WO2024088386A1 - Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
Definitions
- the present invention relates to antibodies, and in particular, to antibodies exhibiting specificity for epidermal growth factor receptor 3, and to use thereof, for example in the treatment of cancer.
- the human epidermal growth factor receptor 3 (ErbB3, also known as HER3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes EGFR (HER1, ErbB1) , HER2 (ErbB2, Neu) , and HER4 (ErbB4) . Additionally, HER3 is a unique HER family member with no or little intracellular tyrosine kinase activity, but still having a role in both tumor progression and drug resistance.
- EGFR epidermal growth factor receptor
- HER3 is a unique HER family member with no or little intracellular tyrosine kinase activity, but still having a role in both tumor progression and drug resistance.
- HER3 is a single transmembrane protein that consists of a ligand-binding extracellular domain, a transmembrane domain, an intracellular kinase domain, and a C-terminal tail. Although HER3 has been reported to have some kinase activity, it is suggested to be 1,000-fold weaker than the kinase activity of the fully activated EGFR. Because HER3 is unable to form homodimers, HER3 is generally activated through extracellular ligand binding (e.g., NRG1) , inducing a conformational change, followed by forming active heterodimers with other members of the ErbB family, most notably the ligand binding-impaired HER2, or the fully functional EGFR.
- extracellular ligand binding e.g., NRG1
- Targeted therapies against HER family members such as EGFR and HER2 are widely and commonly used in cancer therapy by using monoclonal antibodies (mAbs) .
- mAbs monoclonal antibodies
- some cancer patients developed resistance after prolonged treatment. For instance, about 70%of patients are resistant to trastuzumab, an anti-HER2 antibody, and some patients even exhibited primary resistance.
- trastuzumab an anti-HER2 antibody
- HER3 becomes a promising target to overcome existing hurdles.
- HER3-directed antibodies have been the most pursued strategy to target HER3 so far.
- Various HER3-directed mAbs have been under preclinical and clinical development.
- the active antibodies are HMBD-001 from Hummingbird Bioscience (NCT05057013) ; ISU104 from ISU Abxis Co., Ltd. (NCT03552406) ; and SIBP-03 from Shanghai Institute of Biological Products (NCT05203601) .
- Seribantumab (MM-121 from Merrimack) , a fully human IgG2 mAb, in combination with paclitaxel or exemestane (aromatase inhibitor) did not reach the phase 2 clinical endpoint of progression-free survival (PFS) in HER3+ ovarian cancer and breast cancer patients (NCT03241810) .
- PFS progression-free survival
- Patritumab (U3-1287/AMG888) from Daiichi-Sankyo was tested in the phase 3 clinical studies assessing its efficacy NSCLC together with erlotinib, patritumab also failed to meet the efficacy criteria (NCT02134015) .
- HER3-targeted therapeutic antibodies have attempted to treat patients in clinical trials, but their efficacy only can be viewed as modest (J Exp Clin Cancer Res. 2022, Oct 21; 41 (1) : 310) . Therefore, it is imperative to incorporate new strategies to improve the HER3 targeted antibody therapies.
- HER3-targeting monoclonal antibody The insufficient internalization of HER3-targeting monoclonal antibody is an urgent problem to be solved. All the previous anti-HER3 antibodies were raised based on binding not on internalizations. Additionally, all the anti-HER3 antibodies were either isolated from phage display technology or traditional mouse hybridoma technology. We utilized fully humanized mouse to screen fully human antibodies for anti-HER3, which is totally different from all the previous anti-HER3 antibodies.
- the present invention encompasses the following aspects:
- the present disclosure provides an anti-HER3 antibody or an antigen-binding fragment thereof, comprising one or more the CDR region sequences selected from the following sequence thereof:
- An anti-HER3 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 as shown in SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; b) HCDR2 as shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO
- the heavy chain variable region sequence comprises: HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 18 and HCDR3 as shown in SEQ ID NO: 37, respectively; or HCDR1 as shown in SEQ ID NO: 02, HCDR2 as shown in SEQ ID NO: 19 and HCDR3 as shown in SEQ ID NO: 38 respectively; or HCDR1 as shown in SEQ ID NO: 03, HCDR2 as shown in SEQ ID NO: 20 and HCDR3 as shown in SEQ ID NO: 39 respectively; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 21 and HCDR3 as shown in SEQ ID NO: 40 respectively; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 22 and HCDR3 as shown in SEQ ID NO: 41 respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID
- the light chain variable region sequence comprises: LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 58, respectively; or LCDR1 as shown in SEQ ID NO: 60, LCDR2 as shown in SEQ ID NO: 78 and LCDR3 as shown in SEQ ID NO: 89, respectively; or LCDR1 as shown in SEQ ID NO: 61, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 90, respectively; or LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 90, respectively; or LCDR1 as shown in SEQ ID NO: 62, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 91, respectively; or LCDR1 as shown in SEQ ID NO: 63, LCDR2 as shown in SEQ ID NO
- the anti-HER3 antibody or antigen-binding fragment comprises: a) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:08, SEQ ID NO: 27 and SEQ ID NO: 46, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively; or b) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 09, SEQ ID NO: 28 and SEQ ID NO: 47, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 79 and SEQ ID NO: 97, respectively; or c) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ
- a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 36 and SEQ ID NO: 57, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 76, SEQ ID NO: 85 and SEQ ID NO: 100, respectively.
- a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 58, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively; or q) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 18 and SEQ ID NO: 37, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 77 and SEQ ID NO: 88, respectively; or r) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 19 and SEQ ID NO: 38, respectively; and a light chain variable region sequence
- the antibody or antigen-binding fragment thereof is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
- the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, 144, 145, 147, 149, 151, 153, 155, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 and 125, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 143, 146, 148, 150, 152, 154, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 and 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith
- the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 128, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or a-2) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or b) the heavy chain variable region as shown in SEQ ID NO: 129, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 129
- the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 128; a-2) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 126; b) the heavy chain variable region as shown in SEQ ID NO: 129; and/or the light chain variable region as shown in SEQ ID NO: 130; c) the heavy chain variable region as shown in SEQ ID NO: 131; and/or the light chain variable region as shown in SEQ ID NO: 132; d) the heavy chain variable region as shown in SEQ ID NO: 133; and/or the light chain variable region as shown in SEQ ID NO: 134; e-1) the heavy chain variable region as shown in SEQ ID NO: 135; and/or the light chain variable region as shown in SEQ ID NO: 136; e-2) the heavy chain
- the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 128; a-2) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 126; b) the heavy chain variable region as shown in SEQ ID NO: 129 and the light chain variable region as shown in SEQ ID NO: 130; c) the heavy chain variable region as shown in SEQ ID NO: 131 and the light chain variable region as shown in SEQ ID NO: 132; d) the heavy chain variable region as shown in SEQ ID NO: 133 and the light chain variable region as shown in SEQ ID NO: 134; e-1) the heavy chain variable region as shown in SEQ ID NO: 135 and the light chain variable region as shown in SEQ ID NO: 136; e-2) the heavy chain variable region as shown in SEQ ID NO: 137 and the light chain
- the antibody is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant regions is selected from ⁇ and ⁇ chain constant regions of human antibody and conservative variants thereof; more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 156 and a human light chain constant region of SEQ ID NO: 157.
- the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.
- the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 181; a-2) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 179; b) the heavy chain as shown in SEQ ID NO: 182 and the light chain as shown in SEQ ID NO: 183; c) the heavy chain as shown in SEQ ID NO: 184 and the light chain as shown in SEQ ID NO: 185; g) the heavy chain as shown in SEQ ID NO: 186 and the light chain as shown in SEQ ID NO: 187; e-1) the heavy chain as shown in SEQ ID NO: 188 and the light chain as shown in SEQ ID NO: 189; e-2) the heavy chain as shown in SEQ ID NO: 190 and the light chain as shown in SEQ ID NO: 191; f) the heavy chain as shown in SEQ ID NO: 192
- the present disclosure provides an isolated nucleic acid molecule encoding any antibody or the antigen-binding fragment.
- the antibody or antigen-binding fragment thereof of the present invention can be used to form a bispecific or multispecific binding molecule.
- the antibody or antigen-binding fragment thereof of the present invention may be part of a bispecific or multispecific binding molecule, the bispecific or multispecific binding molecule comprising a second functional module (e.g., a second antibody) having a different binding specificity as compared to the antibody or antigen-binding fragment thereof of the present invention, thereby capable of binding to at least two different binding sites and/or target molecules.
- a second functional module e.g., a second antibody
- the present disclosure provides a bispecific or multispecific binding molecule comprising the antibody or antigen-binding fragment thereof of the present invention.
- the antibody or antigen-binding fragment thereof of the present invention can be linked to a therapeutic agent to form an immunoconjugate. Because the immunoconjugate has an ability to selectively deliver one or more therapeutic agents to a target tissue.
- the immunoconjugate is an antibody-drug conjugate (ADC)
- the therapeutic agent is a cytotoxic agent.
- the present disclosure provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof of the present invention.
- the present disclosure provides a chimeric antigen receptor, which comprises an antigen-binding domain of the antibody or antigen-binding fragment thereof of the present invention.
- the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
- the antigen-binding domain is a scFv
- the chimeric antigen receptor is expressed by an immune effector cell (for example, T cell) .
- the present disclosure also provides a recombinant vector comprising the above-mentioned isolated nucleic acid molecule.
- the present disclosure also provides a host cell transformed with the above-mentioned recombinant vector, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
- the present disclosure also provides a method for producing the above-mentioned antibody or the antigen-binding fragment in a medium to produce and accumulate the antibody or the antigen-binding fragment thereof, and harvesting the antibody or the antigen-binding fragment thereof from the culture.
- the present disclosure also provides a method for immunologically detecting or measuring HER3, wherein the method comprises detecting the HER3 by contacting with the above-mentioned antibody or the antigen-binding fragment.
- the present disclosure also provides a method for diagnosing a disease related to a human HER3 positive cell, wherein the method comprises a detecting or measuring the HER3 or HER3 positive cell by contacting with the above-mentioned antibody or the antigen-binding fragment.
- the present disclosure provides a pharmaceutical composition, which comprises a therapeutically effective amount of the above-mentioned antibody or the antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluents or excipients.
- the present disclosure also provides a method of treatment or prevention of a disease related to overexpression of HER3, comprising a step of administering a therapeutically effective amount of the antibody or its antigen-binding fragment above, or the pharmaceutical composition above, to a subject in need of treatment or prevention of the disease.
- the disease related to overexpression of HER3 is cancer; preferably the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
- the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma,
- the present disclosure also provides the use of the antibody or its antigen-binding fragment above, or the pharmaceutical composition above, in the manufacture of a medicament for treating or preventing a disease related to human HER3.
- the disease related to human HER3 is the cancer with HER3 expression; preferably the cancer is breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
- the cancer is breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schw
- the obtained antibodies have a series of excellent characteristics:
- variable region sequences are different from the existing antibody; All our antibodies are fully human, which have less tendency to cause immunogenicity in the human body.
- the obtained antibodies have capacity of binding to human with high affinity, which is confirmed by flow cytometry and ELISA.
- the obtained antibodies have better internalization properties.
- the obtained antibodies are only marginally inhibited by the equal molar concentration of NRG1 molecules.
- Figure 1 In vitro binding characterization of HER3 hybridoma clones to HER3+ (T47D) and HER3-(Jurkat E6.1) cell lines using flow cytometry analysis.
- Figure 3 Internalization assay of anti-HER3 recombinant antibodies in HER3+ CHO-K1-huHER3 cells.
- the present invention is based on the development of an antibody which can specifically bind to HER3.
- the titles used in the present section are for convenience of specification only, and do not limit the present invention.
- antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
- Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
- Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
- VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
- CDR complementarity determining regions
- Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
- the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
- antigen-binding fragment of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., HER3) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
- binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) , and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
- a Fab fragment a monovalent fragment consisting of the VL, VH, CL and
- the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883) .
- Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
- human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
- the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
- the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
- recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below) , (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
- Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
- such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
- CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
- One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.
- the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (LCDR1, LCDR2, LCDR3 or L1, L2, L3) , as well as CDR1, CDR2 and CDR3 of heavy chain variable domain (HCDR1, HCDR2, HCDR3 or H1, H2, H3) .
- Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
- Exemplary conventions that can be used to identify the boundaries of CDRs including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
- Note 1 some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note 2 : any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note 3 : the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.
- nucleic acid molecule refers to a DNA molecule and a RNA molecule.
- the nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA.
- a nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
- the preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.
- the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog.
- the homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked.
- the vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segment can be ligated.
- the vector is a viral vector, wherein an additional DNA segment can be ligated into viral genome.
- the vectors disclosed herein are capable of self-replicating in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial replication origin and a episomal mammalian vector) or can be integrated into the genome of a host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector) .
- the recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thus being constructed.
- the expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule.
- the vector preferably includes: various plasmids, cosmids, phage or virus vectors and the like.
- transfectoma includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.
- sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening.
- sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.
- the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
- a relevant sequence can be synthesized artificially, especially when the fragment is short in length.
- several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.
- DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis.
- the DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art.
- mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.
- the host cell obtained is cultured.
- the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
- the monoclonal antibody obtained can be identified by conventional means.
- the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) .
- the binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980) ) .
- the antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.
- protein precipitant such as salt precipitation
- centrifugation such as salt precipitation
- cell lysis by osmosis cell lysis by osmosis
- ultrasonic treatment supercentrifugation
- molecular sieve chromatography gel
- variants of a polypeptide such as for example, an antigen-binding fragment, a protein or an antibody is a polypeptide in which one or more amino acid residues are inserted, deleted, added and/or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide.
- a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintaining biological activity of the antibody disclosed herein, for example, binding to HER3 and specificity.
- the variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%identical to the sequence of the antibody or its antigen-binding fragment disclosed herein.
- the percent identity (%) or homology may be calculated with reference to the following description.
- the percent homology or identity may be calculated as 100 x [ (identical position) /min (TGA, TGB) ] , and in the formula, TGA, TGB are the sum of the number of residues of sequences A and B compared and the internal gap position (Russell et al., J. Mol Biol., 244: 332-350 (1994) .
- the antibody of the present invention also includes a conservative variant thereof, which means that, compared to the amino acid sequence of the antibody of the present invention, there are up to 10, preferably up to 8 and more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
- conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
- K D (M)
- M molar concentration
- K D values for antibodies can be determined using methods in the art in view of the present disclosure.
- the K D of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
- affinity is the strength of interaction between an antibody or its antigen-binding fragment and an antigen, and it is determined by properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment.
- properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment.
- the antibody or its antigen-binding fragment is called "specifically binding" to its target such as an antigen, when a dissociation constant (K D ) is ⁇ l0 6 M.
- the antibody specifically binds to a target with "high affinity” , when K D is ⁇ l0 9 M.
- the term "Pharmaceutical composition” is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological /pharmaceutically acceptable carriers and excipients.
- the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.
- administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.
- administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
- Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell.
- administering and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell.
- Treatment when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
- the present disclosure includes a medicament for treating a disease associated with HER3, comprising an antibody or an antigen-binding fragment thereof of the present disclosure as an active ingredient.
- the molecules of the present disclosure are very useful for those who suffer a tumor, cancer or infectious disease when in preparations and formulations suitable for therapeutic applications.
- the present disclosure relates to a method for immunologically detecting or measuring HER3, a reagent for immunologically detecting or measuring HER3, a method for immunologically detecting or measuring cells expressing HER3, and a diagnostic reagent for diagnosis of disease related to HER3 positive cells, comprising the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human HER3, as an active ingredient.
- the method for detecting or determining the amount of HER3 may be any known method.
- it includes immunodetection or assay.
- the immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody.
- immunodetection or assay include a radioactive substance labeled immunological antibody method (RIA) , an enzyme immunoassay (EIA or ELISA) , a fluorescent immunoassay (FIA) , a luminescent immunoassay, a western blotting method, physicochemical methods, etc.
- the above-mentioned diseases related to HER3 positive cells can be diagnosed by detecting or measuring cells expressing HER3 by using the antibodies or antibody fragments thereof of the present invention.
- a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemically staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Bio system) can be used.
- the room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.
- a combination of recombinant protein antigen huHER3-His and rhesus HER3-His were used to immunize humanized mice (Alloy GK MIX strain) . Briefly, 10 ⁇ g is the typical amount of a protein antigen used in subcutaneous or intraperitoneal injections in experiments performed with ATX-Gx mice. The antigens were mixed with proprietary adjuvants for immunizations. For the subcutaneous injections, 2 sites were used (50-100 ⁇ l per site) ; and for the intraperitoneal injection we typically use 200 ⁇ l.
- Anti-HER3 antibodies were obtained by two-armed immunization schemes using genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions by RIMMS (Repetitive Immunization at Multiple Sites) protocol.
- One group of mice were immunized and boosted with recombinant protein antigen huHER3-His (AcroBio, catalog number ER3-H5223) , and the other group of mice were immunized with the same huHER3-His but boosted with rhesus HER3-His (Sino Bio, catalog number 90043-K08H) .
- the antibody immune response was monitored by an HER3-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for HER3 specificity
- the spleen lymphocytes and myeloma cells Sp2/0 were fused to obtain hybridoma cells by electrofusion or PEG fusion.
- PEG fusion was performed using Clonacell TM HY technology (STEMCELL technologies) , following manufacturer’s instructions.
- the primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10: 1 for PEG fusion.
- EXAMPLE 1-4 Screening of hybridoma clones specifically binding to HER3 protein by ELISA
- ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) .
- ELISA plates were coated with 1 ⁇ g/ml of human HER3 (Acro Bio, catalog number ER3-H5223) rhesus HER3 (Sino Bio, catalog number 90043-K08H) , mouse HER3 (Acro Bio, catalog number ER3-M52H5) , rat HER3 (Sino Bio, catalog number 80111-r08H) or BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature.
- HER3 hybridoma supernatant 50 ⁇ l HER3 hybridoma supernatant was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off and 50 ⁇ l of 1: 20000 diluted secondary antibody Goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for another 1 hour. Plates were washed before the addition 50 ⁇ L of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25 ⁇ L of 2N sulfuric acid. Optical density at 450nm of samples was measured by a microplate reader (PerkinElmer) . All tested clones showed selective binding to human HER3 but not to BSA, demonstrating HER3 specificity.
- EXAMPLE 1-5 Screening of hybridoma clones specifically binding to HER3+ cancer cells by Flow cytometry
- Hybridoma supernatants were subjected to binding tests on HER3+ cell line T-47D (ATCC, HTB-133) , and HER3-cell line Jurkat E6.1 (ATCC, TIB-152) using flow cytometry analysis. Briefly, 50 ⁇ L of cells in cell staining buffer (2 ⁇ 10 6 cells/mL) was mixed with 50 ⁇ L undiluted hybridoma supernatants. The mixture was incubated on ice for 20 min and then washed with ice cold staining buffer twice. The cells were subsequently stained with 50 ⁇ L of PE labelled secondary antibody (1: 400 dilution, BioLegend, Cat#405307) for 20 min.
- FIG. 1 shows examples of selected cell binding signals measured by flow cytometry.
- EXAMPLE 1-6 Screening of hybridoma clones with cell internalization activity by indirect killing assay
- CHO-K1-huHER3 cells were seeded in 96 well plate at 3,000 cells/well and incubated overnight.
- the hybridoma supernatants from each hybridoma clone were diluted with hybridoma culture medium and mixed with Fab anti-mouse IgG Fc-MMAF conjugates with cleavable linker (Moradec, AM-202AF) , then added to each well.
- the final concentrations of mouse IgG were roughly 10 nM, 2 nM, and 0.4 nM.
- the final concentration of the Fab anti-mouse IgG Fc-MMAF conjugates in each well was 20 nM. With the presence of secondary Fab-vc-MMAF, the internalized antibody/Fac-vc-MMAF conjugates complex will release the cytotoxic payload and kill the cells. After 3 days incubation, the viable cells in each well were detected by Cell Titer Glo 2.0 assay (Promega, G9243) . A purified anti-human HER3 antibody was used as positive control. A purified mouse IgG1 antibody was used as isotype control (Biolegend, Cat#400102) . Cells treated with selected hybridoma supernatants showed reduced viability, as shown in Figure 2, indicating the internalization of the antibodies.
- the process of cloning sequences from positive hybridomas are as follows.
- the logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification.
- Amplified cDNA library from each clone were subjected to next-generation sequencing.
- the amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies of lead candidates were obtained.
- several mutations were made in the FR region, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following tables.
- the amino acid residues of the CDRs in VH/VL are numbered and annotated according to the Kabat &Wu numbering system.
- Example 3-1 Molecular cloning of recombinant antibodies
- the cDNA sequences that encode VH and VL regions of selected clones were directly synthesized as DNA fragments with 5’ -end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into selected vectors using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) . VH region was cloned into pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) using EcoRI site and NheI site, which in-frame with constant region of hIgG1 heavy chain in the vector.
- VL region was cloned into pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) using AgeI site and BsiWI site, which in-frame with constant region of hIg kappa light chain in the vector.
- the IgG form of antibodies were disclosed as the following heavy chain and light chain full lengths.
- Example 3-2 Expression and purification of recombinant antibodies
- the heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-Scells (ThermoFisher #A29127) using ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 1: 1. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 C, 8%CO2.
- Example 4 Binding characterization of anti-HER3 recombinant antibody to HER3 + cell lines by flow cytometry
- Binding of the hlgGl mAbs to the cell surface HER3 was determined by FACS analysis using T-47D cell, the cancer cell lines positive for HER3 expression. Experimental procedures refer to Example 1-5.
- Example 5 Characterization of anti-HER3 recombinant antibody cell internalization in HER3 expressing cells
- CHO-K1-huHER3 cells were seeded in 96 well plate at 20,000 cells/well and incubated overnight.
- the recombinant antibodies were diluted with cell culture medium and mixed with Fab anti-human Fc-pHast conjugates with pH dependent fluorescent reporter pHast (Advanced Targeting Systems, PH-01) , then added to each well.
- the final concentrations of anti-HER3 recombinant antibodies were 1 nM, 3 nM, and 9 nM.
- the final concentration of the Fab anti-human Fc-pHast conjugates in each well was 35 nM. With the presence of secondary Fab-pHast, the internalized antibody/Fac-pHast conjugates complex will show increased fluorescence in the acidic environment inside a cell. After 17 hours of incubation, the fluorescence from all the wells were measured using a plate reader. A purified anti-human HER3 antibody, Patritumab, was used as positive control. A purified human IgG1 antibody was used as negative control. As shown in Figure 3, strong internalization signals in CHO-K1-huHER3 cells were observed for anti-HER3 antibodies.
- Example 6-1 ELISA binding to human HER3 subdomain proteins
- ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) .
- 96 well ELISA plates were coated with 1 ⁇ g/well of human HER3 protein (HER3-His) or human HER3 subdomains 1&2 (HER3 D1-2, a.a. 20-329) , HER3 subdomain 2 (HER3 D2, a.a. 185-329) , HER3 subdomain 3&4 (HER3 D3-4, a.a. 330-643) and HER3 subdomain 4 (HER3 D4, a.a. 496-643) overnight at 4°C.
- human HER3 protein HER3-His
- human HER3 subdomains 1&2 HER3 D1-2, a.a. 20-329)
- HER3 subdomain 2 HER3 D2, a.a. 185-329)
- HER3 subdomain 3&4 HER3 D3-4, a.a. 330-643
- the leading three clones (18E11-1, 20E1-3, 23F6-1) were subjected to epitope binning and compared to reference anti-HER3 antibodies patritumab.
- Antibody epitope binning was performed using an Octet Red384 system equipped with Ni-NTA biosensors from Pall Life Sciences (Menlo Park, CA) . The experiment was performed as an in-tandem binning assay.
- the assay is comprised of a five-step binding cycle: 1) A buffer baseline was established for 30 seconds, 2) 5 nM HER3 antigen (HER3-His) was coupled to Ni-NTA octet sensors using a standard 1x assay buffer (PBS + 0.02%Tween20, 0.1%BSA, 0.05%sodium azide) diluted from a 10x kinetic buffer stock (ForteBio) for 5 minutes, 3) 25 nM of each antibody (saturating mAb) was loaded to saturate the immobilized antigen for 10 minutes, 4) 25 nM of each antibody (competing mAb) was bound for 5 minutes, and 5) capture sensors were regenerated for 30 seconds.
- a standard 1x assay buffer PBS + 0.02%Tween20, 0.1%BSA, 0.05%sodium azide
- all the four anti-HER3 antibodies can be divided into 2 different epitope bins.
- 18E11-1, 20E1-3 and 23F6-1 are in the same bin, while patritumab is in a different bin.
- patritumab After binding of 18E11-1, 20E1-3, or 23F6-1, patritumab can still bind to HER3 with a great association curve, indicating different epitopes.
- NRG1 has been shown to have high affinity ( [Kd] ⁇ 100 pM) for HER3 as a natural ligand (Am J Respir Cell Mol Biol. 2000 Apr; 22 (4) : 432-40. ) , it is hard for an antibody to compete with. However, antibodies could bind the antigen on different epitope which could be different than where NRG1 could bind, we then tested our antibodies on SKBr3 cell
- HER3 recombinant antibodies to HER3+ cell line by flow cytometry were inhibited in the presence of NRG1 in SKBr3 (HER3+, EGFR+ and HER2+) cells.
- some HER3 recombinant antibodies (18E11-1, 20E1-3 and 23F6-1) were not affected by NRG1 and still showed binding to HER3+ SKBr3 (HER3+, EGFR+ and HER2+) cells in the presence of NRG1, HER3 recombinant antibody 20B5-1 is only marginally inhibited by the equal molar concentration of NRG1 molecules, whereas the binding of patritumab is significantly affected.
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Abstract
The present application discloses the antibodies or antigen-binding fragments, and their pharmaceutical use. In particular, a monoclonal antibody according to the present application suppresses tumors, and can thus be useful as a cancer therapeutic agent and in targeted cancer treatments including the detection of various cancers, and the delivery of drugs to specific cancers, etc
Description
The present invention relates to antibodies, and in particular, to antibodies exhibiting specificity for epidermal growth factor receptor 3, and to use thereof, for example in the treatment of cancer.
The human epidermal growth factor receptor 3 (ErbB3, also known as HER3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes EGFR (HER1, ErbB1) , HER2 (ErbB2, Neu) , and HER4 (ErbB4) . Additionally, HER3 is a unique HER family member with no or little intracellular tyrosine kinase activity, but still having a role in both tumor progression and drug resistance. HER3 is a single transmembrane protein that consists of a ligand-binding extracellular domain, a transmembrane domain, an intracellular kinase domain, and a C-terminal tail. Although HER3 has been reported to have some kinase activity, it is suggested to be 1,000-fold weaker than the kinase activity of the fully activated EGFR. Because HER3 is unable to form homodimers, HER3 is generally activated through extracellular ligand binding (e.g., NRG1) , inducing a conformational change, followed by forming active heterodimers with other members of the ErbB family, most notably the ligand binding-impaired HER2, or the fully functional EGFR.
Targeted therapies against HER family members such as EGFR and HER2 are widely and commonly used in cancer therapy by using monoclonal antibodies (mAbs) . However, due to the adaptive characteristics of cancer treatments, some cancer patients developed resistance after prolonged treatment. For instance, about 70%of patients are resistant to trastuzumab, an anti-HER2 antibody, and some patients even exhibited primary resistance. Several studies reported that strong expression of HER3 was observed after tumor grows resistance to trastuzumab treatment, hence HER3 becomes a promising target to overcome existing hurdles.
Because HER3 has only minimal kinase activity, HER3-directed antibodies have been the most pursued strategy to target HER3 so far. Various HER3-directed mAbs have been under preclinical and clinical development. Currently, the active antibodies are HMBD-001 from Hummingbird Bioscience (NCT05057013) ; ISU104 from ISU Abxis Co., Ltd. (NCT03552406) ; and SIBP-03 from Shanghai Institute of Biological Products (NCT05203601) . However, Seribantumab (MM-121 from Merrimack) , a fully human IgG2 mAb, in combination with paclitaxel or exemestane (aromatase inhibitor) did not reach the phase 2 clinical endpoint of progression-free survival (PFS) in HER3+ ovarian cancer and breast cancer patients (NCT03241810) . Additionally, Patritumab (U3-1287/AMG888) from Daiichi-Sankyo was tested in the phase 3 clinical studies assessing its efficacy NSCLC together with erlotinib, patritumab also failed to meet the efficacy criteria (NCT02134015) . Many HER3-targeted therapeutic antibodies have attempted to treat patients in clinical trials, but their efficacy only can be viewed as modest (J Exp Clin Cancer Res. 2022, Oct 21; 41 (1) : 310) . Therefore, it is imperative to incorporate new strategies to improve the HER3 targeted antibody therapies.
The insufficient internalization of HER3-targeting monoclonal antibody is an urgent problem to be solved. All the previous anti-HER3 antibodies were raised based on binding not on internalizations. Additionally, all the anti-HER3 antibodies were either isolated from phage display technology or traditional mouse hybridoma technology. We utilized fully humanized mouse to screen fully human antibodies for anti-HER3, which is totally different from all the previous anti-HER3 antibodies.
Specifically, the present invention encompasses the following aspects:
The present disclosure provides an anti-HER3 antibody or an antigen-binding fragment thereof, comprising one or more the CDR region sequences selected from the following sequence thereof: An anti-HER3 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 as shown in SEQ ID NO:
01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; b) HCDR2 as shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36; c) HCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 or SEQ ID NO: 58; d) LCDR1 as shown in SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76; e) LCDR2 as shown in SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87; f) LCDR3 as shown in SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 or SEQ ID NO: 104.
In some embodiments, the heavy chain variable region sequence comprises: HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 18 and HCDR3 as shown in SEQ ID NO: 37, respectively; or HCDR1 as shown in SEQ ID NO: 02, HCDR2 as shown in SEQ ID NO: 19 and HCDR3 as shown in SEQ ID NO: 38 respectively; or HCDR1 as shown in SEQ ID NO: 03, HCDR2 as shown in SEQ ID NO: 20 and HCDR3 as shown in SEQ ID NO: 39 respectively; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 21 and HCDR3 as shown in SEQ ID NO: 40 respectively; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 22 and HCDR3 as shown in SEQ ID NO: 41 respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 23 and HCDR3 as shown in SEQ ID NO: 42 respectively; or HCDR1 as shown in SEQ ID NO: 07, HCDR2 as shown in SEQ ID NO: 24 and HCDR3 as shown in SEQ ID NO: 43 respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 25 and HCDR3 as shown in SEQ ID NO: 44, respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 26 and HCDR3 as shown in SEQ ID NO: 45 respectively; or HCDR1 as shown in SEQ ID NO: 08, HCDR2 as shown in SEQ ID NO: 27 and HCDR3 as shown in SEQ ID NO: 46 respectively; or HCDR1 as shown in SEQ ID NO: 09, HCDR2 as shown in SEQ ID NO: 28 and HCDR3 as shown in SEQ ID NO: 47 respectively; or HCDR1 as shown in SEQ ID NO: 10, HCDR2 as shown in SEQ ID NO: 29 and HCDR3 as shown in SEQ ID NO: 48 respectively; or HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 30 and HCDR3 as shown in SEQ ID NO: 49 respectively; or HCDR1 as shown in SEQ ID NO: 12, HCDR2 as shown in SEQ ID NO: 31, and HCDR3 as shown in SEQ ID NO: 50 respectively; or HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 21 and HCDR3 as shown in SEQ ID NO: 51 respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 32 and HCDR3 as shown in SEQ ID NO: 52 respectively; or HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 33, and HCDR3 as shown in SEQ ID NO: 53 respectively; or HCDR1 as shown in SEQ ID NO: 14, HCDR2 as shown in SEQ ID NO: 33 and HCDR3 as shown in SEQ ID NO: 54 respectively; or HCDR1 as shown in SEQ ID NO: 15, HCDR2 as shown in SEQ ID NO: 34, and HCDR3 as shown in SEQ ID NO: 55 respectively; or HCDR1 as shown in SEQ ID NO: 16, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 56 respectively; or HCDR1 as shown in SEQ ID NO: 17, HCDR2 as shown in SEQ ID NO: 36, and HCDR3 as shown in SEQ ID NO: 57, respectively; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 32 and HCDR3 as shown in SEQ ID NO: 58 respectively.
In some embodiments, the light chain variable region sequence comprises: LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 58, respectively; or LCDR1 as shown in SEQ ID NO: 60, LCDR2 as shown in SEQ ID NO: 78 and LCDR3 as shown in SEQ ID NO: 89, respectively; or LCDR1 as shown in SEQ ID NO: 61, LCDR2 as shown
in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 90, respectively; or LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 90, respectively; or LCDR1 as shown in SEQ ID NO: 62, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 91, respectively; or LCDR1 as shown in SEQ ID NO: 63, LCDR2 as shown in SEQ ID NO: 79, and LCDR3 as shown in SEQ ID NO: 92, respectively; or LCDR1 as shown in SEQ ID NO: 60, LCDR2 as shown in SEQ ID NO: 80 and LCDR3 as shown in SEQ ID NO: 93, respectively; or LCDR1 as shown in SEQ ID NO: 64, LCDR2 as shown in SEQ ID NO: 81, and LCDR3 as shown in SEQ ID NO: 94, respectively; or LCDR1 as shown in SEQ ID NO: 65, LCDR2 as shown in SEQ ID NO: 82 and LCDR3 as shown in SEQ ID NO: 95, respectively; or LCDR1 as shown in SEQ ID NO: 60, LCDR2 as shown in SEQ ID NO: 80 and LCDR3 as shown in SEQ ID NO: 89, respectively; or LCDR1 as shown in SEQ ID NO: 66, LCDR2 as shown in SEQ ID NO: 83 and LCDR3 as shown in SEQ ID NO: 96, respectively; or LCDR1 as shown in SEQ ID NO: 67, LCDR2 as shown in SEQ ID NO: 79 and LCDR3 as shown in SEQ ID NO: 97, respectively; or LCDR1 as shown in SEQ ID NO: 68, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 98, respectively; or LCDR1 as shown in SEQ ID NO: 69, LCDR2 as shown in SEQ ID NO: 84, and LCDR3 as shown in SEQ ID NO: 99, respectively; or LCDR1 as shown in SEQ ID NO: 70, LCDR2 as shown in SEQ ID NO: 85 and LCDR3 as shown in SEQ ID NO: 100, respectively; or LCDR1 as shown in SEQ ID NO: 71, LCDR2 as shown in SEQ ID NO: 85 and LCDR3 as shown in SEQ ID NO: 100, respectively; or LCDR1 as shown in SEQ ID NO: 61, LCDR2 as shown in SEQ ID NO: 77 and LCDR3 as shown in SEQ ID NO: 90, respectively; or LCDR1 as shown in SEQ ID NO: 72, LCDR2 as shown in SEQ ID NO: 86, and LCDR3 as shown in SEQ ID NO: 101, respectively; or LCDR1 as shown in SEQ ID NO: 73, LCDR2 as shown in SEQ ID NO: 82 and LCDR3 as shown in SEQ ID NO: 103, respectively; or LCDR1 as shown in SEQ ID NO: 64, LCDR2 as shown in SEQ ID NO: 82 and LCDR3 as shown in SEQ ID NO: 103, respectively; or LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 79 and LCDR3 as shown in SEQ ID NO: 92, respectively; or LCDR1 as shown in SEQ ID NO: 75, LCDR2 as shown in SEQ ID NO: 82 and LCDR3 as shown in SEQ ID NO: 104, respectively; or LCDR1 as shown in SEQ ID NO: 65, LCDR2 as shown in SEQ ID NO: 82 and LCDR3 as shown in SEQ ID NO: 95, respectively; or LCDR1 as shown in SEQ ID NO: 76, LCDR2 as shown in SEQ ID NO: 85 and LCDR3 as shown in SEQ ID NO: 100, respectively; or LCDR1 as shown in SEQ ID NO: 66, LCDR2 as shown in SEQ ID NO: 83 and LCDR3 as shown in SEQ ID NO: 96, respectively.
In a preferable embodiment, the anti-HER3 antibody or antigen-binding fragment comprises: a) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:08, SEQ ID NO: 27 and SEQ ID NO: 46, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively; or b) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 09, SEQ ID NO: 28 and SEQ ID NO: 47, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 79 and SEQ ID NO: 97, respectively; or c) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 29 and SEQ ID NO: 48, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 68, SEQ ID NO: 77 and SEQ ID NO: 98, respectively; or d) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 30 and SEQ ID NO: 49, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 69, SEQ ID NO: 84 and SEQ ID NO: 99, respectively; or e) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: 50, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 85 and SEQ ID NO: 100, respectively; or f) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: 50, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 85 and SEQ ID NO: 100, respectively; or g) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 21 and SEQ ID NO: 51, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2
and LCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 77 and SEQ ID NO: 90, respectively; or h) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 52, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 72, SEQ ID NO: 86 and SEQ ID NO: 101, respectively; or i) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 52, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 87 and SEQ ID NO: 102, respectively; or j) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 53, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 82 and SEQ ID NO: 103, respectively; or k) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 33 and SEQ ID NO: 54, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 82 and SEQ ID NO: 103, respectively; or l) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 34 and SEQ ID NO: 55, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 79 and SEQ ID NO: 92, respectively; or m) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 35 and SEQ ID NO: 56, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 75, SEQ ID NO: 82 and SEQ ID NO: 104, respectively; or n) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 35 and SEQ ID NO: 56, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SEQ ID NO: 82 and SEQ ID NO: 95, respectively. or o) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 36 and SEQ ID NO: 57, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 76, SEQ ID NO: 85 and SEQ ID NO: 100, respectively. or p) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 58, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively; or q) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 18 and SEQ ID NO: 37, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 77 and SEQ ID NO: 88, respectively; or r) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 19 and SEQ ID NO: 38, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ ID NO: 78 and SEQ ID NO: 89, respectively; or s) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 20 and SEQ ID NO: 39, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 77 and SEQ ID NO: 90, respectively; or t) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 21 and SEQ ID NO: 40, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 77 and SEQ ID NO: 90, respectively; or u) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 22 and SEQ ID NO: 41, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 77 and SEQ ID NO: 91, respectively; or v) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 23 and SEQ ID NO: 42, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 63, SEQ ID NO: 79 and SEQ ID NO: 92, respectively; or w) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 24 and SEQ ID NO: 43, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ
ID NO: 80 and SEQ ID NO: 93, respectively; or x) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 25 and SEQ ID NO: 44, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 81 and SEQ ID NO: 94, respectively; or y) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 26 and SEQ ID NO: 45, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SEQ ID NO: 82 and SEQ ID NO: 95, respectively; or z) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 08, SEQ ID NO: 27 and SEQ ID NO: 46, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ ID NO: 80 and SEQ ID NO: 89, respectively.
In a preferable embodiment, the antibody or antigen-binding fragment thereof is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
In some embodiments, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, 144, 145, 147, 149, 151, 153, 155, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 and 125, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 143, 146, 148, 150, 152, 154, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 and 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
In some embodiments, the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 128, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or a-2) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or b) the heavy chain variable region as shown in SEQ ID NO: 129, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 130, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or c) the heavy chain variable region as shown in SEQ ID NO: 131, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 132, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or d) the heavy chain variable region as shown in SEQ ID NO: 133, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 134, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or e-1) the heavy chain variable region as shown in SEQ ID NO: 135, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 136, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or e-2) the heavy chain variable region as shown in SEQ ID NO: 137, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 138, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or f) the heavy chain variable region as shown in SEQ ID NO: 139, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 140, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or g-1) the heavy chain variable region as shown in SEQ ID NO: 141, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 142, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or g-2) the heavy chain variable region as shown in SEQ ID NO: 141, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 143, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or g-3) the heavy chain variable region as shown in SEQ ID NO: 144, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 142, or having at least 80%, 85%, 90%, 95%
or 99%sequence identity therewith; or h) the heavy chain variable region as shown in SEQ ID NO: 145, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 146, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or i) the heavy chain variable region as shown in SEQ ID NO: 147, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 148, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or j) the heavy chain variable region as shown in SEQ ID NO: 149, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 150, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or k-1) the heavy chain variable region as shown in SEQ ID NO: 151, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 152, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or k-2) the heavy chain variable region as shown in SEQ ID NO: 151, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 122, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or l) the heavy chain variable region as shown in SEQ ID NO: 153, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 154, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or m) the heavy chain variable region as shown in SEQ ID NO: 155, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or n) the heavy chain variable region as shown in SEQ ID NO: 105, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 106, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or o) the heavy chain variable region as shown in SEQ ID NO: 107, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 108, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or p) the heavy chain variable region as shown in SEQ ID NO: 109, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 110, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or q) the heavy chain variable region as shown in SEQ ID NO: 111, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 112, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or r) the heavy chain variable region as shown in SEQ ID NO: 113, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 114, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or s) the heavy chain variable region as shown in SEQ ID NO: 115, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 116, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or t) the heavy chain variable region as shown in SEQ ID NO: 117, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 118, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or u) the heavy chain variable region as shown in SEQ ID NO: 119, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 120, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or v) the heavy chain variable region as shown in SEQ ID NO: 121, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 122, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or w-1) the heavy chain variable region as shown in SEQ ID NO: 123, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 124, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or w-2) the heavy chain variable region as shown in SEQ ID NO: 125, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or x) the heavy chain variable region as shown in SEQ ID NO: 123, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or
the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
In some embodiments, the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 128; a-2) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 126; b) the heavy chain variable region as shown in SEQ ID NO: 129; and/or the light chain variable region as shown in SEQ ID NO: 130; c) the heavy chain variable region as shown in SEQ ID NO: 131; and/or the light chain variable region as shown in SEQ ID NO: 132; d) the heavy chain variable region as shown in SEQ ID NO: 133; and/or the light chain variable region as shown in SEQ ID NO: 134; e-1) the heavy chain variable region as shown in SEQ ID NO: 135; and/or the light chain variable region as shown in SEQ ID NO: 136; e-2) the heavy chain variable region as shown in SEQ ID NO: 137; and/or the light chain variable region as shown in SEQ ID NO: 138; f) the heavy chain variable region as shown in SEQ ID NO: 139; and/or the light chain variable region as shown in SEQ ID NO: 140; g-1) the heavy chain variable region as shown in SEQ ID NO: 141; and/or the light chain variable region as shown in SEQ ID NO: 142; g-2) the heavy chain variable region as shown in SEQ ID NO: 141; and/or the light chain variable region as shown in SEQ ID NO: 143; g-3) the heavy chain variable region as shown in SEQ ID NO: 144; and/or the light chain variable region as shown in SEQ ID NO: 142; h) the heavy chain variable region as shown in SEQ ID NO: 145; and/or the light chain variable region as shown in SEQ ID NO: 146; i) the heavy chain variable region as shown in SEQ ID NO: 147; and/or the light chain variable region as shown in SEQ ID NO: 148; j) the heavy chain variable region as shown in SEQ ID NO: 149; and/or the light chain variable region as shown in SEQ ID NO: 150; k-1) the heavy chain variable region as shown in SEQ ID NO: 151; and/or the light chain variable region as shown in SEQ ID NO: 152; k-2) the heavy chain variable region as shown in SEQ ID NO: 151; and/or the light chain variable region as shown in SEQ ID NO: 122; l) the heavy chain variable region as shown in SEQ ID NO: 153; and/or the light chain variable region as shown in SEQ ID NO: 154; m) the heavy chain variable region as shown in SEQ ID NO: 155; and/or the light chain variable region as shown in SEQ ID NO: 126; n) the heavy chain variable region as shown in SEQ ID NO: 105; and/or the light chain variable region as shown in SEQ ID NO: 106; o) the heavy chain variable region as shown in SEQ ID NO: 107; and/or the light chain variable region as shown in SEQ ID NO: 108; p) the heavy chain variable region as shown in SEQ ID NO: 109; and/or the light chain variable region as shown in SEQ ID NO: 110; q) the heavy chain variable region as shown in SEQ ID NO: 111; and/or the light chain variable region as shown in SEQ ID NO: 112; r) the heavy chain variable region as shown in SEQ ID NO: 113; and/or the light chain variable region as shown in SEQ ID NO: 114; s) the heavy chain variable region as shown in SEQ ID NO: 115; and/or the light chain variable region as shown in SEQ ID NO: 116; t) the heavy chain variable region as shown in SEQ ID NO: 117; and/or the light chain variable region as shown in SEQ ID NO: 118; u) the heavy chain variable region as shown in SEQ ID NO: 119; and/or the light chain variable region as shown in SEQ ID NO: 120; v) the heavy chain variable region as shown in SEQ ID NO: 121; and/or the light chain variable region as shown in SEQ ID NO: 122; w-1) the heavy chain variable region as shown in SEQ ID NO: 123; and/or the light chain variable region as shown in SEQ ID NO: 124; w-2) the heavy chain variable region as shown in SEQ ID NO: 125; and/or the light chain variable region as shown in SEQ ID NO: 126; x) the heavy chain variable region as shown in SEQ ID NO: 123; and/or the light chain variable region as shown in SEQ ID NO: 126.
In a preferable embodiment, the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 128; a-2) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 126; b) the heavy chain variable region as shown in SEQ ID NO: 129 and the light chain variable region as shown in SEQ ID NO: 130; c) the heavy chain variable region as shown in SEQ ID NO: 131 and the light chain variable region as shown in SEQ ID NO: 132; d) the heavy chain variable region as shown in SEQ ID NO: 133 and the light chain variable region as shown in SEQ ID NO: 134; e-1) the heavy chain variable region as shown in SEQ ID NO: 135 and the light chain variable region as shown in SEQ ID NO: 136; e-2) the heavy chain variable region as shown in SEQ ID NO: 137 and the light chain variable region as shown in SEQ ID NO: 138; f) the heavy chain
variable region as shown in SEQ ID NO: 139 and the light chain variable region as shown in SEQ ID NO: 140; g-1) the heavy chain variable region as shown in SEQ ID NO: 141 and the light chain variable region as shown in SEQ ID NO: 142; g-2) the heavy chain variable region as shown in SEQ ID NO: 141 and the light chain variable region as shown in SEQ ID NO: 143; g-3) the heavy chain variable region as shown in SEQ ID NO: 144 and the light chain variable region as shown in SEQ ID NO: 142; h) the heavy chain variable region as shown in SEQ ID NO: 145 and the light chain variable region as shown in SEQ ID NO: 146; i) the heavy chain variable region as shown in SEQ ID NO: 147 and the light chain variable region as shown in SEQ ID NO: 148; j) the heavy chain variable region as shown in SEQ ID NO: 149 and the light chain variable region as shown in SEQ ID NO: 150; k-1) the heavy chain variable region as shown in SEQ ID NO: 151 and the light chain variable region as shown in SEQ ID NO: 152; k-2) the heavy chain variable region as shown in SEQ ID NO: 151 and the light chain variable region as shown in SEQ ID NO: 122; l) the heavy chain variable region as shown in SEQ ID NO: 153 and the light chain variable region as shown in SEQ ID NO: 154; m) the heavy chain variable region as shown in SEQ ID NO: 155 and the light chain variable region as shown in SEQ ID NO: 126; n) the heavy chain variable region as shown in SEQ ID NO: 105 and the light chain variable region as shown in SEQ ID NO: 106; o) the heavy chain variable region as shown in SEQ ID NO: 107 and the light chain variable region as shown in SEQ ID NO: 108; p) the heavy chain variable region as shown in SEQ ID NO: 109 and the light chain variable region as shown in SEQ ID NO: 110; q) the heavy chain variable region as shown in SEQ ID NO: 111 and the light chain variable region as shown in SEQ ID NO: 112; r) the heavy chain variable region as shown in SEQ ID NO: 113 and the light chain variable region as shown in SEQ ID NO: 114; s) the heavy chain variable region as shown in SEQ ID NO: 115 and the light chain variable region as shown in SEQ ID NO: 116; t) the heavy chain variable region as shown in SEQ ID NO: 117 and the light chain variable region as shown in SEQ ID NO: 118; u) the heavy chain variable region as shown in SEQ ID NO: 119 and the light chain variable region as shown in SEQ ID NO: 120; v) the heavy chain variable region as shown in SEQ ID NO: 121 and the light chain variable region as shown in SEQ ID NO: 122; w-1) the heavy chain variable region as shown in SEQ ID NO: 123 and the light chain variable region as shown in SEQ ID NO: 124; w-2) the heavy chain variable region as shown in SEQ ID NO: 125 and the light chain variable region as shown in SEQ ID NO: 126; x) the heavy chain variable region as shown in SEQ ID NO: 123 and the light chain variable region as shown in SEQ ID NO: 126.
In some embodiments, the antibody is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant regions is selected from κ and λchain constant regions of human antibody and conservative variants thereof; more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 156 and a human light chain constant region of SEQ ID NO: 157.
In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.
In a preferable embodiment, the antibody or antigen-binding fragment thereof comprising: a-1) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 181; a-2) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 179; b) the heavy chain as shown in SEQ ID NO: 182 and the light chain as shown in SEQ ID NO: 183; c) the heavy chain as shown in SEQ ID NO: 184 and the light chain as shown in SEQ ID NO: 185; g) the heavy chain as shown in SEQ ID NO: 186 and the light chain as shown in SEQ ID NO: 187; e-1) the heavy chain as shown in SEQ ID NO: 188 and the light chain as shown in SEQ ID NO: 189; e-2) the heavy chain as shown in SEQ ID NO: 190 and the light chain as shown in SEQ ID NO: 191; f) the heavy chain as shown in SEQ ID NO: 192 and the light chain as shown in SEQ ID NO: 193; g-1) the heavy chain as shown in SEQ ID NO: 194 and the light chain as shown in SEQ ID NO: 195; g-2) the heavy chain as shown in SEQ ID NO: 194 and the light chain as shown in SEQ ID NO: 196; g-3) the heavy chain as shown in SEQ ID NO: 197 and the light chain as shown in SEQ ID NO: 195; h) the heavy chain as shown in SEQ ID NO: 198 and the light chain as shown in SEQ ID NO: 199; i) the heavy chain
as shown in SEQ ID NO: 200 and the light chain as shown in SEQ ID NO: 201; j) the heavy chain as shown in SEQ ID NO: 202 and the light chain as shown in SEQ ID NO: 203; k-1) the heavy chain as shown in SEQ ID NO: 204 and the light chain as shown in SEQ ID NO: 205; k-2) the heavy chain as shown in SEQ ID NO: 204 and the light chain as shown in SEQ ID NO: 175; l) the heavy chain as shown in SEQ ID NO: 206 and the light chain as shown in SEQ ID NO: 207; m) the heavy chain as shown in SEQ ID NO: 208 and the light chain as shown in SEQ ID NO: 179; n) the heavy chain as shown in SEQ ID NO: 158 and the light chain as shown in SEQ ID NO: 159; o) the heavy chain as shown in SEQ ID NO: 160 and the light chain as shown in SEQ ID NO: 161; p) the heavy chain as shown in SEQ ID NO: 162 and the light chain as shown in SEQ ID NO: 163; q) the heavy chain as shown in SEQ ID NO: 164 and the light chain as shown in SEQ ID NO: 165; r) the heavy chain as shown in SEQ ID NO: 166 and the light chain as shown in SEQ ID NO: 167; s) the heavy chain as shown in SEQ ID NO: 168 and the light chain as shown in SEQ ID NO: 169; t) the heavy chain as shown in SEQ ID NO: 170 and the light chain as shown in SEQ ID NO: 171; u) the heavy chain as shown in SEQ ID NO: 172 and the light chain as shown in SEQ ID NO: 173; v) the heavy chain as shown in SEQ ID NO: 174 and the light chain as shown in SEQ ID NO: 175; w-1) the heavy chain as shown in SEQ ID NO: 176 and the light chain as shown in SEQ ID NO: 177; w-2) the heavy chain as shown in SEQ ID NO: 178 and the light chain as shown in SEQ ID NO: 179; x) the heavy chain as shown in SEQ ID NO: 176 and the light chain as shown in SEQ ID NO: 179.
In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding any antibody or the antigen-binding fragment.
The antibody or antigen-binding fragment thereof of the present invention can be used to form a bispecific or multispecific binding molecule. The antibody or antigen-binding fragment thereof of the present invention may be part of a bispecific or multispecific binding molecule, the bispecific or multispecific binding molecule comprising a second functional module (e.g., a second antibody) having a different binding specificity as compared to the antibody or antigen-binding fragment thereof of the present invention, thereby capable of binding to at least two different binding sites and/or target molecules.
Accordingly, in one aspect, the present disclosure provides a bispecific or multispecific binding molecule comprising the antibody or antigen-binding fragment thereof of the present invention.
The antibody or antigen-binding fragment thereof of the present invention can be linked to a therapeutic agent to form an immunoconjugate. Because the immunoconjugate has an ability to selectively deliver one or more therapeutic agents to a target tissue. In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) , the therapeutic agent is a cytotoxic agent.
Accordingly, in one aspect, the present disclosure provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof of the present invention.
In another aspect, the present disclosure provides a chimeric antigen receptor, which comprises an antigen-binding domain of the antibody or antigen-binding fragment thereof of the present invention.
In certain embodiments, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
In certain embodiments, the antigen-binding domain is a scFv;
In certain embodiments, the chimeric antigen receptor is expressed by an immune effector cell (for example, T cell) .
In one aspect, the present disclosure also provides a recombinant vector comprising the above-mentioned isolated nucleic acid molecule.
In another aspect, the present disclosure also provides a host cell transformed with the above-mentioned recombinant vector, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
In one aspect, the present disclosure also provides a method for producing the above-mentioned antibody or the antigen-binding fragment in a medium to produce and accumulate the antibody or the antigen-binding fragment thereof, and harvesting the antibody or the antigen-binding fragment thereof from the culture.
In one aspect, the present disclosure also provides a method for immunologically detecting or measuring HER3, wherein the method comprises detecting the HER3 by contacting with the above-mentioned antibody or the antigen-binding fragment.
In one aspect, the present disclosure also provides a method for diagnosing a disease related to a human HER3 positive cell, wherein the method comprises a detecting or measuring the HER3 or HER3 positive cell by contacting with the above-mentioned antibody or the antigen-binding fragment.
In some embodiments, the present disclosure provides a pharmaceutical composition, which comprises a therapeutically effective amount of the above-mentioned antibody or the antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluents or excipients.
In some embodiments, the present disclosure also provides a method of treatment or prevention of a disease related to overexpression of HER3, comprising a step of administering a therapeutically effective amount of the antibody or its antigen-binding fragment above, or the pharmaceutical composition above, to a subject in need of treatment or prevention of the disease.
In some embodiments, the disease related to overexpression of HER3 is cancer; preferably the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
In some embodiments, the present disclosure also provides the use of the antibody or its antigen-binding fragment above, or the pharmaceutical composition above, in the manufacture of a medicament for treating or preventing a disease related to human HER3.
In some embodiments, the disease related to human HER3 is the cancer with HER3 expression; preferably the cancer is breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
The obtained antibodies have a series of excellent characteristics:
i) the variable region sequences are different from the existing antibody; All our antibodies are fully human, which have less tendency to cause immunogenicity in the human body.
ii) the obtained antibodies have capacity of binding to human with high affinity, which is confirmed by flow cytometry and ELISA.
iii) the obtained antibodies have better internalization properties.
iv) the obtained antibodies are only marginally inhibited by the equal molar concentration of NRG1 molecules.
Figure 1. In vitro binding characterization of HER3 hybridoma clones to HER3+ (T47D) and HER3-(Jurkat E6.1) cell lines using flow cytometry analysis.
Figure 2. Cell internalization activity of selected hybridoma clones were characterized using indirect killing assay.
Figure 3. Internalization assay of anti-HER3 recombinant antibodies in HER3+ CHO-K1-huHER3 cells.
Figure 4. NRG1 effect on the binding to SKBr3 Cell.
The present invention is based on the development of an antibody which can specifically bind to HER3. The titles used in the present section are for convenience of specification only, and do not limit the present invention.
Unless otherwise defined herein, the scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Further, unless the context specifically requires, the singular includes the plural, and the plural includes the singular.
DEFINITIONS
Before the present invention is detailed below, it is to be understood that the present invention is not limited to the particular methodologies, protocols and reagents described herein, as those may vary. It is also to be understood that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
For interpretation of the specification, the following definitions will be applied and wherever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
The term "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., HER3) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) , and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883) . Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
The term "human antibody" , as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term "human antibody" , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "recombinant human antibody" , as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an
animal (e.g., a mouse) that is transgenic or trans chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below) , (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (LCDR1, LCDR2, LCDR3 or L1, L2, L3) , as well as CDR1, CDR2 and CDR3 of heavy chain variable domain (HCDR1, HCDR2, HCDR3 or H1, H2, H3) .
Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883) , Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) , Contact (University College London) , International ImMunoGeneTics database (IMGT) (imgt. cines. fr/on the World Wide Web) , and North CDR definition based on the affinity propagation clustering using a large number of crystal structures. Those skilled in the art can easily identify the CDRs defined by each numbering system.
A useful comparison of CDR numbering is as below:
Note 1: some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note2: any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note3: the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.
The term "nucleic acid molecule" as used herein refers to a DNA molecule and a RNA molecule. The nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA. A nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.
Those skilled in the art know that the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. In one embodiment, the vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segment can be ligated. In another embodiment, the vector is a viral vector, wherein an additional DNA segment can be ligated into viral genome. The vectors disclosed herein are capable of self-replicating in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial replication origin and a episomal mammalian vector) or can be integrated into the genome of a host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector) .
The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thus being constructed. The expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phage or virus vectors and the like.
The term "transfectoma" , as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.
The sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening. In addition, the sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.
Once a relevant sequence is obtained, the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
In addition, a relevant sequence can be synthesized artificially, especially when the fragment is short in length. Usually, several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.
At present, it is possible to obtain a DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.
In general, under conditions suitable for expression of the antibody according to the present invention, the host cell obtained is cultured. Then, the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
The monoclonal antibody obtained can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) . The binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980) ) .
The antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by
various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.
The term "variant" of a polypeptide such as for example, an antigen-binding fragment, a protein or an antibody is a polypeptide in which one or more amino acid residues are inserted, deleted, added and/or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide. In addition, a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintaining biological activity of the antibody disclosed herein, for example, binding to HER3 and specificity. The variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%identical to the sequence of the antibody or its antigen-binding fragment disclosed herein. The percent identity (%) or homology may be calculated with reference to the following description.
In one embodiment, the percent homology or identity may be calculated as 100 x [ (identical position) /min (TGA, TGB) ] , and in the formula, TGA, TGB are the sum of the number of residues of sequences A and B compared and the internal gap position (Russell et al., J. Mol Biol., 244: 332-350 (1994) .
In the present invention, the antibody of the present invention also includes a conservative variant thereof, which means that, compared to the amino acid sequence of the antibody of the present invention, there are up to 10, preferably up to 8 and more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
Table A
The term "KD" (M) , as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) . KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example,
the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
The term "affinity" is the strength of interaction between an antibody or its antigen-binding fragment and an antigen, and it is determined by properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment. The methods for determining the affinity are known in the art, and the followings can be referred.
The antibody or its antigen-binding fragment is called "specifically binding" to its target such as an antigen, when a dissociation constant (KD) is < l06 M. The antibody specifically binds to a target with "high affinity" , when KD is < l09 M.
The term "Pharmaceutical composition" , as used herein, is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological /pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.
"Administration" and "treatment" , when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell. “Administration” and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell. "Treatment" , when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
In addition, the present disclosure includes a medicament for treating a disease associated with HER3, comprising an antibody or an antigen-binding fragment thereof of the present disclosure as an active ingredient.
There is no limitation on the diseases related to HER3, as long as it is a disease associated with HER3, for example, the therapeutic response induced by the molecules disclosed in the present disclosure can be reduced by binding human HER3. Therefore, the molecules of the present disclosure are very useful for those who suffer a tumor, cancer or infectious disease when in preparations and formulations suitable for therapeutic applications.
In addition, the present disclosure relates to a method for immunologically detecting or measuring HER3, a reagent for immunologically detecting or measuring HER3, a method for immunologically detecting or measuring cells expressing HER3, and a diagnostic reagent for diagnosis of disease related to HER3 positive cells, comprising the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human HER3, as an active ingredient.
In the present disclosure, the method for detecting or determining the amount of HER3 may be any known method. For example, it includes immunodetection or assay.
The immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody. Examples of immunodetection or assay include a radioactive substance labeled immunological antibody method (RIA) , an enzyme immunoassay (EIA or ELISA) , a fluorescent immunoassay (FIA) , a luminescent immunoassay, a western blotting method, physicochemical methods, etc.
The above-mentioned diseases related to HER3 positive cells can be diagnosed by detecting or measuring cells expressing HER3 by using the antibodies or antibody fragments thereof of the present invention.
In order to detect cells expressing the polypeptide, a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemically staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Bio system) can be used.
EXAMPLE
The invention is further illustrated by the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods without detailed conditions in the following examples are generally in accordance with the conditions described in the conventional conditions such as Sambrook. J et al. "Guide to Molecular Cloning Laboratory" (translated by Huang Peitang et al., Beijing: Science Press, 2002) , or in accordance with the conditions recommended by the manufacturer (for example, product manuals) . Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are commercially available unless otherwise specified.
The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30℃.
EXAMPLE 1: Immunization and screening of antibody
EXAMPLE 1-1: Preparation of immunogen
A combination of recombinant protein antigen huHER3-His and rhesus HER3-His were used to immunize humanized mice (Alloy GK MIX strain) . Briefly, 10 μg is the typical amount of a protein antigen used in subcutaneous or intraperitoneal injections in experiments performed with ATX-Gx mice. The antigens were mixed with proprietary adjuvants for immunizations. For the subcutaneous injections, 2 sites were used (50-100 μl per site) ; and for the intraperitoneal injection we typically use 200 μl.
EXAMPLE 1-2: Immunization
Immunization protocol:
Anti-HER3 antibodies were obtained by two-armed immunization schemes using genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions by RIMMS (Repetitive Immunization at Multiple Sites) protocol. One group of mice were immunized and boosted with recombinant protein antigen huHER3-His (AcroBio, catalog number ER3-H5223) , and the other group of mice were immunized with the same huHER3-His but boosted with rhesus HER3-His (Sino Bio, catalog number 90043-K08H) . The antibody immune response was monitored by an HER3-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for HER3 specificity
EXAMPLE 1-3: Spleen cell fusion
The spleen lymphocytes and myeloma cells Sp2/0 (CRL-158) were fused to obtain hybridoma cells by electrofusion or PEG fusion. PEG fusion was performed using ClonacellTM HY technology (STEMCELL technologies) , following manufacturer’s instructions. The primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10: 1 for PEG fusion.
EXAMPLE 1-4: Screening of hybridoma clones specifically binding to HER3 protein by ELISA
ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1μg/ml of human HER3 (Acro Bio, catalog number ER3-H5223) rhesus HER3 (Sino Bio, catalog number 90043-K08H) , mouse HER3 (Acro Bio, catalog number ER3-M52H5) , rat HER3 (Sino Bio, catalog number 80111-r08H) or BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50μl HER3 hybridoma supernatant was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off and 50μl of 1: 20000 diluted secondary antibody Goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for another 1 hour. Plates were washed before the addition 50 μL of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25μL of 2N sulfuric acid. Optical density at 450nm of samples was measured by a microplate reader (PerkinElmer) . All tested clones showed selective binding to human HER3 but not to BSA, demonstrating HER3 specificity.
Table 1. Binding characterization of hybridoma clones by ELISA assay (OD450)
EXAMPLE 1-5: Screening of hybridoma clones specifically binding to HER3+ cancer cells by Flow cytometry
Hybridoma supernatants were subjected to binding tests on HER3+ cell line T-47D (ATCC, HTB-133) , and HER3-cell line Jurkat E6.1 (ATCC, TIB-152) using flow cytometry analysis. Briefly, 50 μL of cells in cell staining buffer (2×106 cells/mL) was mixed with 50 μL undiluted hybridoma supernatants. The mixture was incubated on ice for 20 min and then washed with ice cold staining buffer twice. The cells were subsequently stained with 50 μL of PE labelled secondary antibody (1: 400 dilution, BioLegend, Cat#405307) for 20 min. After washing by staining buffer and fixing by 4%PFA, cells were analyzed by flow cytometry. A purified anti-human HER3 antibody was used as positive control.
A purified mouse IgG1 antibody was used as isotype control (R&D Systems, Cat#MAB3481) . Figure 1 shows examples of selected cell binding signals measured by flow cytometry.
EXAMPLE 1-6: Screening of hybridoma clones with cell internalization activity by indirect killing assay
Cell internalization activity of anti-HER3 antibodies from hybridoma supernatants were measured by using an indirect killing assay in CHO-K1-huHER3 cells (KYinno, cat#KC-1511) . The CHO-K1-huHER3 cells were seeded in 96 well plate at 3,000 cells/well and incubated overnight. The hybridoma supernatants from each hybridoma clone were diluted with hybridoma culture medium and mixed with Fab anti-mouse IgG Fc-MMAF conjugates with cleavable linker (Moradec, AM-202AF) , then added to each well. The final concentrations of mouse IgG were roughly 10 nM, 2 nM, and 0.4 nM. The final concentration of the Fab anti-mouse IgG Fc-MMAF conjugates in each well was 20 nM. With the presence of secondary Fab-vc-MMAF, the internalized antibody/Fac-vc-MMAF conjugates complex will release the cytotoxic payload and kill the cells. After 3 days incubation, the viable cells in each well were detected by Cell Titer Glo 2.0 assay (Promega, G9243) . A purified anti-human HER3 antibody was used as positive control. A purified mouse IgG1 antibody was used as isotype control (Biolegend, Cat#400102) . Cells treated with selected hybridoma supernatants showed reduced viability, as shown in Figure 2, indicating the internalization of the antibodies.
EXAMPLE 2 Sequencing of positive hybridoma clones
The process of cloning sequences from positive hybridomas are as follows. The logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification. Amplified cDNA library from each clone were subjected to next-generation sequencing. The amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies of lead candidates were obtained. Upon the manufacturability assessment evaluation, several mutations were made in the FR region, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following tables. The amino acid residues of the CDRs in VH/VL are numbered and annotated according to the Kabat &Wu numbering system.
Table 2. CDR sequence of heavy chain variable domain for HER3 hybridoma clones
Table 3. CDR sequence of light chain variable domain for HER3 hybridoma clones
Table 4. Sequences of heavy chain and light chain variable domains for HER3 hybridoma clones
Example 3 Construction and Expression of anti-HER3 Recombinant Antibody
Example 3-1: Molecular cloning of recombinant antibodies
The cDNA sequences that encode VH and VL regions of selected clones were directly synthesized as DNA fragments with 5’ -end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into selected vectors using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) . VH region was cloned into pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) using EcoRI site and NheI site, which in-frame with constant region of hIgG1 heavy chain in the vector. VL region was cloned into pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) using AgeI site and BsiWI site, which in-frame with constant region of hIg kappa light chain in the vector.
The IgG form of antibodies were disclosed as the following heavy chain and light chain full lengths.
Table 5. Sequences of recombinant antibodies IgG constant regions
Table 6. Sequences of heavy chain and light chain full lengths for anti-HER3 recombinant antibodies
Example 3-2: Expression and purification of recombinant antibodies
The heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-Scells (ThermoFisher #A29127) using ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 1: 1. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 C, 8%CO2. Conditioned medium was collected, and antibodies were purified using HiTrap MabSelect SuRe column (Cytiva, #17549112) on AKTA Pure 25 machine (Cytiva) . Eluted antibodies were neutralized with Tris Buffer (pH 9.0) and subjected to PBS buffer exchange. Product concentration was measured by UV absorption, and quality was determined by SDS-PAGE and HPLC.
Example 4: Binding characterization of anti-HER3 recombinant antibody to HER3+ cell lines by flow cytometry
Binding of the hlgGl mAbs to the cell surface HER3 was determined by FACS analysis using T-47D cell, the cancer cell lines positive for HER3 expression. Experimental procedures refer to Example 1-5.
Table 7. KD values of anti-HER3 recombinant antibodies binding to HER3+ T-47D cells
Example 5: Characterization of anti-HER3 recombinant antibody cell internalization in HER3 expressing cells
Cell internalization activity of anti-HER3 recombinant antibodies were measured using an indirect fluorescence internalization assay in CHO-K1-huHER3 cells (KYinno, cat#KC-1511) . The CHO-K1-huHER3 cells were seeded in 96 well plate at 20,000 cells/well and incubated overnight. The recombinant antibodies were diluted with cell culture medium and mixed with Fab anti-human Fc-pHast conjugates with pH dependent fluorescent reporter pHast (Advanced Targeting Systems, PH-01) , then
added to each well. The final concentrations of anti-HER3 recombinant antibodies were 1 nM, 3 nM, and 9 nM. The final concentration of the Fab anti-human Fc-pHast conjugates in each well was 35 nM. With the presence of secondary Fab-pHast, the internalized antibody/Fac-pHast conjugates complex will show increased fluorescence in the acidic environment inside a cell. After 17 hours of incubation, the fluorescence from all the wells were measured using a plate reader. A purified anti-human HER3 antibody, Patritumab, was used as positive control. A purified human IgG1 antibody was used as negative control. As shown in Figure 3, strong internalization signals in CHO-K1-huHER3 cells were observed for anti-HER3 antibodies.
Example 6: Epitope characterization of anti-HER3 recombinant antibodies
Example 6-1: ELISA binding to human HER3 subdomain proteins
ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . 96 well ELISA plates were coated with 1μg/well of human HER3 protein (HER3-His) or human HER3 subdomains 1&2 (HER3 D1-2, a.a. 20-329) , HER3 subdomain 2 (HER3 D2, a.a. 185-329) , HER3 subdomain 3&4 (HER3 D3-4, a.a. 330-643) and HER3 subdomain 4 (HER3 D4, a.a. 496-643) overnight at 4℃. Excess unbound proteins were washed off by washing the plates three times with the wash buffer by a plate washer before blocking for 1 hour at room temperature. 100 μl of 10 μg/mL HER3 recombinant antibodies was added in duplicate wells and incubated for 1 hour at room temperature. Excess unbound antibodies were washed off by a plate washer and 100 μl of 1: 5000 diluted secondary antibody Goat anti-human IgG Fc-HRP (ab6858) was added to each well to incubate for 30 min at room temperature. Plates were washed again by a plate washer before the addition of 100μL of chemiluminescence agents (TMB) according to manufacturer's protocol. The reactions were stopped using 100 μL of 2 N sulfuric acid. Optical density at 450nm of samples were measured by microplate reader (PerkinElmer) . All recombinant antibodies tested bound to full length human HER3 protein. Recombinant antibody 20B5-1 showed binding to human HER3 subdomain 3-4 and subdomain 4, indicating binding to subdomain 4. All the remaining recombinant antibodies (see table 8 below) showed binding to human HER3 subdomain 1-2 and subdomain 1, indicating binding to subdomain 1.
Table 8. Binding characterization of HER3 recombinant antibodies to human HER3 full length or subdomain proteins by ELISA (OD450)
Example 6-2: Octet binning
The leading three clones (18E11-1, 20E1-3, 23F6-1) were subjected to epitope binning and compared to reference anti-HER3 antibodies patritumab. Antibody epitope binning was performed using an Octet Red384 system equipped with Ni-NTA biosensors from Pall Life Sciences (Menlo Park, CA) . The experiment was performed as an in-tandem binning assay. The assay is comprised of a five-step binding cycle: 1) A buffer baseline was established for 30 seconds, 2) 5 nM HER3 antigen (HER3-His) was coupled to Ni-NTA octet sensors using a standard 1x assay buffer (PBS + 0.02%Tween20, 0.1%BSA, 0.05%sodium azide) diluted from a 10x kinetic buffer stock (ForteBio) for 5 minutes, 3) 25 nM of each antibody (saturating mAb) was loaded to saturate the immobilized antigen for 10 minutes, 4) 25 nM of each antibody (competing mAb) was bound for 5 minutes, and 5) capture sensors were regenerated for 30 seconds. As shown in Table 9 and Table 10, all the four anti-HER3 antibodies can be divided into 2 different epitope bins. 18E11-1, 20E1-3 and 23F6-1 are in the same bin, while patritumab is in a different bin. After binding of 18E11-1, 20E1-3, or 23F6-1, patritumab can still bind to HER3 with a great association curve, indicating different epitopes.
Table 9. Epitope binning for recombinant antibodies binding to human HER3 protein determined by Octet Red384 system.
Signal shift unit: nm
Table 10. Epitope bins of anti-HER3 recombinant antibodies to human HER3
Example 7: NRG1 effect on the Binding to SKBr3 Cell
NRG1 has been shown to have high affinity ( [Kd] <100 pM) for HER3 as a natural ligand (Am J Respir Cell Mol Biol. 2000 Apr; 22 (4) : 432-40. ) , it is hard for an antibody to compete with. However, antibodies could bind the antigen on different epitope which could be different than where NRG1 could bind, we then tested our antibodies on SKBr3 cell
In vitro cell binding of HER3 recombinant antibodies to HER3+ cell line by flow cytometry were inhibited in the presence of NRG1 in SKBr3 (HER3+, EGFR+ and HER2+) cells. However, some HER3 recombinant antibodies (18E11-1, 20E1-3 and 23F6-1) were not affected by NRG1 and still showed binding to HER3+ SKBr3 (HER3+, EGFR+ and HER2+) cells in the presence of NRG1, HER3 recombinant antibody 20B5-1 is only marginally inhibited by the equal molar concentration of NRG1 molecules, whereas the binding of patritumab is significantly affected.
Claims (22)
- An anti-HER3 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein:the heavy chain variable region sequence comprises:HCDR1 as shown in SEQ ID NO: 08,HCDR2 as shown in SEQ ID NO: 27, andHCDR3 as shown in SEQ ID NO: 46 respectively;orHCDR1 as shown in SEQ ID NO: 09,HCDR2 as shown in SEQ ID NO: 28, andHCDR3 as shown in SEQ ID NO: 47 respectively;orHCDR1 as shown in SEQ ID NO: 10,HCDR2 as shown in SEQ ID NO: 29, andHCDR3 as shown in SEQ ID NO: 48 respectively;orHCDR1 as shown in SEQ ID NO: 11,HCDR2 as shown in SEQ ID NO: 30, andHCDR3 as shown in SEQ ID NO: 49 respectively;orHCDR1 as shown in SEQ ID NO: 12,HCDR2 as shown in SEQ ID NO: 31, andHCDR3 as shown in SEQ ID NO: 50 respectively;orHCDR1 as shown in SEQ ID NO: 01,HCDR2 as shown in SEQ ID NO: 21, andHCDR3 as shown in SEQ ID NO: 51 respectively;orHCDR1 as shown in SEQ ID NO: 06,HCDR2 as shown in SEQ ID NO: 32, andHCDR3 as shown in SEQ ID NO: 52 respectively;orHCDR1 as shown in SEQ ID NO: 13,HCDR2 as shown in SEQ ID NO: 33, andHCDR3 as shown in SEQ ID NO: 53 respectively;orHCDR1 as shown in SEQ ID NO: 14,HCDR2 as shown in SEQ ID NO: 33, andHCDR3 as shown in SEQ ID NO: 54 respectively;orHCDR1 as shown in SEQ ID NO: 15,HCDR2 as shown in SEQ ID NO: 34, andHCDR3 as shown in SEQ ID NO: 55 respectively;orHCDR1 as shown in SEQ ID NO: 16,HCDR2 as shown in SEQ ID NO: 35, andHCDR3 as shown in SEQ ID NO: 56 respectively;orHCDR1 as shown in SEQ ID NO: 17,HCDR2 as shown in SEQ ID NO: 36, andHCDR3 as shown in SEQ ID NO: 57, respectively;orHCDR1 as shown in SEQ ID NO: 06,HCDR2 as shown in SEQ ID NO: 32, andHCDR3 as shown in SEQ ID NO: 58 respectively;orHCDR1 as shown in SEQ ID NO: 01,HCDR2 as shown in SEQ ID NO: 18, andHCDR3 as shown in SEQ ID NO: 37, respectively;orHCDR1 as shown in SEQ ID NO: 02,HCDR2 as shown in SEQ ID NO: 19, andHCDR3 as shown in SEQ ID NO: 38 respectively;orHCDR1 as shown in SEQ ID NO: 03,HCDR2 as shown in SEQ ID NO: 20, andHCDR3 as shown in SEQ ID NO: 39 respectively;orHCDR1 as shown in SEQ ID NO: 04,HCDR2 as shown in SEQ ID NO: 21, andHCDR3 as shown in SEQ ID NO: 40 respectively;orHCDR1 as shown in SEQ ID NO: 05,HCDR2 as shown in SEQ ID NO: 22, andHCDR3 as shown in SEQ ID NO: 41 respectively;orHCDR1 as shown in SEQ ID NO: 06,HCDR2 as shown in SEQ ID NO: 23, andHCDR3 as shown in SEQ ID NO: 42 respectively;orHCDR1 as shown in SEQ ID NO: 07,HCDR2 as shown in SEQ ID NO: 24, andHCDR3 as shown in SEQ ID NO: 43 respectively;orHCDR1 as shown in SEQ ID NO: 06,HCDR2 as shown in SEQ ID NO: 25, andHCDR3 as shown in SEQ ID NO: 44, respectively;orHCDR1 as shown in SEQ ID NO: 06,HCDR2 as shown in SEQ ID NO: 26, andHCDR3 as shown in SEQ ID NO: 45 respectively;and/orthe antibody light chain variable region comprises:LCDR1 as shown in SEQ ID NO: 66,LCDR2 as shown in SEQ ID NO: 83, andLCDR3 as shown in SEQ ID NO: 96, respectively;orLCDR1 as shown in SEQ ID NO: 67,LCDR2 as shown in SEQ ID NO: 79, andLCDR3 as shown in SEQ ID NO: 97, respectively;orLCDR1 as shown in SEQ ID NO: 68,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 98, respectively;orLCDR1 as shown in SEQ ID NO: 69,LCDR2 as shown in SEQ ID NO: 84, andLCDR3 as shown in SEQ ID NO: 99, respectively;orLCDR1 as shown in SEQ ID NO: 70,LCDR2 as shown in SEQ ID NO: 85, andLCDR3 as shown in SEQ ID NO: 100, respectively;orLCDR1 as shown in SEQ ID NO: 71,LCDR2 as shown in SEQ ID NO: 85, andLCDR3 as shown in SEQ ID NO: 100, respectively;orLCDR1 as shown in SEQ ID NO: 61,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 90, respectively;orLCDR1 as shown in SEQ ID NO: 72,LCDR2 as shown in SEQ ID NO: 86, andLCDR3 as shown in SEQ ID NO: 101, respectively;orLCDR1 as shown in SEQ ID NO: 73,LCDR2 as shown in SEQ ID NO: 82, andLCDR3 as shown in SEQ ID NO: 103, respectively;orLCDR1 as shown in SEQ ID NO: 64,LCDR2 as shown in SEQ ID NO: 82, andLCDR3 as shown in SEQ ID NO: 103, respectively;orLCDR1 as shown in SEQ ID NO: 74,LCDR2 as shown in SEQ ID NO: 79, andLCDR3 as shown in SEQ ID NO: 92, respectively;orLCDR1 as shown in SEQ ID NO: 75,LCDR2 as shown in SEQ ID NO: 82, andLCDR3 as shown in SEQ ID NO: 104, respectively;orLCDR1 as shown in SEQ ID NO: 65,LCDR2 as shown in SEQ ID NO: 82, andLCDR3 as shown in SEQ ID NO: 95, respectively;orLCDR1 as shown in SEQ ID NO: 76,LCDR2 as shown in SEQ ID NO: 85, andLCDR3 as shown in SEQ ID NO: 100, respectively;orLCDR1 as shown in SEQ ID NO: 66,LCDR2 as shown in SEQ ID NO: 83, andLCDR3 as shown in SEQ ID NO: 96, respectively;orLCDR1 as shown in SEQ ID NO: 59,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 58, respectively;orLCDR1 as shown in SEQ ID NO: 60,LCDR2 as shown in SEQ ID NO: 78, andLCDR3 as shown in SEQ ID NO: 89, respectively;orLCDR1 as shown in SEQ ID NO: 61,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 90, respectively;orLCDR1 as shown in SEQ ID NO: 59,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 90, respectively;orLCDR1 as shown in SEQ ID NO: 62,LCDR2 as shown in SEQ ID NO: 77, andLCDR3 as shown in SEQ ID NO: 91, respectively;orLCDR1 as shown in SEQ ID NO: 63,LCDR2 as shown in SEQ ID NO: 79, andLCDR3 as shown in SEQ ID NO: 92, respectively;orLCDR1 as shown in SEQ ID NO: 60,LCDR2 as shown in SEQ ID NO: 80, andLCDR3 as shown in SEQ ID NO: 93, respectively;orLCDR1 as shown in SEQ ID NO: 64,LCDR2 as shown in SEQ ID NO: 81, andLCDR3 as shown in SEQ ID NO: 94, respectively;orLCDR1 as shown in SEQ ID NO: 65,LCDR2 as shown in SEQ ID NO: 82, andLCDR3 as shown in SEQ ID NO: 95, respectively;orLCDR1 as shown in SEQ ID NO: 60,LCDR2 as shown in SEQ ID NO: 80, andLCDR3 as shown in SEQ ID NO: 89, respectively.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claim 1, wherein:a) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 08, SEQ ID NO: 27 and SEQ ID NO: 46, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively;orb) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 09, SEQ ID NO: 28 and SEQ ID NO: 47, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 79 and SEQ ID NO: 97, respectively;orc) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 29 and SEQ ID NO: 48, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 68, SEQ ID NO: 77 and SEQ ID NO: 98, respectively;ord) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 30 and SEQ ID NO: 49, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 69, SEQ ID NO: 84 and SEQ ID NO: 99, respectively;ore) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: 50, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 85 and SEQ ID NO: 100, respectively;orf) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: 50, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 71, SEQ ID NO: 85 and SEQ ID NO: 100, respectively;org) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 21 and SEQ ID NO: 51, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 77 and SEQ ID NO: 90, respectively;orh) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 52, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 72, SEQ ID NO: 86 and SEQ ID NO: 101, respectively;ori) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 52, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 87 and SEQ ID NO: 102, respectively;orj) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 53, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 82 and SEQ ID NO: 103, respectively;ork) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 33 and SEQ ID NO: 54, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 82 and SEQ ID NO: 103, respectively;orl) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 34 and SEQ ID NO: 55, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 79 and SEQ ID NO: 92, respectively;orm) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 35 and SEQ ID NO: 56, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 75, SEQ ID NO: 82 and SEQ ID NO: 104, respectively;orn) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 35 and SEQ ID NO: 56, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SEQ ID NO: 82 and SEQ ID NO: 95, respectively.oro) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 36 and SEQ ID NO: 57, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 76, SEQ ID NO: 85 and SEQ ID NO: 100, respectively.orp) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 32 and SEQ ID NO: 58, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 83 and SEQ ID NO: 96, respectively;orq) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 18 and SEQ ID NO: 37, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 77 and SEQ ID NO: 88, respectively;orr) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 19 and SEQ ID NO: 38, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ ID NO: 78 and SEQ ID NO: 89, respectively;ors) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 20 and SEQ ID NO: 39, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 77 and SEQ ID NO: 90, respectively;ort) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 21 and SEQ ID NO: 40, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 77 and SEQ ID NO: 90, respectively;oru) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 22 and SEQ ID NO: 41, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 77 and SEQ ID NO: 91, respectively;orv) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 23 and SEQ ID NO: 42, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 63, SEQ ID NO: 79 and SEQ ID NO: 92, respectively;orw) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 24 and SEQ ID NO: 43, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ ID NO: 80 and SEQ ID NO: 93, respectively;orx) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 25 and SEQ ID NO: 44, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 81 and SEQ ID NO: 94, respectively;ory) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 26 and SEQ ID NO: 45, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SEQ ID NO: 82 and SEQ ID NO: 95, respectively;orz) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 08, SEQ ID NO: 27 and SEQ ID NO: 46, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, SEQ ID NO: 80 and SEQ ID NO: 89, respectively.
- An anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody or antigen-binding fragment thereof comprising:a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, 144, 145, 147, 149, 151, 153, 155, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 and 125, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;and/ora light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 143, 146, 148, 150, 152, 154, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 and 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprising:a-1) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 128, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ora-2) the heavy chain variable region as shown in SEQ ID NO: 127, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orb) the heavy chain variable region as shown in SEQ ID NO: 129, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 130, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orc) the heavy chain variable region as shown in SEQ ID NO: 131, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 132, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ord) the heavy chain variable region as shown in SEQ ID NO: 133, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 134, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ore-1) the heavy chain variable region as shown in SEQ ID NO: 135, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 136, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ore-2) the heavy chain variable region as shown in SEQ ID NO: 137, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 138, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orf) the heavy chain variable region as shown in SEQ ID NO: 139, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 140, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;org-1) the heavy chain variable region as shown in SEQ ID NO: 141, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 142, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;org-2) the heavy chain variable region as shown in SEQ ID NO: 141, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 143, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;org-3) the heavy chain variable region as shown in SEQ ID NO: 144, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 142, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orh) the heavy chain variable region as shown in SEQ ID NO: 145, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 146, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ori) the heavy chain variable region as shown in SEQ ID NO: 147, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 148, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orj) the heavy chain variable region as shown in SEQ ID NO: 149, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 150, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ork-1) the heavy chain variable region as shown in SEQ ID NO: 151, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 152, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ork-2) the heavy chain variable region as shown in SEQ ID NO: 151, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 122, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orl) the heavy chain variable region as shown in SEQ ID NO: 153, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 154, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orm) the heavy chain variable region as shown in SEQ ID NO: 155, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orn) the heavy chain variable region as shown in SEQ ID NO: 105, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 106, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;oro) the heavy chain variable region as shown in SEQ ID NO: 107, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 108, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orp) the heavy chain variable region as shown in SEQ ID NO: 109, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 110, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orq) the heavy chain variable region as shown in SEQ ID NO: 111, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 112, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orr) the heavy chain variable region as shown in SEQ ID NO: 113, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 114, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ors) the heavy chain variable region as shown in SEQ ID NO: 115, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 116, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;ort) the heavy chain variable region as shown in SEQ ID NO: 117, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 118, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;oru) the heavy chain variable region as shown in SEQ ID NO: 119, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 120, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orv) the heavy chain variable region as shown in SEQ ID NO: 121, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 122, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orw-1) the heavy chain variable region as shown in SEQ ID NO: 123, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 124, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orw-2) the heavy chain variable region as shown in SEQ ID NO: 125, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;orx) the heavy chain variable region as shown in SEQ ID NO: 123, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 126, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprising:a-1) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 128;a-2) the heavy chain variable region as shown in SEQ ID NO: 127; and/or the light chain variable region as shown in SEQ ID NO: 126;b) the heavy chain variable region as shown in SEQ ID NO: 129; and/or the light chain variable region as shown in SEQ ID NO: 130;c) the heavy chain variable region as shown in SEQ ID NO: 131; and/or the light chain variable region as shown in SEQ ID NO: 132;d) the heavy chain variable region as shown in SEQ ID NO: 133; and/or the light chain variable region as shown in SEQ ID NO: 134;e-1) the heavy chain variable region as shown in SEQ ID NO: 135; and/or the light chain variable region as shown in SEQ ID NO: 136;e-2) the heavy chain variable region as shown in SEQ ID NO: 137; and/or the light chain variable region as shown in SEQ ID NO: 138;f) the heavy chain variable region as shown in SEQ ID NO: 139; and/or the light chain variable region as shown in SEQ ID NO: 140;g-1) the heavy chain variable region as shown in SEQ ID NO: 141; and/or the light chain variable region as shown in SEQ ID NO: 142;g-2) the heavy chain variable region as shown in SEQ ID NO: 141; and/or the light chain variable region as shown in SEQ ID NO: 143;g-3) the heavy chain variable region as shown in SEQ ID NO: 144; and/or the light chain variable region as shown in SEQ ID NO: 142;h) the heavy chain variable region as shown in SEQ ID NO: 145; and/or the light chain variable region as shown in SEQ ID NO: 146;i) the heavy chain variable region as shown in SEQ ID NO: 147; and/or the light chain variable region as shown in SEQ ID NO: 148;j) the heavy chain variable region as shown in SEQ ID NO: 149; and/or the light chain variable region as shown in SEQ ID NO: 150;k-1) the heavy chain variable region as shown in SEQ ID NO: 151; and/or the light chain variable region as shown in SEQ ID NO: 152;k-2) the heavy chain variable region as shown in SEQ ID NO: 151; and/or the light chain variable region as shown in SEQ ID NO: 122;l) the heavy chain variable region as shown in SEQ ID NO: 153; and/or the light chain variable region as shown in SEQ ID NO: 154;m) the heavy chain variable region as shown in SEQ ID NO: 155; and/or the light chain variable region as shown in SEQ ID NO: 126;n) the heavy chain variable region as shown in SEQ ID NO: 105; and/or the light chain variable region as shown in SEQ ID NO: 106;o) the heavy chain variable region as shown in SEQ ID NO: 107; and/or the light chain variable region as shown in SEQ ID NO: 108;p) the heavy chain variable region as shown in SEQ ID NO: 109; and/or the light chain variable region as shown in SEQ ID NO: 110;q) the heavy chain variable region as shown in SEQ ID NO: 111; and/or the light chain variable region as shown in SEQ ID NO: 112;r) the heavy chain variable region as shown in SEQ ID NO: 113; and/or the light chain variable region as shown in SEQ ID NO: 114;s) the heavy chain variable region as shown in SEQ ID NO: 115; and/or the light chain variable region as shown in SEQ ID NO: 116;t) the heavy chain variable region as shown in SEQ ID NO: 117; and/or the light chain variable region as shown in SEQ ID NO: 118;u) the heavy chain variable region as shown in SEQ ID NO: 119; and/or the light chain variable region as shown in SEQ ID NO: 120;v) the heavy chain variable region as shown in SEQ ID NO: 121; and/or the light chain variable region as shown in SEQ ID NO: 122;w-1) the heavy chain variable region as shown in SEQ ID NO: 123; and/or the light chain variable region as shown in SEQ ID NO: 124;w-2) the heavy chain variable region as shown in SEQ ID NO: 125; and/or the light chain variable region as shown in SEQ ID NO: 126;x) the heavy chain variable region as shown in SEQ ID NO: 123; and/or the light chain variable region as shown in SEQ ID NO: 126.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprising:a-1) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 128;a-2) the heavy chain variable region as shown in SEQ ID NO: 127 and the light chain variable region as shown in SEQ ID NO: 126;b) the heavy chain variable region as shown in SEQ ID NO: 129 and the light chain variable region as shown in SEQ ID NO: 130;c) the heavy chain variable region as shown in SEQ ID NO: 131 and the light chain variable region as shown in SEQ ID NO: 132;d) the heavy chain variable region as shown in SEQ ID NO: 133 and the light chain variable region as shown in SEQ ID NO: 134;e-1) the heavy chain variable region as shown in SEQ ID NO: 135 and the light chain variable region as shown in SEQ ID NO: 136;e-2) the heavy chain variable region as shown in SEQ ID NO: 137 and the light chain variable region as shown in SEQ ID NO: 138;f) the heavy chain variable region as shown in SEQ ID NO: 139 and the light chain variable region as shown in SEQ ID NO: 140;g-1) the heavy chain variable region as shown in SEQ ID NO: 141 and the light chain variable region as shown in SEQ ID NO: 142;g-2) the heavy chain variable region as shown in SEQ ID NO: 141 and the light chain variable region as shown in SEQ ID NO: 143;g-3) the heavy chain variable region as shown in SEQ ID NO: 144 and the light chain variable region as shown in SEQ ID NO: 142;h) the heavy chain variable region as shown in SEQ ID NO: 145 and the light chain variable region as shown in SEQ ID NO: 146;i) the heavy chain variable region as shown in SEQ ID NO: 147 and the light chain variable region as shown in SEQ ID NO: 148;j) the heavy chain variable region as shown in SEQ ID NO: 149 and the light chain variable region as shown in SEQ ID NO: 150;k-1) the heavy chain variable region as shown in SEQ ID NO: 151 and the light chain variable region as shown in SEQ ID NO: 152;k-2) the heavy chain variable region as shown in SEQ ID NO: 151 and the light chain variable region as shown in SEQ ID NO: 122;l) the heavy chain variable region as shown in SEQ ID NO: 153 and the light chain variable region as shown in SEQ ID NO: 154;m) the heavy chain variable region as shown in SEQ ID NO: 155 and the light chain variable region as shown in SEQ ID NO: 126;n) the heavy chain variable region as shown in SEQ ID NO: 105 and the light chain variable region as shown in SEQ ID NO: 106;o) the heavy chain variable region as shown in SEQ ID NO: 107 and the light chain variable region as shown in SEQ ID NO: 108;p) the heavy chain variable region as shown in SEQ ID NO: 109 and the light chain variable region as shown in SEQ ID NO: 110;q) the heavy chain variable region as shown in SEQ ID NO: 111 and the light chain variable region as shown in SEQ ID NO: 112;r) the heavy chain variable region as shown in SEQ ID NO: 113 and the light chain variable region as shown in SEQ ID NO: 114;s) the heavy chain variable region as shown in SEQ ID NO: 115 and the light chain variable region as shown in SEQ ID NO: 116;t) the heavy chain variable region as shown in SEQ ID NO: 117 and the light chain variable region as shown in SEQ ID NO: 118;u) the heavy chain variable region as shown in SEQ ID NO: 119 and the light chain variable region as shown in SEQ ID NO: 120;v) the heavy chain variable region as shown in SEQ ID NO: 121 and the light chain variable region as shown in SEQ ID NO: 122;w-1) the heavy chain variable region as shown in SEQ ID NO: 123 and the light chain variable region as shown in SEQ ID NO: 124;w-2) the heavy chain variable region as shown in SEQ ID NO: 125 and the light chain variable region as shown in SEQ ID NO: 126;x) the heavy chain variable region as shown in SEQ ID NO: 123 and the light chain variable region as shown in SEQ ID NO: 126.
- An anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant regions is selected from κ and λ chain constant regions of human antibody and conservative variants thereof;more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 156 and a human light chain constant region of SEQ ID NO: 157.
- An anti-HER3 antibody or antigen-binding fragment thereof according to claims 1-8, wherein the antibody or antigen-binding fragment thereof comprising:a-1) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 181;a-2) the heavy chain as shown in SEQ ID NO: 180 and the light chain as shown in SEQ ID NO: 179;b) the heavy chain as shown in SEQ ID NO: 182 and the light chain as shown in SEQ ID NO: 183;c) the heavy chain as shown in SEQ ID NO: 184 and the light chain as shown in SEQ ID NO: 185;g) the heavy chain as shown in SEQ ID NO: 186 and the light chain as shown in SEQ ID NO: 187;e-1) the heavy chain as shown in SEQ ID NO: 188 and the light chain as shown in SEQ ID NO: 189;e-2) the heavy chain as shown in SEQ ID NO: 190 and the light chain as shown in SEQ ID NO: 191;f) the heavy chain as shown in SEQ ID NO: 192 and the light chain as shown in SEQ ID NO: 193;g-1) the heavy chain as shown in SEQ ID NO: 194 and the light chain as shown in SEQ ID NO: 195;g-2) the heavy chain as shown in SEQ ID NO: 194 and the light chain as shown in SEQ ID NO: 196;g-3) the heavy chain as shown in SEQ ID NO: 197 and the light chain as shown in SEQ ID NO: 195;h) the heavy chain as shown in SEQ ID NO: 198 and the light chain as shown in SEQ ID NO: 199;i) the heavy chain as shown in SEQ ID NO: 200 and the light chain as shown in SEQ ID NO: 201;j) the heavy chain as shown in SEQ ID NO: 202 and the light chain as shown in SEQ ID NO: 203;k-1) the heavy chain as shown in SEQ ID NO: 204 and the light chain as shown in SEQ ID NO: 205;k-2) the heavy chain as shown in SEQ ID NO: 204 and the light chain as shown in SEQ ID NO: 175;l) the heavy chain as shown in SEQ ID NO: 206 and the light chain as shown in SEQ ID NO: 207;m) the heavy chain as shown in SEQ ID NO: 208 and the light chain as shown in SEQ ID NO: 179;n) the heavy chain as shown in SEQ ID NO: 158 and the light chain as shown in SEQ ID NO: 159;o) the heavy chain as shown in SEQ ID NO: 160 and the light chain as shown in SEQ ID NO: 161;p) the heavy chain as shown in SEQ ID NO: 162 and the light chain as shown in SEQ ID NO: 163;q) the heavy chain as shown in SEQ ID NO: 164 and the light chain as shown in SEQ ID NO: 165;r) the heavy chain as shown in SEQ ID NO: 166 and the light chain as shown in SEQ ID NO: 167;s) the heavy chain as shown in SEQ ID NO: 168 and the light chain as shown in SEQ ID NO: 169;t) the heavy chain as shown in SEQ ID NO: 170 and the light chain as shown in SEQ ID NO: 171;u) the heavy chain as shown in SEQ ID NO: 172 and the light chain as shown in SEQ ID NO: 173;v) the heavy chain as shown in SEQ ID NO: 174 and the light chain as shown in SEQ ID NO: 175;w-1) the heavy chain as shown in SEQ ID NO: 176 and the light chain as shown in SEQ ID NO: 177;w-2) the heavy chain as shown in SEQ ID NO: 178 and the light chain as shown in SEQ ID NO: 179;x) the heavy chain as shown in SEQ ID NO: 176 and the light chain as shown in SEQ ID NO: 179.
- An anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.
- A bispecific or multispecific binding molecule, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-10.
- An immunoconjugate, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-10.
- A chimeric antigen receptor, which comprises an antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
- An isolated nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof according to any one of claims 1-10.
- A recombinant vector comprising the isolated nucleic acid molecule according to claim 14.
- A host cell comprising the recombinant vector according to claim 15, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
- A method for producing the antibody or the antigen-binding fragment thereof according to any one of claims 1-10, wherein the method comprises culturing the host cell according to claim 16 in a medium to produce and accumulate the antibody or the antigen-binding fragment thereof according to any one of claims 1-10, and harvesting the antibody or the antigen-binding fragment thereof from the culture.
- A method for immunologically detecting or measuring HER3, wherein the method comprises detecting the HER3 by contacting with the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1-10.
- A method for diagnosing a disease related to a human HER3 positive cell, wherein the method comprises a detecting or measuring the HER3 or HER3 positive cell by contacting with the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1-10.
- A pharmaceutical composition, which comprises a therapeutically effective amount of the anti-HER3 antibody or the antigen-binding fragment thereof according to any one of claims 1-10, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
- Use of the antibody or its antigen-binding fragment according to any one of claims 1-10, or the pharmaceutical composition of claim 20, in the manufacture of a medicament for treating or preventing a disease related to human HER3.
- The use according to claim 21, characterized in that it is for the manufacture of a medicament for treating or preventing a cancer with HER3 expression; preferably the cancer is breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
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Citations (6)
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WO2011044311A2 (en) * | 2009-10-09 | 2011-04-14 | Merck Sharp & Dohme Corp. | Generation, characterization and uses thereof of anti-her 3 antibodies |
WO2012156532A1 (en) * | 2011-05-19 | 2012-11-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Anti-human-her3 antibodies and uses thereof |
WO2014159915A1 (en) * | 2013-03-14 | 2014-10-02 | The Board Of Regents Of The University Of Texas System | Her3 specific monoclonal antibodies for diagnostic and therapeutic use |
US20150210766A1 (en) * | 2014-01-29 | 2015-07-30 | Samsung Electronics Co., Ltd. | Anti-her3 scfv fragment and bispecific anti-c-met/anti-her3 antibodies comprising the same |
WO2016040622A1 (en) * | 2014-09-11 | 2016-03-17 | Bulldog Pharmaceuticals, Inc. | Uses of anti-her3 antibodies for treating cancer |
WO2019241893A2 (en) * | 2018-06-22 | 2019-12-26 | Crd Pharmaceuticals Inc | Anti-her3 antibody and uses thereof |
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WO2011044311A2 (en) * | 2009-10-09 | 2011-04-14 | Merck Sharp & Dohme Corp. | Generation, characterization and uses thereof of anti-her 3 antibodies |
WO2012156532A1 (en) * | 2011-05-19 | 2012-11-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Anti-human-her3 antibodies and uses thereof |
WO2014159915A1 (en) * | 2013-03-14 | 2014-10-02 | The Board Of Regents Of The University Of Texas System | Her3 specific monoclonal antibodies for diagnostic and therapeutic use |
US20150210766A1 (en) * | 2014-01-29 | 2015-07-30 | Samsung Electronics Co., Ltd. | Anti-her3 scfv fragment and bispecific anti-c-met/anti-her3 antibodies comprising the same |
WO2016040622A1 (en) * | 2014-09-11 | 2016-03-17 | Bulldog Pharmaceuticals, Inc. | Uses of anti-her3 antibodies for treating cancer |
WO2019241893A2 (en) * | 2018-06-22 | 2019-12-26 | Crd Pharmaceuticals Inc | Anti-her3 antibody and uses thereof |
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