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表A所示：
表A

在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体糖基化的程度。
可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。例如，可以消除恒定区(例如Fc区)中的岩藻糖基化，例如获得低或无岩藻糖基化。
这类改变的糖基化模式已显示可增加抗体的ADCC能力。可通过例如在具有改变的糖基化体系的宿主细胞中表达抗体来实现这类糖类修饰。举例而言，在α-1,6-岩藻糖基转移酶8(Fut8)敲除的宿主细胞表达无岩藻糖基化的抗体(WO2000061739)。举例而言，通过在引入编码与糖链修饰相关的酶RMD的宿主细胞中获得低或者无盐藻糖基化的抗体(GlymaxX技术，ProBioGen AG，参见专利公开号：WO2011035884A1)。备选地，可使用岩藻糖苷酶切除抗体的岩藻糖残基；举例而言，岩藻糖苷酶α-L-岩藻糖苷酶自抗体去除岩藻糖基残基(Tarentino等人(1975)Biochem.14:5516-23)。
示例性双特异性抗体分子
在一些实施方案中，本发明的抗B7-H3/EGFR双特异性抗体具有如下一种或多种性质：
(1)本发明的双特异性抗体一方面可以阻断EGFR配体与EGFR的结合，抑制生物信号传递，阻断肿瘤相应生物活性；另一方面会刺激EGFR内吞而最终被细胞内溶酶体等降解；
(2)本发明的双特异性抗体采用了对EGFR低亲和力的EGFR抗体亲本序列，极大降低了系列EGFR单克隆抗体对皮肤等正常上皮组织带来的毒副作用；
(3)本发明的双特异性抗体在EGER低亲和力的基础上引用B7-H3高亲和力的抗体亲本序列，极大提高了EGFR信号阻断活性，提高了本发明双特异性抗体的药效生物活性和药效安全窗口；
(4)本发明的双特异性抗体为低岩藻糖基化的抗体；
(5)本发明的双特异性抗体具有较高的药效生物活性和安全性；
(6)本发明的双特异性抗体具有优良的肿瘤杀伤和抑制效果；
(7)本发明的双特异性抗体具有优良的ADCC体内外药效活性；
(8)本发明的双特异性抗体与KRAS小分子抑制剂联用具有优良的抗肿瘤效果，特别是协同效果。
在一些实施方案中，在本发明的抗体分子中，特异性结合EGFR的抗原结合区与重链恒定区CH连接，例如其中的重链可变区与重链恒定区CH连接，例如其中的重链可变区的C末端与重链恒定区CH的N末端连接。在一些实施方案中，在本发明的抗体分子中，特异性结合EGFR的抗原结合区与轻链恒定区连接，例如其中的轻链可变区与轻链恒定区CL连接,例如其中的轻链可变区的C末端与轻链恒定区CL的N末端连接。在一些实施方案中，在本发明的抗体分子中，特异性结合EGFR的抗原结合区中，重链可变区与重链恒定区CH连接且轻链可变区与轻链恒定区CL连接。
在一些实施方案中，在本发明的抗体分子中，特异性结合B7-H3的抗原结合区与重链恒定区CH连接，例如其中的重链可变区与重链恒定区CH连接，例如其中的重链可变区的C末端与重链恒定区CH的N末端连接。在一些实施方案中，在本发明的抗体分子中，特异性结合B7-H3的抗原结合区与轻链恒定区连接，例如其中的轻链可变区与轻链恒定区CL连接,例如其中的轻链可变区的C末端与轻链恒定区CL的N末端连接。在一些实施方案中，在本发明的抗体分子中，特异性结合B7-H3的抗原结合区中，重链可变区与重链恒定区CH连接且轻链可变区与轻链恒定区CL连接。
在一些实施方案中，所述重链恒定区包含CH1和Fc区，经由或不经由铰链区连接。
在一些实施方案中，所述重链恒定区为IgG1,IgG2,IgG3，或IgG4的重链恒定区，例如人IgG1,人IgG2,人IgG3，或人IgG4的重链恒定区。在一些方案中，所述CH1包含SEQ ID NO:42所示的氨基酸序列或与SEQ ID NO:42的氨基酸 序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述轻链恒定区为kappa轻链恒定区或lambda轻链恒定区，例如人Kappa或人Lambda轻链恒定区。在一些实施方案中，所述轻链恒定区包含SEQ ID NO:54所示的氨基酸序列或与SEQ ID NO:54的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些优选的实施方案中，本发明提供了一种双特异性抗体分子，其包含一个特异性结合EGFR的Fab片段，一个特异性结合B7-H3的Fab片段，以及Fc二聚体，其中特异性结合EGFR的Fab片段与一个Fc形成特异性结合EGFR的半抗体，且特异性结合B7-H3的Fab片段与一个Fc形成特异性结合B7-H3的半抗体。
在一些实施方案中，所述双特异性抗体为IgG样抗体，其具有如图1所示的构型。
在一些可实施方案中，所述双特异性抗体包含重链1和轻链1，以及重链2和轻链2，其中重链1和轻链1构成第一半抗体，且重链2和轻链2构成第二半抗体；其中重链1包含第一抗原结合区的重链可变区和第一重链恒定区；轻链1包含第一抗原结合区的轻链可变区和第一轻链恒定区；且重链2包含第二抗原结合区的重链可变区和第二重链恒定区；轻链2包含第二抗原结合区的轻链可变区和第二轻链恒定区。
在一些可实施方案中，所述双特异性抗体中，重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％。90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，重链2包含SEQ ID NO:35、37或39所示的氨基酸序列或与SEQ ID NO:35、37或39所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，轻链2包含SEQ ID NO:36、38或40所示的氨基酸序列或与SEQ ID NO:36、38或40所示的氨基酸序列具 有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，
(1)重链2包含SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:36所示的氨基酸序列或与SEQ ID NO:36所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(2)重链2包含SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:38所示的氨基酸序列或与SEQ ID NO:38所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(3)重链2包含SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:40所示的氨基酸序列或与SEQ ID NO:40所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在一些可实施方案中，所述双特异性抗体中，
重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且重链2和轻链2分别包含如下的SEQ ID NO:所示的氨基酸序列，或包含与所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，或由如下SEQ ID NO所示的序列组成：
i)SEQ ID NO:35和SEQ ID NO:36；
ii)SEQ ID NO:37和SEQ ID NO:38；
iii)SEQ ID NO:39和SEQ ID NO:40。
在一些可实施方案中，所述双特异性抗体中，
(i)重链1包含SEQ ID NO:33所示的氨基酸序列的重链，且轻链1包含SEQ ID NO:34所示的氨基酸序列，
重链2包含SEQ ID NO:35所示的氨基酸序列，且轻链2包含SEQ ID NO:36所示的氨基酸序列；或，
(ii)重链1包含SEQ ID NO:33所示的氨基酸序列，且轻链1包含SEQ ID NO:34所示的氨基酸序列，
重链2包含SEQ ID NO:37所示的氨基酸序列，且轻链2包含SEQ ID NO:38所示的氨基酸序列；或，
(iii)重链1包含SEQ ID NO:33所示的氨基酸序列，且轻链1包含SEQ ID NO:34所示的氨基酸序列，
重链2包含SEQ ID NO:39所示的氨基酸序列，且轻链2包含SEQ ID NO:40所示的氨基酸序列。
III.免疫缀合物
在一些实施方案中，本发明还涵盖与其他物质缀合的抗体。因此，本发明涉及免疫缀合物，其包含与其他物质缀合的抗体。(“免疫缀合物”)。在一些实施方案中，其它物质例如治疗剂或标记，如细胞毒性剂、免疫调节剂(例如免疫激动剂)或化疗剂。细胞毒性剂包括任何对细胞有害的药剂。适合于形成免疫缀合物的细胞毒性剂(例如化疗剂)的例子是本领域中已知的。例如，细胞毒性剂包括但不限于：放射性同位素；生长抑制剂；酶及其片段如核酸水解酶；抗生素；毒素如小分子毒素或细菌、真菌、植物或动物起源的酶促活性毒素，包括其片段和/或变体；和已知的各种抗肿瘤或抗癌剂。
另外，本发明的抗体分子可以与标记序列(如肽)缀合以促进纯化。
在其他实施方案中，本发明的抗体分子与诊断剂或可检测剂缀合。这类抗体可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测物质偶联实现这类诊断和检测，所述可检测物质包括但不限于多种酶；辅基；荧光物质；发光物质；放射性物质；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
另外，本发明的抗体分子可以与调节给定生物学反应的治疗性部分或药物部分缀合。治疗性部分或药物部分包括但不限于经典的化学治疗药。例如，药物部分可以是拥有所需生物学活性的蛋白质、肽或多肽。
另外，本发明的抗体分子可以缀合至治疗性部分如放射性金属离子。
抗体也可以连接至固相支持物，所述支持物特别可用于免疫测定法或靶抗原的纯化。
在一些实施方案中，所述免疫缀合物用于预防或治疗疾病，如急性和慢性炎性疾病、感染(例如，慢性感染)、肿瘤等。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤是上皮来源的癌症，例如胃肠道肿瘤或肺部肿瘤或皮肤肿瘤，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)。在一些实施方案中，感染是慢性感染。在一些实施方案中，感染是例如细菌感染、病毒感 染、真菌感染、原生动物感染等。
IV.本发明的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何抗体或其片段或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
例如，本发明的核酸包含编码选自SEQ ID NO:3-8和33-40中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:3-8和33-40中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。编码本发明分子的核酸序列可以采用本领域熟知的方法，例如通过从头固相DNA合成，或通过PCR扩增而产生。
在一方面，本发明提供编码以上任何抗体或任何抗体链的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示人EGFR/或B7-H3抗原结合能力。
在再一方面，本发明提供编码以上任何双特异性抗体的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示人EGFR/或B7-H3抗原结合能力。在一个实施方案中，编码双特异性抗体的各条链的核酸可以在相同的载体中或在不同的载体中。在另一个实施方案中，编码双特异性抗体的各条链的核酸可以引入相同或不同的宿主细胞以表达。因此，在一些实施方案中，本发明的双特异性抗体的生产方法包括步骤：在适于表达所述分子的各条链的条件下，培养包含编码所述各条链的核酸的宿主细胞，产生本发明双特异性抗体。
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是例如pcDNA载体，例如pcDNA3.1。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。
在一个实施方案中，提供了包含一种或多种本发明多核苷酸的宿主细胞。在 一些实施方案中，提供了包含本发明表达载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞例如CHO细胞(例如CHO-S，如ExpiCHO-S)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在一个实施方案中，宿主细胞是原核的，例如是细菌，例如大肠杆菌。
合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、293细胞、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、中国仓鼠卵巢细胞(CHO细胞)、CHOK1SV细胞、CHOK1SV GS-KO细胞、CHOS细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO细胞，例如CHOS细胞CHOK1SV细胞或CHOK1SV GS-KO，或所述宿主细胞是293细胞，例如HEK293细胞。在一些优选的实施方案中，所述宿主细胞是CHO细胞。在一个实施方案中，本发明的宿主细胞为糖基化改造的宿主细胞，优选为糖基化改造的CHO细胞。在一个实施方案中，所述宿主细胞被工程化而表达RMD酶。在一个实施方案中，所述宿主细胞包含编码RMD酶的核酸。在一个实施方案中，所述RMD酶包含SEQ ID NO:41所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，优选地，所述RMD酶来自铜绿假单胞菌。
在一个实施方案中，本发明的宿主细胞包含编码本发明的抗体分子的一个或多个或全部的链的核酸和编码RMD酶的核酸。
V.本发明的抗体分子的生产和纯化
在一个实施方案中，本发明提供制备本发明抗体分子的方法，其中所述方法包括在适于表达编码本发明抗体分子的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体。在某个实施方案中，所述方法还包括从宿主细胞回收本发明抗体分子。
在一个实施方案中，提供了制备本发明抗体分子的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体(例如任意一条多肽链和/或多条多肽链)的核酸或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。
为了重组产生本发明抗体分子，分离编码抗体(例如上文所描述的抗体，例如任意一条多肽链和/或多条多肽链)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序 (例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
如本文所述制备的抗体分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。
VI.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗体分子鉴定，筛选，或表征其物理/化学特性和/或生物学活性。一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹等来进行。可使用本领域已知方法来测定对所结合抗原的结合，本文中公开了例示性方法，例如生物膜层干涉技术和SPR。
本发明还提供了用于鉴定具有生物学活性的抗体的测定法。生物学活性可以包括例如结合抗原，对细胞表面抗原的结合、对抗原的抑制或激活作用等。还提供在体内和/或在体外具有此类生物学活性的抗体。
在某些实施方案中，对本发明的抗体测试此类生物学活性。
本发明还提供用于鉴定抗体的性质，例如成药性相关性质的方法。所述成药性相关性质包括例如热稳定性，例如长期热稳定性。
供任何上述体外测定法使用的细胞包括天然表达抗原或经改造而表达抗原的细胞系。此类细胞还包括表达抗原的细胞系和并非正常情况下表达抗原的编码抗原的DNA转染的细胞系。
可以理解的是，能够使用本发明的免疫缀合物替换或补充本发明的抗体分子来进行任何上述测定法。
可以理解的是，能够使用本发明的抗体分子和别的活性剂来进行任何上述测定法。
在一些实施方案中，所述抗原为EGFR(例如人EGFR)和/或B7-H3(例如人B7-H3)。
VII.药物组合物和药物制剂
在一些实施方案中，本发明提供包含本文所述的任何抗体分子或其片段(优选地其抗原结合片段)或其免疫缀合物的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的抗体分子或其片段或其免疫缀合物，以及一种或多种其它治疗剂的组合。
在一些实施方案中，其它治疗剂选自抗血管发生剂、化疗剂、其它抗体、细 胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂为小分子药物，优选地，所述小分子药物选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
在一些实施方案中，所述组合物用于预防或治疗疾病，如急性和慢性炎性疾病、感染(例如，慢性感染)、肿瘤等。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤是上皮来源的癌症，例如胃肠道肿瘤或肺部肿瘤或皮肤肿瘤，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)。在一些实施方案中，感染是慢性感染。在一些实施方案中，感染是例如细菌感染、病毒感染、真菌感染、原生动物感染等。
本发明还包括包含本发明抗体或其免疫缀合物的组合物(包括药物组合物或药物制剂)和/或包含编码本发明抗体的多核苷酸的组合物(包括药物组合物或药物制剂)。在某些实施方案中，组合物包含一种或多种本发明抗体或其片段或一种或多种编码一种或多种本发明抗体或其片段的多核苷酸。
这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。
合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,PharmaceuticalPress,London,Chicago。
若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准药用载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、分散体剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如，静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中，通过静脉内输注或注射施用抗体分子。在另一个优选实施方案中，通过肌内、腹腔或皮下注射施用抗体分子。
可以通过将具有所需纯度的本发明的抗体与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本文所述的抗体的药物制剂，优选地以冻干制剂或水溶液的形式。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂。在一些实施方案中，其它治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂为小分子药物，优选地，所述小分子药物选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明的药物组合物适于静脉内、肌内、皮下、肠胃外、直肠、脊髓或表皮施用(例如，通过注射或输注)。
治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散体、脂质体或冻干形式。可以通过将活性化合物(即抗体分子)以要求的量加入适宜的溶剂中，随后过滤消毒，制备无菌可注射溶液剂。通常，通过将所述活性化合物并入无菌溶媒中来制备分散体，所述无菌溶媒含有基础分散介质和其他成分。可以使用包衣剂如卵磷脂等。在分散体的情况下，可以通过使用表面活性剂来维持溶液剂的适宜流动性。可以通过在组合物中包含延迟吸收的物质例如单硬脂酸盐和明胶而引起可注射组合物的延长吸收。
包含本文所述抗体分子的试剂盒也处于本发明的范围内。试剂盒可以包含一个或多个其他要素，例如包括：包装插页；其他试剂，例如标记物或用于偶联的试剂；可药用载体；和用于施用至受试者的装置或其他材料。
VIII.组合产品或试剂盒
在一些实施方案中，本发明还提供了组合产品，其包含本发明的抗体或其抗原结合片段，或其免疫缀合物，以及一种或多种其它治疗剂。在一些实施方案中，其它治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂为小分子药物，优选地，所述小分子药物选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
在一些实施方案中，所述组合产品用于预防或治疗疾病，如急性和慢性炎性疾病、感染(例如，慢性感染)、肿瘤等。例如，所述疾病是肿瘤(例如癌症)或感染。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤是上皮来源的癌症，例如胃肠道肿瘤或肺部肿瘤或皮肤肿瘤，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)。在一些实 施方案中，感染是慢性感染。在一些实施方案中，感染是例如细菌感染、病毒感染、真菌感染、原生动物感染等。
在一些方案中，所述组合产品中的两种或多种成分可以依次、分开或同时联合施用给受试者。
在一些实施方案中，本发明还提供了包含本发明的抗体、药物组合物、免疫缀合物或组合产品的试剂盒，以及任选的指导施用的包装插页。
在一些实施方案中，本发明还提供了包含本发明的抗体、药物组合物、免疫缀合物、组合产品的药物制品，任选地，所述药物制品还包括指导施用的包装插页。
IX.本发明的抗体分子的用途
在另一方面中，本发明涉及预防或治疗受试者的肿瘤(例如癌症)的方法，所述方法包括向所述受试者施用有效量的本文公开的抗体分子或药物组合物或免疫缀合物或组合产品或试剂盒。在一些实施方案中，本发明受试者中的肿瘤包括实体肿瘤和血液肿瘤。在一些实施方案中，肿瘤是肿瘤免疫逃逸。在一些实施方案中，所述肿瘤是癌症。
在一些实施方案中，相比同一受试者相邻的同一组织的正常细胞或临近正常组织的正常细胞，或相比健康受试者相同组织的正常细胞，所述肿瘤的肿瘤细胞具有以下一种或多种特征：
(i)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型EGFR(例如具有升高的核酸或蛋白质水平的野生型EGFR)，和/或表达突变的EGFR，例如包含Passaro A,et al.Nat Cancer.2021中列出的突变的EGFR，优选地，所述突变的EGFR包含选自R521K、L858R、T790M、G719X、C797S、Y1069C、Exon19缺失(Del19)、Exon20ins(例如S768_D770dup)中的一个或多个突变，优选地，所述突变的EGFR包含R521K/Y1069C、R521K、L858R/T790M/C797S、Del19/T790M/C797S或S768_D770dup；
(ii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型KRAS(例如具有升高的核酸或蛋白质水平的野生型KRAS)，，或表达突变的KRAS，优选地，所述突变的KRAS包含G12位或G13位突变，例如G12D或G12C；
(iii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，具有升高的核酸水平或蛋白水平的B7-H3；和/或
(iv)所述肿瘤细胞对于酪氨酸激酶抑制剂，例如对一代(厄洛替尼Erlotinib)和三代(奥希替尼Osimertinib)耐药，例如对于奥希替尼耐药。
在另一方面中，本发明涉及预防或治疗受试者的感染性疾病的方法，所述方法包括向所述受试者施用有效量的本文公开的抗体分子或药物组合物或免疫缀合物或组合产品或试剂盒。在一个实施方案中，感染性疾病是慢性感染。
受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类 (例如，患有本文所述疾病或具有患有本文所述疾病的风险的患者)。在一个实施方案中，受试者患有本文所述疾病(例如，如本文所述的肿瘤或感染或自身免疫性疾病)或具有患有本文所述疾病的风险。在某些实施方案中，受试者接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法。
在其他方面，本发明提供抗体分子或其片段或其免疫缀合物或组合物或组合产品或试剂盒在生产或制备药物中的用途，所述药物用于治疗本文提及的相关疾病或病症。
在一些实施方案中，本发明的抗体或抗体片段或免疫缀合物或组合物或组合产品或试剂盒会延迟病症和/或与病症相关的症状的发作。
在一些实施方案中，本发明的抗体或药物组合物或免疫缀合物或组合产品或试剂盒还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的预防和/或治疗。
在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)或放射疗法。
在一些实施方案中，治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂。
在一些实施方案中，所述小分子药物选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
免疫调节剂包括免疫检查点分子抑制剂和共刺激性分子激活剂。
在进一步的一些实施方案中，本发明的抗体或其片段与KRAS小分子抑制剂组合使用，所述小分子抑制剂例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
在一些实施方案中，本发明的抗体或其片段可以与包含过继转移表达嵌合抗原受体(CAR)的T细胞(例如细胞毒性T细胞或CTL)的治疗联合施用。
在一些实施方案中，本发明的抗体或其片段可以与抗肿瘤剂联合施用。
在一些实施方案中，本发明的抗体或其片段可以与细胞因子联合施用。细胞因子可以作为与本发明的抗体分子的融合分子施用，或作为单独的组合物施用。在一个实施方案中，本发明的抗体与一种、两种、三种或更多种细胞因子(例如，作为融合分子或作为单独组合物)组合施用。
在一些实施方案中，本发明的抗体或其片段可以与本领域常规的癌症疗法组合，常规的癌症疗法包括但不限于：(i)放射疗法；(ii)化学疗法，或应用细胞毒药物，其一般影响快速分裂的细胞；(iii)靶向疗法，或特异性影响癌细胞蛋白去调节的药剂；(iv)免疫疗法，或增强宿主免疫应答(例如，疫苗)；(v)激素疗法，或阻断激素(例如，当肿瘤是激素敏感的时候)，(vi)血管发生抑制剂，或阻断血管形成和生长，和(vii)姑息护理。
在一些实施方案中，本发明的抗体或其片段可以与增强宿主免疫功能的常规方法组合。
上文所述的各种组合疗法可以进一步组合以用于治疗。
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。
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
可以由技术人员确定本发明抗体分子的剂量和治疗方案。在一些实施方案中，调整剂量方案以提供最佳的所需反应(例如，治疗反应)。
在一些实施方案中，本发明的抗体(以及包含其的药物组合物或免疫缀合物)(单独或与其他治疗剂组合)可以在一周两次，或者一周一次，或者两周一次施用。
可以理解的是，能够使用本发明的免疫缀合物或组合物或组合产品或试剂盒替换或补充本发明的抗体来进行任何治疗。
实施例1.抗B7-H3/EGFR双特异性抗体构建
本发明的抗B7-H3/EGFR双特异性抗体分子是通过信达生物制药(苏州)有限公司蛋白科学Innobody技术平台(申请号：PCT/CN2021/143141，发明名称：含异二聚体抗体Fc的蛋白以及其制备方法)或者采用本领域常规方法，将抗B7-H3抗体亲本和抗EGFR抗体亲本组装成IgG1的抗体形式(如图1所示)。该双特异性抗体形式含有四条多肽链，并且可以和两种抗原结合，抗原A为EGFR，抗原B为B7-H3。
其中，用于构建双特异性抗体的亲本抗体为抗EGFR单克隆抗体抗Zalutumumab(后文简称Zalu,公开号：WO02100348A2，发明名称：Human monoclonal antibodies to epidermal growth factor receptor(EGFR))和抗B7-H3单克隆抗体，其来源于信达生物杂交瘤技术筛选平台并经抗体人源化得到抗B7-H3单克隆抗体Hz20G5.26、Hz19A2.25的抗原结合区序列(申请号：PCT/CN2021/140449，发明名称：抗B7-H3抗体及其用途中的Hz20G5、Hz19A2) 和来源于信达生物杂交瘤技术筛选平台并经抗体人源化得到抗B7-H3单克隆抗体Hz5C2.9的抗原结合区序列；同时，采用Innobody技术在Fc区进行突变提高特异性抗体异源二聚体形成(申请号：PCT/CN2021/143141，发明名称：含异二聚体抗体Fc的蛋白以及其制备方法)。筛选获得了三种同时结合B7H3和EGFR的双特异性抗体，分别编号为Hz5C2.9/Zalu bsAb、Hz19A2.25/Zalu bsAb和Hz20G5.26/Zalu bsAb双特异性抗体分子，具体的氨基酸序列编号参见表1和表A-序列信息，三种双特异性抗体均采用IgG样式的结构，具体结构示意图参见图1。
表1.B7H3/EGFR bsAb分子以及阳性对照Gp120/Zalu序列详情

1Zhou,T.,Xu,L.,Dey,B.et al.Structural definition of a conserved neutralization epitope on 
HIV-1 gp120.Nature 445,732–737(2007)
实施例2.抗B7-H3/EGFR双特异性抗体分子的制备
1、双特异性抗体分子质粒构建
分别将表1所列的抗EGFR抗体的重链序列，抗EGFR抗体的轻链序列，抗B7-H3抗体的重链序列，抗B7-H3抗体的轻链序列插入到载体pcDNA3.1(Invitrogen,V790-20)中，分别得到抗EGFR端的重链质粒、轻链质粒，抗B7-H3端的重链质粒、轻链质粒。
2、双特异性抗体制备过程
利用GlymaxX技术(ProBioGenAG，参见专利公开号：WO2011035884A1，该申请的所有内容为本发明之目的完整并入本文作为参考)，通过PEI MAX(Polysciences，A804355)将RMD酶(GDP-6-deoxy-D-lyxo-4-hexylose reductase，序列：SEQ ID NO：41)质粒分别与抗EGFR亲本抗体的重链质粒、轻链质粒和抗B7-H3亲本抗体的重链质粒和轻链质粒共同瞬时转染到ExpiCHO(Invitrogen,A29133)细胞中，表达低岩藻糖含量的抗EGFR和抗B7-H3端的抗体亲本。7天后收获细胞发酵液，将其过滤澄清，分别用Hitrap Mabselect Sure层析柱(GE Healthcare，11-0034-95)进行捕获，得到抗EGFR亲本和B7H3亲本的抗体。
采用A280法检测浓度之后，将抗体亲本按摩尔比例1:1混合，加入适量还原剂GSH，室温反应过夜，超滤去除还原剂，终止反应。采用MonoS阳离子交换层析柱(GE Healthcare,17-5168-01)进行精细纯化，抗B7-H3亲本液为20mM磷酸钠缓冲液(pH 6.6)，抗EGFR亲本液为含有1M氯化钠的20mM磷酸钠缓冲液(pH 6.6)，洗脱梯度为0-50％(30柱体积)。将洗脱得到的蛋白溶液超滤并换液至PBS(Gibco，70011-044)，用SEC-HPLC进行纯度检测。
实施例3.Hz20G5.26/Zalu bsAb双特异性抗体分子亲和力检测
采用生物膜薄层干涉测定技术(BLI)测定本发明双特异性抗体结合B7H3和EGFR的亲和力(KD)。
实验开始前半个小时，根据样品数量，取合适数量的AHC传感器(18-5060，Sartorius)浸泡于SD缓冲液(1x PBS，0.1％BSA，0.05％Tween-20)中。将上述制备的抗体和人B7H3(B73-H52E2，Acro Biosystem)、人EGFR(EGR-H5222，Acro Biosystem)分别稀释至100nM。
分别将SD缓冲液、抗体溶液、人B7H3、人EGFR分别加入到96孔黑色聚苯乙烯微孔板(Greiner，655209)中。使用Fortebio Octet Red96e进行检测，根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：平衡基线120s、加样固化抗体100s、平衡基线120s、结合抗原100s和解离120s，转速为1000rpm，温度为30℃。实验完成后，使用ForteBio Octet分析软件分析KD值，结果见下表2。
表2：B7H3/EGFR bs双特异性抗体分子亲和力检测

实施例4.抗B7-H3/EGFR双特异性抗体分子筛选
采用Innobody技术平台，将抗B7-H3和抗EGFR抗体亲本序列表达和组装成Hz5C2.9/Zalu bsAb、Hz19A2.25/Zalu bsAb和Hz20G5.26/Zalu bsAb双特异性抗体分子。通过抗体对细胞的增殖抑制实验(Growth inhibition assay)和抗体依赖的细胞介导的细胞毒性实验(ADCC cell report)，对B7H3/Zalu bsAb双特异性抗体分子在非小细胞肺癌(NSCLC)和头颈鳞癌(HNSCC)进行体外活性检测，同时在人皮肤鳞癌细胞系(A431)进行安全性验证。
细胞系来源和培养基：
PC9(NSCLC):上海钰博生物,YB-H3210D；培养基：MEM+10％FBS+1％Pen/strep
TE-1(HNSCC):CoBioer,CBP60655；培养基：RPMI 1640+10％FBS+1％Pen/strep
SK-MES-1(NSCLC):CoBioer,CBP60152；培养基：MEM+1％NEAA+1mM Sodium Pyruvate+10％FBS+1％Pen/strep
A431:皮肤鳞癌细胞，ATCC,CRL-1555；培养基：DMEM+10％FBS+1％Pen/strep
PC9+hB7H3和CHO-S+hB7H3细胞系构建：构建和包装Lentvirus+hB7H3慢病毒(hB7H3(UniProt,Q5ZPR3-1)，Lentvirus(PPL质粒与蛋白共享库，BC000141))，用Lentvirus+hB7H3分别感染PC9(NSCLC,上海钰博生物,YB-H3210)和CHO-S(Thermo)，通过加压筛选和分选获得出PC9+hB7H3和CHO-S+hB7H3稳转细胞系。
实验方法：
1.增殖抑制实验(Growth inhibition assay)
(1)、NSCLC:1500-2500cells/100ul铺96孔低吸附板(Corning,CLS7007-24EA),进行3D细胞培养。
HNSCC:1500-2000cells/100ul铺96孔白底板(NUNC,136101),进行2D细胞培养。
(2)、将提前稀释准备好的抗体分子(最高浓度300nM，3.16倍稀释)加到相 应细胞孔板中，混合均匀，37℃5％CO2培养5天。
(3)、增殖抑制实验持续培养5天后，将提前准备好的Cell-Titer试剂(Promega,G7572)加入相应细胞孔中，室温避光静止15-25分钟,HNSCC细胞直接进行多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
(4)、NSCLC:将Cell-Titer和细胞混合液转入96孔白底板(NUNC,136101)，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
结果见图2。其中存活细胞百分比(％of surviving cells)(图2A、B、C、D和F)是检测的细胞相对存活率，相应细胞标注以及所属类别参见图2以及附图说明，如PC9是NSCLC一种，TE-1是HNSCC一种。其中A431是皮肤鳞癌细胞，其不响应抗体治疗，目的是用来衡量药物副反应。
图2E中的生长抑制百分比(Growth inhibition％)是根据图2结果(％of surviving cells)进行分析和统计后得出的，具体如下：在抗体300nM最高浓度下，药物在每种细胞的最大细胞抑制率，计算方法：300nM,(1-％of surviving cells)×100％。
在HNSCC和NSCLC细胞系中，Hz5C2.9/Zalu bsAb、Hz19A2.25/Zalu bsAb和Hz20G5.26/Zalu bsAb三种双抗特异性分子增值抑制实验结果(图2A、B和C)显示，三种特异性双抗分子在HNSCC和NSCLC中均有明显肿瘤杀伤作用，其中Hz20G5.26/Zalu bsAb这个双抗特异性分子不仅药效优异，而且在A431体外药效差(图2D)，预示Hz20G5.26/Zalu bsAb可减少或降低EGFR抗体系列临床用药的皮肤毒性等副反应。
2.ADCC报告实验(ADCC cell report assay)
(1)、按照效靶比(10:1)，将靶细胞(上文构建的CHO-S+hB7H3或NCI-H522(CoBioer,CBP60140))和ADCC效应细胞(Promega,G7102)混合均匀至96孔白底板(NUNC,136101)。
(2)、将提前稀释准备好的抗体分子(对于CHO-S+HB7H3的检测:抗体稀释后终浓度中，最高6.25nM，4倍系列稀释；对于NCI-H522的检测：抗体稀释后终浓度中,最高100nM，4倍系列稀释)加到相应细胞孔板中，混合均匀，37℃5％CO2培养20小时。
(3)、将提前准备好的Bio-glo试剂(Promega,G755B)加入细胞孔板中，室温避光静止10-15分钟，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
实验结果见图3，其中纵坐标是以IgG1为对照获取的针对IgG1的倍数
在NSCLC和HNSCC肿瘤细胞系中，通过细胞增殖抑制(图2)和ADCC报告实验(图3)，实验结果发现，Hz20G5.26/Zalu bsAb体外药效整体最佳；在A431皮肤鳞癌细胞系中，Hz20G5.26/Zalu bsAb体外毒性活性最低(用于衡量EGFR抑制剂对与人体皮肤的毒性)。因此，本发明的双抗，特别是Hz20G5.26/Zalu bsAb对人体皮肤毒性较小，且在不引起或弱引起毒副反应时，有更大的有效安全药物剂量，即其具有更宽的药效选择窗口，药效更安全。
实施例5.Hz20G5.26/Zalu bsAb双特异性抗体分子增殖抑制体外药效活性
EGFR配体与肿瘤细胞表面的EGFR结合后，EGFR会形成同源二聚体，将信号传递到细胞内，通过各种级联反应，将使肿瘤细胞产生增殖、侵袭、转移和抗凋亡等生物活性。
Hz20G5.26/Zalu bsAb中抗EGFR的抗原结合部分与肿瘤细胞表面的EGFR结合后，一方面可以阻断EGFR配体与EGFR的结合，抑制生物信号传递，阻断肿瘤相应生物活性；另一方面会刺激EGFR内吞而最终被细胞内溶酶体等降解。EGFR是一个上皮源广谱酪氨酸激酶受体，Hz20G5.26/Zalu bs Ab采用了对EGFR低亲和力的EGFR抗体亲本序列，极大降低了EGFR单克隆抗体对皮肤等正常上皮组织带来的毒副作用。B7-H3在多种肿瘤细胞系高表达，在正常组织器官低表达，是一个选择性极高和表达谱广泛的TAA，Hz20G5.26/Zalu bs Ab在EGER低亲和力的基础上引用B7-H3高亲和力Hz20G5.26亲本，极大提高了EGFR信号阻断活性，提高了Hz20G5.26/Zalu bs Ab药效生物活性和药效安全窗口。
实施例5.1.肿瘤细胞系B7-H3和EGFR表达检测
Hz20G5.26/Zalu bsAb对肿瘤细胞系的药物敏感性与肿瘤细胞表面的受体表达丰度有一定的正相关关系。从NSCLC(非小细胞肺癌)、HNSCC(头颈部鳞状细胞癌)、CRC(结肠癌)和正常细胞系中筛选部分细胞系进行B7-H3和EGFR相对表达检测，根据B7-H3和EGFR在细胞表面的表达丰度进行体外Hz20G5.26/Zalu bsAb药效活性检测和验证，实验结果如图4所示。
实验方法：
1、将各种细胞用FACS缓冲液重悬，取100000-200000细胞铺96孔板(Corning,CLS3799-50EA)，将10ug/ml EGFR抗体(Cetuximab)、10ug/ml B7-H3抗体(Hz20G5.26mAb)和10ug/ml IgG加入细胞孔板，4度孵育1小时。
2、PBS洗涤两次，将准备好的APC-anti humanFc抗体(Biolegend,410712)，加入相应细胞孔板，4℃孵育30-40分钟。
3、PBS洗涤两次，FACS检测(BD,Celesta)，结果见图4。
图4显示了不同细胞系中的B7H3表达以及EGFR的表达情况，其中B7H3表达较高且依赖于EGFR信号通路的细胞系(例如后续通过测序分析得到的包含有EGFR突变的细胞系(结果未显示)或者EGFR过表达的细胞系)可以被选择用于双特异性抗体的活性检测。
实施例5.2.Hz20G5.26/Zalu bsAb双特异性抗体分子在多种肿瘤细胞系增殖抑制实验
EGFR在多种实体瘤细胞表面高表达，如肺癌(30％-80％)、头颈鳞癌(36％-100％)、结直肠癌(25％-77％)、食管癌(43％-89％)等，是一个广谱的抗肿瘤酪氨酸激酶靶点蛋白。EGFR作为一个广谱抗肿瘤靶点蛋白，在非小细胞肺癌患者突变类型中突变率也最高(全球平均突变率约为35％，中国达到40％)，在携带EGFR突变的NSCLC患者中，经典突变(L858R,T790M或Exon19 deletion)占85-90％；EGFR exon 20ins、T790M原发点突变和复合突变以及以G719X为代表的位于外显子18-21间的其他点突变及其序列重复突变等非常见突变占 EGFR突变比例约为10％。
H1975-EGFRL858R/T790M/C797S、PC9+B7H3-EGFRDel19/T790M/C797S和H322-EGFRS768_D770dup细胞系构建：分别构建和包装Lentvirus+EGFRL858R/T790M/C797S、Lentvirus+EGFRDel19/T790M/C797S和Lentvirus+EGFRS768_D770dup病毒，分别用Lentvirus+EGFRL858R/T790M/C797S、Lentvirus+EGFRDel19/T790M/C797S和Lentvirus+EGFRS768_D770du病毒感染NCI-H1975(ATCC,CRL-5908)、PC9+hB7H3(如上所述构建)和H322，通过加压筛选，获得H1975-EGFRL858R/T790M/C797S、PC9+B7H3-EGFRDel19/T790M/C797S和H322-EGFRS768_D770dup稳转细胞系。
实验方法：
增殖抑制实验
(1)、NSCLC:1500-2500cells/100ul铺96孔低吸附板(Corning,CLS7007-24EA),进行3D细胞培养，其中应用了如下细胞系和具体的量：
NSCLC:2000cells/100μl/well：
NCI-H292:中科院细胞库，SCSP-582
NCI-H322:Cobioer,CBP60134
NCI-H1650:ATCC,CRL-5883
NCI-H1975:ATCC,CRL-5908
SK-MES-1:CoBioer,CBP60152
NCI-H1703:ATCC,HTB-43
PC9+hB7H3:如上文所构建
CRC和HNSCC:1500-2000cells/100ul铺96孔白底板(NUNC,136101),进行2D细胞培养，其中应用了如下细胞系和具体的量：
CRC:1500cells/100ul/well
CCK-81:CoBioer,CBP60581
HT-55:CoBioer,CBP60012
H508:CoBioer,CBP60795
LS180:CoBioer,CBP60034
HNSCC:1500cells/100ul/well
TE-1:CoBioer,CBP60655
Colo680:CoBioer,CBP60452。(2)、将提前稀释准备好的抗体分子加到相应细胞孔板中，混合均匀，37℃5％CO2培养箱培养5天。
(3)、增殖抑制实验持续培养5天后，将提前准备好的Cell-Titer试剂(Promega, G7572)加入细胞孔中，室温避光静止15-25分钟,CRC和HNSCC细胞直接进行多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
(4)、NSCLC:将Cell-Titer和细胞混合液转入96孔白底板(NUNC,136101)，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
EGFR作为一个广谱抗肿瘤靶点蛋白，从体外增殖抑制抗肿瘤药效发现，Hz20G5.26/Zalu bsAb在食管癌肿瘤细胞系中有抗肿瘤效果(如图6所示)，其中在TE-1食管癌肿瘤细胞系，Hz20G5.26/Zalu bsAb比JNJ372具有更好的抗肿瘤效果。在结肠癌等肿瘤细胞系中(如图5所示)，Hz20G5.26/Zalu bsAb能达到EGFR单抗抗肿瘤效果，特别在LS180结肠癌肿瘤细胞系中，其体外抗肿瘤效果要优异于EGFR单抗；在NCI-H508结肠癌肿瘤细胞系中，抗肿瘤最大杀伤率达到84％。
在肺癌中，并非所有癌症肿瘤患者都是由于EGFR原发基因突变而发展演变的，在非小细胞肺癌患者EGFR平均突变率约为35％；作为治疗标准的EGFR-TKI小分子(例如Erlotinib(Selleck Chemicals,cat:S7786)或Osimertinib(Selleck Chemicals,cat:S7297))主要针对敏感基因突变患者，但也常因基因突变而造成耐药失效，需要不断针对新突变位点而开发新的靶向药物。在肺癌中，Hz20G5.26/Zalu bsAb不仅对EGFR突变的NSCLC肿瘤细胞系有效，而且对野生型EGFR和异常扩增型EGFR非小细胞肺癌肿瘤细胞系都有抗肿瘤体外效果。在NSCLC肿瘤细胞系中，Hz20G5.26/Zalu bsAb相比JNJ-372体外抗肿瘤效果，其体外整体抗肿瘤药效要远优异于JNJ-372。
在NSCLC-EGFRWT肿瘤细胞系(图7.1)，NCI-H292体外最大抗肿瘤杀伤率为74.2±2.8％；在NSCLC-EGFR经典突变(L858R,T790M或Exon19 deletion)肿瘤细胞系(图7.2)，针对NCI-H1975(EGFRL858R/T790M)(图7.2B)体外最大抗肿瘤抑制杀伤率为51.2±1.4％，IC50为0.99nM,体外药效优异于JNJ-372和EGFR单抗；在NSCLC-EGFR异常扩增肿瘤细胞系(图7.3)，针对SK-MES-1体外最大抗肿瘤杀伤率为60.7±15.9％，IC50为0.40nM,整体体外药效优异于JNJ-372和EGFR单抗，并且SK-MES-1和NCI-H1703这些异常扩增NSCLC-EGFR的肿瘤细胞系是对一代(厄洛替尼Erlotinib)和三代(奥希替尼Osimertinib)非敏感耐药的。已上市和用于T790M突变治疗的第三代EGFR-TKI小分子抑制剂奥希替尼是对EGFRexon20ins罕见突变药效较差，为探究Hz20G5.26/Zalu bsAb对EGFRexon20ins肿瘤的药效，我们在NCI-H322-EGFRWT细胞基础上，构建H322-EGFRS768_D770dup稳转细胞系，体外增殖抑制抗肿瘤药效显示(图7.4)，Hz20G5.26/Zalu bsAb是对EGFRexon20ins有体外药效活性的。随着EGFR-TKI小分子抑制剂的服用和治疗，NSCLC患者不断出现两重(如Del19/T790M，L858R/T790M)或三重(如Del19/T790M/C797S，L858R/T790M/C797S)耐药突变，这也是下一代EGFR-TKI重要研发方向。为验证Hz20G5.26/Zalu bsAb对EGFR-TKI三重耐药突变的药效活性，我们构建了H1975-EGFRL858R/T790M/C797S和PC9+B7H3-EGFRDel19/T790M/C797S稳转细胞系，体外增殖抗肿瘤药效显示(图7.4)，Hz20G5.26/Zalu bsAb对EGFRL858R/T790M/C797S和EGFRDel19/T790M/C797S有一定的体外药效活性。
为进一步验证Hz20G5.26/Zalu bsAb体外药效活性，将Hz20G5.26/Zalu bsAb与gp120/Zalu和gp120/Hz20G5.26联合进行体外增殖抑制药效比对分析(图7.5)，结果发现在Hz20G5.26/Zalu bsAb在NCI-H292(Lung adeno,EGFRWT)和SK-MES-1(sqNSCLC，EGFRAmp)NSCLC肿瘤细胞系以及LS180 CRC肿瘤细胞 系中，Hz20G5.26/Zalu bsAb体外药效远优异于gp120/Zalu和gp120/Hz20G5.26联合用药。Hz20G5.26/Zalu bsAb增殖抑制肿瘤杀伤实验结果如表3所示：
表3.Hz20G5.26/Zalu bsAb增殖抑制肿瘤杀伤实验结果
实施例5.3.Hz20G5.26/Zalu bsAb双特异性抗体分子与KRAS小分子抑制剂联合协同作用
在癌症肿瘤患者中，除了EGFR异常激活会导致癌症发生外，KRAS基因突变激活也是癌症肿瘤常见驱动诱因。在人类癌症中约有25％是KRAS突变，KRAS突变与癌症预后治疗有很大关系。在CRC和NSCLC肿瘤中，KRAS-G12D和KRAS-G12C是常见异常突变，现在随着KRAS小分子抑制剂的使用，预期后面会产生KRAS获得性耐药性。KRAS小分子抑制剂与其他药物联合使用(如免疫治疗药物疗或靶向治疗药物)或是新药研究和开发方向。
实验方法：
增殖抑制实验
(1)、NSCLC(H358)(Cobioer,CBP60136)和CRC(LS180)(Cobioer,CBP60034):1500-2500cells/100ul铺96孔低吸附板(Corning,CLS7007-24EA),进行3D细胞培养。
(2)、将提前稀释准备好的抗体分子和小分子抑制剂加到相应细胞孔板中，混合均匀，37℃5％CO2培养5天。
(3)、增殖抑制实验持续培养5天后，将提前准备好的Cell-Titer试剂(Promega,G7572)加入细胞孔中，室温避光静止15-25分钟。
(4)、将Cell-Titer和上述细胞混合液转入96孔白底板(NUNC,136101)，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
从NCI-H358(EGFRWT,KRASG12C)NSCLC肿瘤细胞系结果发现(图8A)，Hz20G5.26/Zalu bsAb在H358体外最大抗肿瘤杀伤率为66.9±1.9％，IC50＝0.48nM，抗肿瘤药效远优异于EGFR单抗Zalu单抗和JNJ-372，优异于Gp120/Zalu和Gp120/Hz20G5.26联合用药；通过与AMG510(KRAS-G12C小分子抑制剂)(MedChem Expresss,HY-114277)联合用药，结果发现Hz20G5.26/Zalu与AMG510有协同效果，0.98nM AMG510在10nM Hz20G5.26/Zalu bsAb作用下，H358细胞杀伤率由22.51％提高到67.12％，在H358细胞中抗肿瘤效果增强(图8B)。为了证明协同效应，进一步基于图8B，在SynergyFinder(https://synergyfinder.fimm.fi/synergy/20210817124619827928/)中计算药效协同得分，结果参见图8C和图8D。图8D中给出了不同维度得分总和，其中总分为11.895，大于10，代表两种药物之间具有协同作用。
从LS180(EGFRR521K,KRASG12D)CRC肿瘤细胞系结果发现，Hz20G5.26/Zalu bsAb的体外抗肿瘤药效不仅优异于EGFR单抗Zalu、JNJ372以及Gp120/Zalu和gp120/Hz20G5.26联合用药(图7.5)；而且与MRTX1133(KRAS-G12D小分子抑制剂)联合用药有明显协同效果，在1.56nM Hz20G5.26/Zalu bsAb条件下，15.62nM MRTX1133抗LS180肿瘤药效有原来36.45％提升到61.56％，抗肿瘤效果明显增强(图9A)。图9B和图9C中进一步应用SynergyFinder计算了协同得分，其中总分为37.714，代表两种药物之间的协同作用。
实施例5.4.Hz20G5.26/Zalu bsAb双特异性抗体分子增殖抑制体外药效机制研究
在增殖抑制实验中，Hz20G5.26/Zalu bsAb是在B7H3抗体帮助下，进一步提升EGFR信号阻断而实现更强抗肿瘤杀伤作用。在NCI-H358细胞上，我们通过Western blotting进行EGFR信号抑制阻断和EGFR配体阻断两个方向进行研究，探究Hz20G5.26/Zalu bsAb体外抗肿瘤效果优异于EGFR单抗以及Gp120/Zalu和Gp120/Hz20G5.26联合用药的原因。
实验方法：
1、EGFR信号阻断实验(Signal blocking)
(1)、细胞处理
<1>、取NCI-H358(Cobioer,CBP60136)，7.0E5cells/孔铺6孔板(NEST,703011)过夜培养。
<2>、去除培养基(RPMI1640完全培养基(RPMI1640+10％FBS))，添加无血清培养基RPMI1640(assay medium)饥饿处理过夜。
<3>、去除<2>中的培养基，分别加入包含200nM Hz20G5.26/Zalu bsAb、200nM Gp120/Zalu、200nM Gp120/Hz20G5.26、200nM Zalu mAb、200nM Gp120/Zalu+200nM Gp120/Hz20G5.26的无血清培养基(assay medium)至相应细胞孔板，设置Blank组(培养基)、bsAb组(200nM Hz20G5.26/Zalu bsAb)、Gp120/Zalu组(200nM Gp120/Zalu)、Gp120/B7H3组(200nM Gp120/Hz20G5.26)、Zalu组(200nM Zalu mAb)、Gp120/Zalu+Gp120/B7H3组(200nM Gp120/Zalu+200nM Gp120/Hz20G5.26)，37℃5％CO2放置处理2小时。
(2)、蛋白提取和总蛋白定量
<1>、提前准备细胞裂解液，细胞裂解液组成：100-200ul/管RIPA(Thermo,89900)+1:100磷酸酶抑制剂(abcam,ab201112)+1:20蛋白酶抑制剂(Roche,11836170001)。
<2>、将提前准备好的细胞裂解液加入上述(1)中获得的细胞以消化细胞，冰上裂解20min。
<3>、离心机预先4℃预冷，12000rpm/min，4℃离心15min，取上清。
<4>、采用BCA试剂(Beyotime,P0012)，对提取的总蛋白进行浓度标定。
<5>、取相应总蛋白用LDS Sample buffer(Invitrogen,2201446)稀释，70℃变性处理10分钟，-40℃保存备用。
(3)、Westernblotting
<1>、将(3)中获得的蛋白样品和蛋白marker(Prestained Protein Ladder(Thermo,26620))加入预制胶(Thermo,NP0321BOX)；在200V电压下，采用电泳仪(BIO-RAD,TRANS SD CELL)跑胶约50分钟。
<2>、将胶片取出，采用转膜仪(Invitrogen,IBCOT2)进行转膜。
<3>、用准备好的封闭液(含5％脱脂奶粉TBST)，缓速封闭1-2小时；加入EGF Receptor Rabbit mAb(CST,4267)、pEGF Receptor Rabbit mAb(CST,3777)和GAPDH Rabbit mAb(CST,2118)抗体，4度孵育过夜。
<4>、用TBST洗涤三次，每次8-10min，摇床转速控制80-100rpm/min。
<5>、用标记的HRP-Goat Anti-Rabbit IgG(abcam,Ab205718)室温孵育1-2小时。
<6>、用TBST洗涤三次，每次8-10min，摇床转速控制80-100rpm/min。
<7>、ECL发光液(Beyotime,P0018AM)显影，显影仪(BIO-RAD,chemi Doc MP)曝光。
结果如图10。
2、EGFR配体阻断实验(Signal blocking)
(1)、细胞处理
<1>、取NCI-H358(H358)，7.0E5cells/孔铺6孔板(NEST,703011)过夜培养。
<2>、去除培养基(RPMI1640完全培养基(RPMI1640+10％FBS))，添加无血清培养基RPMI1640饥饿处理过夜。
<3>、去除培养基，分别加入培养基(2个细胞孔)、包含200nM Hz20G5.26/Zalu bsAb、200nM Gp120/Zalu、200nM Gp120/Hz20G5.26、200nM Zalu mAb、200nM Gp120/Zalu+200nM Gp120/Hz20G5.26的培养基至相应细胞孔板，设置Blank组(培养基)、Control组(培养基)、bsAb组(200nM Hz20G5.26/Zalu bsAb)、Gp120/Zalu组(200nM Gp120/Zalu)、Gp120/B7H3组(200nM Gp120/Hz20G5.26)、Zalu组(200nM Zalu mAb)、Gp120/Zalu+Gp120/B7H3组(200nM Gp120/Zalu+200nM Gp120/Hz20G5.26)，37℃5％CO2放置处理1小时。
<4>、待一小时后，在Blank组(培养基)、bsAb组(200nM Hz20G5.26/Zalu bsAb)、Gp120/Zalu组(200nM Gp120/Zalu)、Gp120/B7H3组(200nM Gp120/Hz20G5.26)、Zalu组(200nM Zalu mAb)、Gp120/Zalu+Gp120/B7H3组(200nM Gp120/Zalu+200nM Gp120/Hz20G5.26)，分别添加30nM EGF(ACRO,EGF-H52b)(图11上图)和30nM TGF-α(R&D,239-A-100)(图11下图)，37℃5％CO2放置处理1小时。
(2)、蛋白提取和总蛋白定量
<1>、提前准备细胞裂解液，细胞裂解液组成：100-200ul/管RIPA(Thermo,89900)+1:100磷酸酶抑制剂(abcam,ab201112)+1:20蛋白酶抑制剂(Roche,11836170001)。
<2>、将提前准备好的细胞裂解液加入上述(1)中获得的细胞以消化细胞，冰上裂解20min。
<3>、离心机预先4℃预冷，12000rpm/min，4℃离心15min，取上清。
<4>、采用BCA试剂(Beyotime,P0012)，对提取的总蛋白进行浓度标定。
<5>、取相应总蛋白用LDS Sample buffer(Invitrogen,2201446)稀释，70℃变性处理10分钟，-40℃保存备用。
(3)、Western blotting
<1>、将蛋白样品和蛋白marker(Prestained Protein Ladder(Thermo,26620))加入预制胶(Thermo,NP0321BOX)；在200V电压下，采用电泳仪(BIO-RAD,TRANS SD CELL)跑胶约50分钟。
<2>、将胶片取出，采用转膜仪(Invitrogen,IBCOT2)进行转膜。
<3>、用准备好的封闭液(含5％脱脂奶粉TBST)，缓速封闭1-2小时；加入EGF Receptor Rabbit mAb(CST,4267)、pEGF Receptor Rabbit mAb(CST,3777)和GAPDH Rabbit mAb(CST,2118)抗体，4度孵育过夜。
<4>、用TBST洗涤三次，每次8-10min，摇床转速控制80-100rpm/min。
<5>、用标记的HRP-Goat Anti-Rabbit IgG(abcam,Ab205718)室温孵育1-2小时。
<6>、用TBST洗涤三次，每次8-10min，摇床转速控制80-100rpm/min。
<7>、ECL发光液(Beyotime,P0018AM)显影，显影仪(BIO-RAD,chemi Doc MP)曝光。
结果参见图11。
从Signal blocking机制研究结果(图10和图11)发现，Hz20G5.26/Zalu bsAb在信号阻断和配体阻断作用均强于EGFR单抗Zalu以及Gp120/Zalu和Gp120/Hz20G5.26联合作用；该结果也揭示了Hz20G5.26/Zalu bsAb在体外抗CRC、NSCLC和HNSCC肿瘤效果比EGFR单抗以及Gp120/Zalu和Gp120/Hz20G5.26更优的原因。
实施例6.Hz20G5.26/Zalu bsAb双特异性抗体分子ADCC体外药效活性
抗体依赖的细胞介导的细胞毒性作用(ADCC)是抗肿瘤抗体发挥抗肿瘤作用的一种重要机制，其原理是利用抗体Fab段与肿瘤细胞表面的抗原表位结合，其Fc段与杀伤免疫(NK细胞、巨噬细胞、中性粒细胞等)表面的FcR结合，通过免疫细胞介导直接杀伤肿瘤细胞，ADCC作用主要是通过抗体Fc与NK表面的FcRⅢa受体来实现的。
Hz20G5.26/Zalu bsAb由于Hz20G5.26亲本的引入，不仅提高了Hz20G5.26/Zalu bsAb中EGFR抗体阻断活性，而且也整体提高了Hz20G5.26/Zalu bsAb ADCC作用(推测由于Hz20G5.26结合于B7H3的近膜端特定的表位，因而引发了强ADCC功能)；同时，采用GlymaxX低岩藻糖技术，进一步增强了Hz20G5.26/Zalu bsAb ADCC作用。
因此，Hz20G5.26/Zalu bsAb双特性抗体分子可通过EGFR信号阻断和ADCC两种作用机制，共同发挥多种抗肿瘤药效。具体实验步骤如下：
实施例6.1.ADCC报告实验
在ADCC报告实验中，ADCC效应细胞((Promega,G7102))是通过工程化将FcRⅢa(V158)受体过表达到Jurkat T细胞，通过Jurkat T细胞内NFAT-RE驱动元件能快速反应ADCC强弱的工程化细胞。
实验方法：
(1)、按照效靶比(10:1)，将肿瘤细胞(表3所列细胞系)和ADCC效应细胞(Promega,G7102)混合均匀(1.5E4cells:1.5E5cells/well/100ul(1.5E5:1.5E6cells/ml))至96孔白底板(NUNC,136101)。
(2)、将提前稀释准备好的抗体分子加到相应细胞孔板中，混合均匀，37℃5％CO2培养20小时。
(3)、将提前准备好的Bio-glo试剂(Promega,G755B)加入细胞孔板中，室温避光静止10-15分钟，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
我们在NSCLC肿瘤细胞系中，挑选不同EGFR类型肿瘤细胞系(EGFR野生型、EGFR异常扩增型、EGFR突变型、EGFR野生和KRAS突变型)，通过ADCC报告实验验证Hz20G5.26/Zalu bsAb ADCC活性，从ADCC报告实验结果(图12和表4)发现，在不同类型NSCLC-EGFR肿瘤细胞系中，Hz20G5.26/Zalu bsAb ADCC活性强于JNJ373和EGFR单抗；同时在H292(NSCLC-EGFR野生型)和H358(NSCLC-EGFR野生和KRAS突变型)实验结果发现，Hz20G5.26/Zalu bsAb ADCC活性不仅强于JNJ372和EGFR单抗，而且ADCC活性强于Gp120/Zalu和Gp120/Hz20G5.26联合ADCC活性。
表4.Hz20G5.26/Zalu bsAb ADCC报告基因实验结果
实施例6.2 huPBMC ADCC实验
通过上述ADCC报告实验结果发现，Hz20G5.26/Zalu bsAb ADCC活性强于JNJ372、EGFR单抗Zalu、Gp120/Zalu和Gp120/Hz20G5.26联合；为更能真实有效反应Hz20G5.26/Zalu bsAb ADCC活性，我们设计huPBMC ADCC实验，采用正常人PBMC进行ADCC活性验证。
实验方法：
(1)、CTS培养基(Gibco,A3021002)37℃预热，取huPBMC(秒通生物科技，PB100C-W)快速水浴融化，将细胞缓慢加入8ml CTS培养基(含有1％DNA酶)中。
(2)、300g，8min离心，去除上清，用30ml CTS(含有10uLDNA酶)重悬， 转移T75培养瓶，37℃培养箱过夜。
(3)、取过夜培养的悬浮细胞，离心300g/8min，去除上清，CTS调整细胞密度；按照效应细胞(huPBMC)和靶细胞(表5的肿瘤细胞)效靶比50:1，将huPBMC和提前准备好的肿瘤细胞(Target:huPBMC＝1.5E4/7.5E5cells/well/100ul)，铺96孔低吸附板(Corning,CLS7007-24EA)。
(4)、将稀释准备好的抗体药物加入相应细胞孔板中，37℃ 5％CO2培养8h。
(5)、300g离心5min，取50ul上清转移至96孔透明平底板((NUNC,136101)；取50ul提前配置和准备好的LDH试剂(Promega,G1780)加入相应孔板中，室温避光静置15-30min。
(6)、取50ul LDH Stop Solution(Promega,G1780)，490nm读数，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
结果参见图13和表5。Hz20G5.26/Zalu bsAb在huPBMC ADCC实验结果更能真实有效反应实际药效结果。从huPBMC ADCC结果(图13和表5)发现，Hz20G5.26/Zalu bsAb在huPBMC ADCC活性结果与ADCC报告活性结果基本一致，在众多不同类型NSCLC-EGFR肿瘤细胞系，Hz20G5.26/Zalu bsAb huPBMC ADCC活性强于JNJ373和EGFR单抗，强于Gp120/Zalu和Gp120/Hz20G5.26联合ADCC活性；通过huPBMCADCC作用，Hz20G5.26/Zalu bsAb虽然在H1975抗肿瘤杀伤率最低，但杀伤率也有36.53％；Hz20G5.26/Zalu bsAb在H322抗肿瘤杀率最高，杀伤率高达100％。
因此，Hz20G5.26/Zalu bsAb在增强EGFR信号阻断的基础上，又通过免疫细胞参与ADCC，可进一步发挥Hz20G5.26/Zalu bsAb在众多癌症肿瘤中的抗肿瘤药效活性。
表5.Hz20G5.26/Zalu bsAb huPBMC ADCC实验结果

实施例6.Hz20G5.26/Zalu bsAb双特异性抗体分子药物安全性体外研究
EGFR是一种上皮源表皮生长因子，除过度表达或异常激活导致癌症肿瘤发生外，在一些上皮细胞和角质细胞也会有EGFR的表达。相比EGFR-TKI小分子抑制剂的不良反应，如包括皮疹、腹泻、甲沟炎、口腔粘膜炎、肝损伤、间质性肺疾病，EGFR单抗也会有一定的不良反应，主要体现皮肤毒性，如疹脓疱型皮疹、甲沟炎及皮肤干燥瘙痒等，这极大影响肿瘤患者的生活质量和治疗依从性。
我们选择人皮肤鳞癌细胞系(A431,ATCC，CRL-1555)和人皮肤角质细胞(HaCat,Cell lines service,300493)细胞作为Hz20G5.26/Zalu bs Ab不良反应研究模型，在体外实验探究Hz20G5.26/Zalu bs Ab耐受性。
实验方法
(1)、将A431和HaCat细胞1500-2000cells/100ul铺96孔低吸附板(Corning,CLS7007-24EA),进行3D细胞培养，其中应用的培养基均为DMEM+10％FBS+1％Pen/strep。
(2)、将提前稀释准备好的抗体分子加到相应细胞孔板中，混合均匀，37℃5％CO2培养箱培养5天。
(3)、增殖抑制实验持续培养5天后，将提前准备好的Cell-Titer试剂(Promega,G7572)加入细胞孔中，室温避光静止15-25分钟。
(4)、将Cell-Titer和细胞混合液转入96孔白底板(NUNC,136101)，多功能酶标仪(Molecular Devices,Spectra MAXi3)检测。
结果参见图14。从实验结果发现，Hz20G5.26/Zalu bs Ab在A431和HaCat细胞上，体外药效远低于EGFR单抗，说明细胞对于Hz20G5.26/Zalu bsAb耐受性很高，不良反应远低于EGFR单抗；同时，细胞对于Hz20G5.26/Zalu bs Ab耐受性也优异于JNJ372。
因此，Hz20G5.26/Zalu bs Ab在降低EGER亲和力的基础上，应用了B7H3高亲和力Hz20G5.26亲本，提高了Hz20G5.26/Zalu bs Ab药效生物活性和药效安全窗口。
实施例7.Hz20G5.26/Zalu bsAb双特异性抗体分子体内药效活性
为了证明Hz20G5.26/Zalu双特异性抗体分子在体内的药效，采用NSCLC-EGFRWT肿瘤细胞系NCI-H292细胞(ATCC)接种Balb/c Nude小鼠测定本发明的Hz20G5.26/Zalu双特异性抗体的抗肿瘤药效。实验使用SPF等级的雌性Balb/c Nude小鼠(购自北京维通利华实验动物技术有限公司)，合格证编号为NO.110011211108430747。
将NCI-H292细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)和Matrigel Matix(Corning)等比例混合重悬NCI-H292细胞，制备成细胞浓度为20×106个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至Balb/c Nude小鼠右侧腹部区域中，建立NCI-H292荷瘤小鼠模型。
肿瘤细胞接种4天后检测各只小鼠瘤体积，进行分组(每组5只小鼠)，给药剂量和方式如表6所示。
表6：体内实验的分组、给药剂量和方式

*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号210112-0356。
h-IgG，JNJ372、Gp120/Zalu、Hz20G5.26/Zalu和Zalu mAb的使用浓度均为0.5mg/ml，每3-4天给药一次，共4次(Q3-4D x4)。分别在NCI-H292细胞接种后第4、8、12、15天给药，每周2次监测小鼠瘤体积与体重，如图15A所示，监测至59天后结束。由于部分组别在较早就出现因肿瘤导致的小鼠死亡，因此按照接种后第22天肿瘤体积计算相对肿瘤抑制率(TGI％)，计算公式如下：
TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照
组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。
采用电子天平测定体重。对于肿瘤体积超过2000mm3或体重下降超过20％的小鼠，进行安乐死处理。
肿瘤抑制率结果如表7所示：在接种后第22天，与h-IgG,5mg/kg组对比，JNJ372、Gp120/Zalu、Hz20G5.26/Zalu和Zalu mAb的肿瘤抑制率分别为100％、72％、108％和107％。Hz20G5.26/Zalu和Zalu mAb组各有1只肿瘤完全缓解的小鼠。小鼠生存曲线如图15B所示，Hz20G5.26/Zalu能显著延长小鼠的生存期。综上所述，Hz20G5.26/Zalu对NCI-H292荷瘤小鼠的抗肿瘤药效与亲本的Zalu  mAb相当，且优于JNJ372和无靶向的GP120/Zalu。
同时对小鼠体重进行监测的结果(图15C)显示，在接种后59天内,Hz20G5.26/Zalu组小鼠体重无明显下降。
表7:抗肿瘤药效统计

*完全缓解率：肿瘤完全消退，肿瘤体积为0.
为了证明Hz20G5.26/Zalu双特异性抗体分子对EGFR异常扩增的NSCLC的体内药效，采用SK-MES-1细胞(南京科佰)接种Balb/c Nude小鼠测定本发明的Hz20G5.26/Zalu双特异性抗体分子抗肿瘤药效。实验使用SPF等级的雌性Balb/c Nude小鼠(购自北京维通利华实验动物技术有限公司)，合格证编号为NO.110011211108966881。
将SK-MES-1细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)和Matrigel Matix(Corning)等比例混合SK-MES-1细胞，制备成细胞浓度为20×106个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至Balb/cNude小鼠右侧腹部区域中来建立SK-MES-1荷瘤小鼠模型。
肿瘤细胞接种6天后检测各只小鼠瘤体积，进行分组(每组7只小鼠)，给药剂量和方式如表8所示。
表8：体内实验的分组、给药剂量和方式

*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号210112-0356。
h-IgG，Hz20G5.26/Zalu，JNJ372，Zalu mAb和Gp120/Zalu的使用浓度均为0.1mg/ml。在SK-MES-1细胞接种后第6天给药1次，每周2次监测小鼠瘤体积与体重，如图16A所示，监测至24天后结束。
接种后第24天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。采用电子天平测定体重。
肿瘤抑制率结果如表9所示：在接种后第24天，与h-IgG，1mg/kg组对比，Hz20G5.26/Zalu，JNJ372，Zalu mAb和Gp120/Zalu的肿瘤抑制率分别为143％、122％、145％和113％。同时对小鼠体重进行监测的结果(图16B)显示，在接种后第24天，小鼠体重无显著差异。
表9：第24天抗肿瘤药效统计

*完全缓解率：肿瘤完全消退，肿瘤体积为0.
为了进一步证明Hz20G5.26/Zalu分子作为双特异性抗体在体内药效中的优势，采用SK-MES-1细胞(南京科佰)接种Balb/c Nude小鼠测定本发明的Hz20G5.26/Zalu双特异性抗体分子抗肿瘤药效。实验使用SPF等级的雌性Balb/c Nude小鼠(购自北京维通利华实验动物技术有限公司)，合格证编号为NO.110011221103705231。
将SK-MES-1细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)和Matrigel Matix(Corning)等比例混合SK-MES-1细胞，制备成细胞浓度为25×106个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至Balb/c Nude小鼠右侧腹部区域中来建立SK-MES-1荷瘤小鼠模型。
肿瘤细胞接种14天后检测各只小鼠瘤体积，进行分组(每组6只小鼠)，给药剂量和方式如表10所示。
表10：体内实验的分组、给药剂量和方式

*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号210112-0356。
h-IgG的使用浓度为0.06mg/ml，Hz20G5.26/Zalu，Gp120/Zalu和Gp120/Hz20G5.26的使用浓度均为0.03mg/ml。在SK-MES-1细胞接种后第14天给药1次，每周2次监测小鼠瘤体积与体重，如图17A所示，监测至35天后结束。
接种后第35天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。采用电子天平测定体重。
肿瘤抑制率结果如表11所示：在接种后第35天,与h-IgG,0.6mg/kg组对比，Hz20G5.26/Zalu，Gp120/Zalu，Gp120/Hz20G5.26和Gp120/Zalu+Gp120/Hz20G5.26的肿瘤抑制率分别为121％、31％、1％和16％。Hz20G5.26/Zalu的抗肿瘤药效优于对照无靶向单抗Gp120/Zalu，Gp120/Hz20G5.26或二者联用，证明了Hz20G5.26/Zalu作为双特异性抗体的独特机制，即Hz20G5.26对于zalu阻断EGFR信号的拉动作用。同时对小鼠体重进行监测的结果(图17B)显示，在接种后第24天，小鼠体重无显著差异。
表11：第35天抗肿瘤药效统计
实施例8.Hz20G5.26/Zalu bsAb双特异性抗体分子联合KRAS小分子抑制剂体内药效活性
为了证明Hz20G5.26/Zalu双特异性抗体分子联合KRAS小分子抑制剂在体内的药效，采用KRASG12C突变的NSCLC肿瘤细胞系NCI-H358(南京科佰)接种NOG小鼠测定本发明的Hz20G5.26/Zalu双特异性抗体联合KRAS小分子抑制剂AMG510的抗肿瘤药效。实验使用SPF等级的雌性NOG小鼠(购自北京维通利华实验动物技术有限公司)，合格证编号为NO.1100112211001153625。
将NCI-H358细胞进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)和Matrigel Matix(Corning)等比例混合重悬NCI-H358细胞，制备成细胞浓度为25×106个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至NOG小鼠右侧腹部区域中，建立NCI-H358荷瘤小鼠模型。
肿瘤细胞接种6天后检测各只小鼠瘤体积，进行分组(每组7只小鼠)，给药剂量和方式如表12所示。
表12：体内实验的分组、给药剂量和方式

*：h-IgG为同种型对照抗体，购自Equitech-Bio，批号210112-0356。
h-IgG的使用浓度为2mg/ml，Hz20G5.26/Zalu，JNJ372和Zalu mAb的使用浓度均为1mg/ml，每3-4天给药1次，共4次(Q3-4D x4)。分别在NCI-H358细胞接种后第6、9、12、15天给药，每周2次监测小鼠瘤体积与体重。AMG510的使用浓度为1mg/ml，在NCI-H358细胞接种后第6天开始给药，每天给药1次，共14次(QD x14)，每周2次监测小鼠瘤体积，在给药期间每天监测小鼠体重，结束给药后每周2次监测小鼠体重。如图18A所示，监测至33天后结束。在接种后第33天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L*W2/2。采用电子天平测定体重。
肿瘤抑制率结果如表13所示：在接种后第33天，与h-IgG，20mg/kg组对比，AMG510，Hz20G5.26/Zalu，AMG510+Hz20G5.26/Zalu，JNJ372和Zalu mAb 的肿瘤抑制率分别为89％、110％、130％、82％和111％。在各组别中，Hz20G5.26/Zalu联合AMG510对NCI-H358荷瘤小鼠的抗肿瘤药效最优，具有协同作用。
同时对小鼠体重进行监测的结果(图18B)显示，AMG510给药造成小鼠体重逐步下降，暂停给药后体重回升，在第33天各组别小鼠体重正常。
表13:第33天肿瘤抑制率
序列表：抗B7-H3/EGFR双特异性抗体的序列信息







Claims (60)

  1. 结合EGFR和B7-H3的双特异性抗体，其包含第一抗原结合区和第二抗原结合区，所述第一抗原结合区特异性结合EGFR，且所述第二抗原结合区特异性结合B7H3。
  2. 权利要求1的双特异性抗体，其中所述第二抗原结合区包含如SEQ ID NO:3、5或7所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:4、6或8所示的轻链可变区的LCDR1、2和3序列。
  3. 权利要求1所述的双特异性抗体，其中所述第二抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
    (i)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:3所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:4所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (ii)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:5所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:6所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；或
    (iii)所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:7所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:8所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  4. 权利要求1所述的双特异性抗体，其中所述第二抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
    (i)HCDR1包含如SEQ ID NO:15所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:16所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:17所示的氨基酸序列或由所述序列组成，
    LCDR1包含如SEQ ID NO:18所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:19所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:20所示的氨基酸序列或由所述序列组成；或，
    (ii)HCDR1包含如SEQ ID NO:21所示的氨基酸序列或由所述序列组成，HCDR2包含如SEQ ID NO:22所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:23所示的氨基酸序列或由所述序列组成，
    LCDR1包含如SEQ ID NO:24所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:25所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:26所示的氨基酸序列或由所述序列组成；或，
    (iii)HCDR1包含如SEQ ID NO:27所示的氨基酸序列或由所述序列组成， HCDR2包含如SEQ ID NO:28所示的氨基酸序列或由所述序列组成，HCDR3包含如SEQ ID NO:29所示的氨基酸序列或由所述序列组成，
    LCDR1包含如SEQ ID NO:30所示的氨基酸序列或由所述序列组成，LCDR2包含如SEQ ID NO:31所示的氨基酸序列或由所述序列组成，LCDR3包含如SEQ ID NO:32所示的氨基酸序列或由所述序列组成。
  5. 权利要求1-4任一项所述的双特异性抗体，其中，所述第二抗原结合区包含重链可变区VH，其中所述VH包含SEQ ID NO:3、5或7所示的氨基酸序列或与SEQ ID NO:3、5或7所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列或由所述氨基酸序列组成。
  6. 权利要求1-5任一项所述的双特异性抗体，其中，所述第二抗原结合区包含轻链可变区VL，其中所述VL包含SEQ ID NO:4、6或8所示的氨基酸序列或与SEQ ID NO:4、6或8所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列或由所述氨基酸序列组成。
  7. 权利要求1-6中任一项的双特异性抗体，其中第二抗原结合区包含重链可变区VH和轻链可变区VL，其中
    (i)所述VH包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
    (ii)所述VH包含SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
    (iii)所述VH包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:8所示的氨基酸序列或与SEQ ID NO:8的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  8. 权利要求1-7中任一项的双特异性抗体，其中第二抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
    SEQ ID NO:3和SEQ ID NO:4；
    SEQ ID NO:5和SEQ ID NO:6；或
    SEQ ID NO:7和SEQ ID NO:8。
  9. 权利要求1-7中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
    所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:2所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  10. 权利要求9的双特异性抗体，其中第一抗原结合区的HCDR1包含SEQ ID NO:9的氨基酸序列或由所述序列组成；HCDR2包含SEQ ID NO:10的氨基酸序列或由所述序列组成；HCDR3包含SEQ ID NO:11的氨基酸序列或由所述序列组成；且第一抗原结合区的LCDR1包含SEQ ID NO:12的氨基酸序列或由所述序列组成；LCDR2包含SEQ ID NO:13的氨基酸序列或由所述序列组成；且LCDR3包含SEQ ID NO:14的氨基酸序列或由所述序列组成。
  11. 权利要求9或10的双特异性抗体，其中第一抗原结合区包含重链可变区VH，其中所述VH包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  12. 权利要求9-11中任一项的双特异性抗体，其中第一抗原结合区包含轻链可变区VL，其中所述VL包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  13. 权利要求9-12中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH和轻链可变区VL，其中所述VH包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且所述VL包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  14. 权利要求9-13中任一项的双特异性抗体，其中第一抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:2。
  15. 权利要求1-14中任一项的双特异性抗体，其中第一抗原结合区特异性结合EGFR，其包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
    HCDR1包含SEQ ID NO:9的氨基酸序列或由所述序列组成；
    HCDR2包含SEQ ID NO:10的氨基酸序列或由所述序列组成；
    HCDR3包含SEQ ID NO:11的氨基酸序列或由所述序列组成；
    LCDR1包含SEQ ID NO:12的氨基酸序列或由所述序列组成；
    LCDR2包含SEQ ID NO:13的氨基酸序列或由所述序列组成；且
    LCDR3包含SEQ ID NO:14的氨基酸序列或由所述序列组成；
    且
    第二抗原结合区特异性结合B7H3，其包含重链可变区VH的HCDR1、HCDR2和HCDR3，以及轻链可变区VL的LCDR1、LCDR2和LCDR3，其中
    (i)HCDR1包含SEQ ID NO:15的氨基酸序列或由所述序列组成；
    HCDR2包含SEQ ID NO:16的氨基酸序列或由所述序列组成；
    HCDR3包含SEQ ID NO:17的氨基酸序列或由所述序列组成；
    LCDR1包含SEQ ID NO:18的氨基酸序列或由所述序列组成；
    LCDR2包含SEQ ID NO:19的氨基酸序列或由所述序列组成；且
    LCDR3包含SEQ ID NO:20的氨基酸序列或由所述序列组成；
    (ii)HCDR1包含SEQ ID NO:21的氨基酸序列或由所述序列组成；
    HCDR2包含SEQ ID NO:22的氨基酸序列或由所述序列组成；
    HCDR3包含SEQ ID NO:23的氨基酸序列或由所述序列组成；
    LCDR1包含SEQ ID NO:24的氨基酸序列或由所述序列组成；
    LCDR2包含SEQ ID NO:25的氨基酸序列或由所述序列组成；且
    LCDR3包含SEQ ID NO:26的氨基酸序列或由所述序列组成；或者
    (iii)HCDR1包含SEQ ID NO:27的氨基酸序列或由所述序列组成；
    HCDR2包含SEQ ID NO:28的氨基酸序列或由所述序列组成；
    HCDR3包含SEQ ID NO:29的氨基酸序列或由所述序列组成；
    LCDR1包含SEQ ID NO:30的氨基酸序列或由所述序列组成；
    LCDR2包含SEQ ID NO:31的氨基酸序列或由所述序列组成；且
    LCDR3包含SEQ ID NO:32的氨基酸序列或由所述序列组成。
  16. 权利要求15的双特异性抗体，其中第一抗原结合区包含含有如SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成的VH和含有如SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成的VL，且第二抗原结合区包含分别含有如下所示的氨基酸序列或由所述氨基酸序列组成的VH和VL:
    SEQ ID NO:3和SEQ ID NO:4；
    SEQ ID NO:5和SEQ ID NO:6；或
    SEQ ID NO:7和SEQ ID NO:8。
  17. 权利要求1-16中任一项的双特异性抗体，其包含Fc区，优选地，所述Fc区具有低岩藻糖基化，例如通过GlymaxX技术处理后获得的低岩藻糖基化。
  18. 权利要求17的双特异性抗体，其包含第一Fc区和第二Fc区，其中第一Fc区和第二Fc区相同或不同。
  19. 权利要求17或18所述的双特异性抗体，其中第一Fc区和第二Fc区分别为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc，或人IgG4 Fc，例如包含氨基酸序列SEQ ID NO:46或47或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。
  20. 权利要求18或19的双特异性抗体，其中在第一Fc区和第二Fc区中引入促进第一Fc区和第二Fc区的异二聚化的突变。
  21. 权利要求20的双特异性抗体，其中所述突变是基于Innobody技术引入的。
  22. 权利要求21的多特异性抗体，其中一个Fc区的CH3包含S364R和D399K突变，且另一个Fc区的CH3突变包含Y349T、K370S和K409D突变。
  23. 权利要求22的双特异性抗体，其中
    a)一个Fc区多肽包含SEQ ID NO:49或50所示的氨基酸序列或由其组成，而另一个Fc区多肽包含SEQ ID NO:52或53所示的氨基酸序列或由其组成；
    b)一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，而另一个Fc区多肽包含与SEQ ID NO:52或53所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列；或
    c)一个Fc区多肽包含与SEQ ID NO:49或50所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变Y349T、K370S和K409D，而另一个Fc区包含与SEQ ID NO:52或53所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列且包含突变S364R和D399K。
  24. 权利要求20的双特异性抗体，其中所述突变是基于Knob-into-Hole技术引入的，其中在第一Fc区和第二Fc区中引入相应的Knob突变和Hole突变。
  25. 权利要求24的双特异性抗体，其中
    a)一个Fc区多肽包含突变T366W，而另一个Fc区多肽包含T366S,L368A和Y407V(编号方式依照EU索引)，或
    b)一个Fc区包含氨基酸替代S354C和T366W,且另一个Fc区包含氨基酸替代Y349C,T366S,L368A和Y407V(编号方式依照EU索引)。
  26. 权利要求1-25中任一项所述的双特异性抗体，其中第一和/或第二抗原结合区(例如其中的重链可变区)还可以与1个或2个重链恒定区(例如人IgG1,人IgG2,人IgG3，或人IgG4的重链恒定区)连接，所述重链恒定区包含CH1 和Fc区，经由或不经由铰链区连接，例如重链可变区的C末端与重链恒定区的CH1的N末端连接。
  27. 权利要求26所述的双特异性抗体，其中所述CH1包含SEQ ID NO:42所示的氨基酸序列或与SEQ ID NO:42的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  28. 权利要求1-27所述的双特异性抗体，其中第一和/或第二抗原结合区(例如其中的轻链可变区)还可以与轻链恒定区连接，例如轻链可变区的C末端与轻链恒定区的N末端连接。
  29. 权利要求28所述的双特异性抗体，其中轻链恒定区为kappa轻链恒定区或lambda轻链恒定区。
  30. 权利要求20所述的双特异性抗体，其中所述轻链恒定区包含SEQ ID NO:54所示的氨基酸序列或与SEQ ID NO:54的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  31. 权利要求1-30中任一项所述的双特异性抗体，其中所述双特异性抗体为IgG样抗体，其具有如图1所示的构型。
  32. 权利要求31所述的双特异性抗体，其中包含重链1和轻链1，以及重链2和轻链2，其中重链1和轻链1构成第一半抗体，且重链2和轻链2构成第二半抗体；其中
    重链1包含第一抗原结合区的重链可变区和第一重链恒定区；轻链1包含第一抗原结合区的轻链可变区和第一轻链恒定区；且重链2包含第二抗原结合区的重链可变区和第二重链恒定区；轻链2包含第二抗原结合区的轻链可变区和第二轻链恒定区。
  33. 权利要求32所述的双特异性抗体，其中重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  34. 权利要求32或33所述的双特异性抗体，其中轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  35. 权利要求32-34中任一项所述的双特异性抗体，其中重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  36. 权利要求32-35中任一项所述的双特异性抗体，其中重链2包含SEQ ID NO:35、37或39所示的氨基酸序列或与SEQ ID NO:35、37或39所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  37. 权利要求32-36中任一项所述的双特异性抗体，其中轻链2包含SEQ ID NO:36、38或40所示的氨基酸序列或与SEQ ID NO:36、38或40所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  38. 权利要求32-37中任一项所述的双特异性抗体，其中
    (1)重链2包含SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:36所示的氨基酸序列或与SEQ ID NO:36所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
    (2)重链2包含SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:38所示的氨基酸序列或与SEQ ID NO:38所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
    (3)重链2包含SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且轻链2包含SEQ ID NO:40所示的氨基酸序列或与SEQ ID NO:40所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  39. 权利要求32-38中任一项所述的双特异性抗体，其中
    重链1包含SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或与SEQ ID NO:34的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且重链2和轻链2分别包含如下的SEQ ID NO:所示的氨基酸序列，或包含与所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性的氨基酸序列，或由如下SEQ ID NO所示的序列组成：
    i)SEQ ID NO:35和SEQ ID NO:36；
    ii)SEQ ID NO:37和SEQ ID NO:38；
    iii)SEQ ID NO:39和SEQ ID NO:40。
  40. 权利要求32-39任一项所述的双特异性抗体，其中，
    (i)重链1包含SEQ ID NO:33所示的氨基酸序列的重链或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
    重链2包含SEQ ID NO:35所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:36所示的氨基酸序列或由其组成；或，
    (ii)重链1包含SEQ ID NO:33所示的氨基酸序列或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
    重链2包含SEQ ID NO:37所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:38所示的氨基酸序列或由其组成；或，
    (iii)重链1包含SEQ ID NO:33所示的氨基酸序列或由其组成，且轻链1包含SEQ ID NO:34所示的氨基酸序列或由其组成，
    重链2包含SEQ ID NO:39所示的氨基酸序列或由其组成，且轻链2包含SEQ ID NO:40所示的氨基酸序列或由其组成。
  41. 权利要求1-40任一项所述的结合EGFR和B7-H3的双特异性抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段具有以下一个或多个特性：
    (i)所述抗体一方面可以阻断EGFR配体与EGFR的结合，抑制生物信号传递，阻断肿瘤相应生物活性；另一方面会刺激EGFR内吞而最终被细胞内溶酶体等降解；
    (ii)所述抗体采用对EGFR低亲和力的EGFR抗体亲本序列，极大降低了系列EGFR单克隆抗体对皮肤等正常上皮组织带来的毒副作用；
    (iii)所述抗体在EGER低亲和力的基础上引用B7-H3高亲和力的抗体亲本序列，极大提高了EGFR信号阻断活性，提高了本发明双特异性抗体的药效生物活性和药效安全窗口；
    (iv)所述抗体为低岩藻糖基化的抗体；
    (ii)所述抗体具有较高的药效生物活性和安全性；
    (v)所述抗体具有优良的肿瘤杀伤和抑制效果；
    (vi)所述抗体具有优良的ADCC体内外药效活性；
    (vii)所述抗体与KRAS小分子抑制剂联用具有优良的抗肿瘤协同效果。
  42. 分离的核酸，其编码权利要求1-41任一项的所述的结合EGFR和B7-H3的双特异性抗体中的任一条链。
  43. 载体，其包含权利要求42的核酸，优选地所述载体是表达载体，优选地，所述表达载体为pcDNA，例如pcDNA3.1。
  44. 宿主细胞，其包含权利要求42的核酸或权利要求43的载体，优选地，所述宿主细胞是原核的或真核的，更优选的酵母细胞或哺乳动物细胞(例如293细胞或CHO细胞，例如293F细胞或293T细胞或CHO-S细胞)。
  45. 权利要求44的宿主细胞，其被糖工程化而表达RMD酶，优选地，所述宿主细胞是CHO细胞。
  46. 权利要求45的宿主细胞，其包含编码RMD酶的核酸。
  47. 权利要求46的宿主细胞，其中所述RMD酶包含SEQ ID NO:41所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成，优选地，所述RMD酶来自铜绿假单胞菌。
  48. 制备结合EGFR和B7-H3的双特异性抗体的方法，所述方法包括在适于表达编码前述权利要求1-41任一项的双特异性抗体的核酸的条件下培养权利要求44-47中任一项所述的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收所述抗体或其抗原结合片段。
  49. 免疫缀合物，其包含与治疗剂或诊断剂缀合的前述权利要求1-41中任一项的双特异性抗体。
  50. 药物组合物，其包含前述权利要求1-41任一项的双特异性抗体或权利要求49的免疫缀合物，以及任选地药用辅料。
  51. 权利要求50的药物组合物，其还包含第二治疗剂；优选地，所述第二治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
  52. 药物组合产品，其包含权利要求1-41任一项的双特异性抗体或权利要求49的免疫缀合物或权利要求50的药物组合物，以及一种或多种第二治疗剂，优选地，所述第二治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
  53. 在受试者中预防或治疗肿瘤或感染性疾病的方法，所述方法包括向所述受试者施用有效量的权利要求1-41中任一项所述的双特异性抗体、或权利要求49所述的免疫缀合物、或权利要求50的药物组合物。
  54. 权利要求53所述的方法，其还包括向所述受试者联合施用一种或多种其它疗法，所述疗法例如包括治疗方式和/或其它治疗剂，优选地，所述治疗方式包括手术治疗和/或放射疗法，或者所述治疗剂选自抗血管发生剂、化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)；优选地，所述第二治疗剂选自KRAS小分子抑制剂，例如KRAS G12C抑制剂(例如AMG510(Sotorasib)或GFH925)、KRAS G12D(例如MRTX1133)或KRAS G12S抑制剂。
  55. 在受试者中预防或治疗肿瘤或感染性疾病的方法，所述方法包括向所述 受试者施用有效量的权利要求51的药物组合物或权利要求52的药物组合产品。
  56. 权利要求53-55中任一项所述的方法，其中所述肿瘤为癌症，例如实体肿瘤或血液肿瘤，包括上皮来源的癌症，例如胃肠道肿瘤或肺部肿瘤或皮肤肿瘤，例如皮肤癌(例如皮肤鳞癌、头颈癌如头颈部鳞状细胞癌)、食管癌(例如：食管鳞状癌)、肠癌(例如：结肠癌、直肠癌、结直肠癌)或肺癌(例如非小细胞肺癌、肺鳞癌、肺腺癌)。
  57. 权利要求53-56中任一项的方法，其中所述肿瘤的肿瘤细胞中
    (i)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型EGFR(例如具有升高的核酸或蛋白质水平的野生型EGFR)，和/或表达突变的EGFR，优选地，所述突变的EGFR包含选自R521K、L858R、T790M、G719X、C797S、Y1069C、Exon19缺失(Del19)、Exon20ins(例如S768_D770dup)中的一个或多个突变，优选地，所述突变的EGFR包含R521K/Y1069C、R521K、L858R/T790M/C797S、Del19/T790M/C797S或S768_D770dup；
    (ii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，过度表达野生型KRAS(例如具有升高的核酸或蛋白质水平的野生型KRAS)，或表达突变的KRAS，优选地，所述突变的KRAS包含G12位或G13位突变，例如G12D或G12C；
    (iii)相比相邻组织正常细胞或相比健康受试者中的相同组织的正常细胞，具有升高的核酸水平或蛋白水平的B7-H3；和/或
    (iv)所述肿瘤细胞对于酪氨酸激酶抑制剂，例如对一代(厄洛替尼Erlotinib)和三代(奥希替尼Osimertinib)耐药，例如对于奥希替尼耐药。
  58. 权利要求57的方法，其中所述肿瘤的肿瘤细胞中表达突变的EGFR和突变的KRAS，例如包含具有R521K的突变的EGFR和具有G120D的突变的KRAS。
  59. 检测样品中抗原EGFR和/或B7-H3的方法，所述方法包括
    (a)将样品与权利要求1-41任一项所述的双特异性抗体接触；和
    (b)检测抗体或其抗原结合片段和EGFR和/或B7-H3间的复合物的形成，任选地，所述抗体被可检测的标记。
  60. 权利要求1-41任一项所述的抗体或其抗原结合片段，和/或权利要求42所述的分离的核酸，和/或权利要求43所述的载体，和/或权利要求44-47中任一项所述的宿主细胞，和/或权利要求49所述的免疫缀合物，和/或权利要求50或51所述的药物组合物或权利要求52的药物组合产品，在制备用于预防和/或治疗受试者疾病的药物中的用途。
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