WO2024088388A1 - Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques - Google Patents

Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques Download PDF

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WO2024088388A1
WO2024088388A1 PCT/CN2023/127136 CN2023127136W WO2024088388A1 WO 2024088388 A1 WO2024088388 A1 WO 2024088388A1 CN 2023127136 W CN2023127136 W CN 2023127136W WO 2024088388 A1 WO2024088388 A1 WO 2024088388A1
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seq
variable region
chain variable
heavy chain
light chain
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PCT/CN2023/127136
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Tsungyi Lin
Peggy Huang
Larry Lo
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Hansoh Bio Llc
Shanghai Hansoh Biomedical Co., Ltd.
Changzhou Hansoh Pharmaceutical Co., Ltd.
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Publication of WO2024088388A1 publication Critical patent/WO2024088388A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a novel HER3 antibody or functional fragments thereof, comprising engineered heavy chains and light chains.
  • the present invention further relates to conjugates of the improved HER3 antibody with small molecule drugs.
  • the present invention further relates to use of the antibody and the conjugates in the manufacture of a drugs for treating cancers.
  • 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.
  • Targeted therapies against HER family members such as EGFR and HER2 are widely and commonly used in cancer therapy by using 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 expression is linked to metastatic events, but also it is implicated in the development of resistance to treatment with other EGFR-targeted therapies (e.g., cetuximab or kinase inhibitors such as Lapatinib) , IGFR-targeted therapies or chemotherapeutic agents.
  • EGFR-targeted therapies e.g., cetuximab or kinase inhibitors such as Lapatinib
  • IGFR-targeted therapies chemotherapeutic agents.
  • 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.
  • ADC antibody-drug conjugates
  • the technical problem to be solved by the present invention is to develop the advanced anti-HER3 ADCs which show very strong antitumor effects in cancers.
  • the antitumor effects may be contributed by improved binding affinities of the antibodies, increased internalizations for robust endocytosis, higher DARs and some potential effects of the ADCs which are free from NRG1 inhibition.
  • the present invention encompasses the following aspects:
  • the present disclosure provides an antibody-drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvate thereof, Ab- (L 2 -L 1 -D) y (A)
  • D is a cytotoxic drug
  • L 1 and L 2 are linking units
  • y is a number of 1 to 20, preferably 2 to10, more preferably 2 to 8, more preferably 2 to 6 or 4 to 8, most preferably 2, 4, 6, 7, 8;
  • Ab is 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,
  • the heavy chain variable region of Ab in general formula (A) 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
  • the light chain variable region of Ab in general formula (A) 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:
  • the Ab in general formula (A) 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
  • 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 Ab in general formula (A) is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
  • the Ab in general formula (A) comprises: a heavy chain variable region (s) of the following sequences: 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.
  • the Ab in general formula (A) comprises: a light chain variable region (s) of the following sequences: 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 Ab in general formula (A) comprises: 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
  • the Ab in general formula (A) comprises: 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
  • the Ab in general formula (A) 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 Ab in general formula (A) comprises: 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
  • the Ab in general formula (A) 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 cytotoxic drug is selected from the group consisting of toxin, chemotherapeutic, antibiotic, radioisotope and nucleolytic enzyme.
  • the cytotoxic drug is selected from the group consisting of tubulin inhibitors or topoisomerase inhibitors; preferably auristatin analogues or camptothecin derivatives; more preferably SN-38, MMAE, MMAF or Exatecan.
  • the antibody-drug conjugate is as shown in general formula (B) :
  • L 1 and L 2 are linking units
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8;
  • Ab is the anti-HER3 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (C) :
  • L 2 are linking units
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8;
  • R 1 is selected from hydrogen, C 1-6 haloalkyl or C 3-8 cycloalkyl
  • R 2 is selected from hydrogen, C 1-6 haloalkyl or C 3-8 cycloalkyl
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-8 cycloalkyl.
  • Ab is the anti-HER3 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (C) :
  • L 2 are linking units
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8;
  • R 1 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl.
  • Ab is the anti-HER3 antibody or antigen-binding fragment above-mentioned.
  • the linking unit L 2 is as shown in general formula (I) :
  • s 1 and s 2 are each independently an integer selected from 0-8, Preferably, s 1 and s 2 are independently selected from 1, 2, 3, 4, 5 or 6;
  • s 1 is an integer from 1-8, s 2 is 0,
  • s 1 is selected from 4, 5, 6, 7 or 8, and s 2 is 0;
  • s 2 is selected from an integer from 2 to 8, s 1 is 2, Preferably, s 2 is selected from 2, 3, 4, 5 or 6, and s 1 is 2.
  • the antibody-drug conjugate is as the following structure:
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8;
  • Ab is the anti-HER3 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is selected from the group consisting of the following compounds:
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8.
  • the present disclosure further provides a method for preparing the atibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, comprising the following step of:
  • Ab is coupled with a compound after reduction to obtain the antibody-drug conjugate; wherein: Ab is the anti-HER3 antibody or antigen-binding fragment above-mentioned;
  • R 1 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl
  • L 2 is the linker above-mentioned
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6 or 4 to 8, further preferably a number of 6 to 8, and most preferably 4, 6, 7, 8.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the ligand-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to the present disclosure, and one or more pharmaceutically acceptable excipient (s) , diluent (s) or carrier (s) .
  • the present disclosure also provides a method of treatment or prevention of a disease related to HER3, comprising a step of administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate, or the pharmaceutical composition according to the present disclosure to a subject in need of treatment or prevention of the disease.
  • the disease is a cancer with HER3 expression
  • 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 present disclosure also provides the use of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate and the pharmaceutical composition in the manufacture of a medicament for treating or preventing a disease related to HER3.
  • the present disclosure also provides the use of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate and the pharmaceutical composition in 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.
  • the cancer is breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia
  • the obtained antibodies or ADCs 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 for constructing ADCs
  • the payload for this ADC has better IC 50 compared to patritumab-DXd
  • the cytotoxicity of this ADC may not be hindered when HER3 is heterodimerized with HER2 under NRG1 presence.
  • 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.
  • Figure 4 In vitro cytotoxicity activity of ADCs against different tumor cell lines (A and B, HCC1569 cells; C, MX-1 cells) , as determined by CellTiter-Glo luminescent viability assay.
  • the present invention is based on the development of an antibody which can specifically bind to HER3.
  • Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, an exatecan or exatecan derivative.
  • 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.
  • ligand is a macromolecular compound able to recognize and bind to the target cell-associated antigens or receptors.
  • the role of the ligand is to deliver the drug to the target cell population bound to the ligand.
  • the ligand includes, but is not limited to, proteinaceous hormones, lectins, growth factors, antibodies and other molecules capable of binding to cells.
  • the ligand is expressed as Ab.
  • a connecting bond can be formed between a hetero atom in the ligand and the connecting unit.
  • linking unit means the part which links the antibody with the drug in the antibody-drug conjugate (i.e. ADC) , which could be cleavable or uncleavable.
  • the cleavable linker i.e., breakable linker or biodegradable linker
  • the linking unit or linker of the present invention has very good stability and greatly decreases the release of the drug during the process of delivering to the target (e.g., in blood) , thereby reducing the side effect and toxicity.
  • the linking unit or linker of the present invention is selected from cleavable linker, such as the linker based on disulfide (which is selectively broken in the tumor cells at a higher thiol concentration) , peptide linker (which is cleaved by the enzyme in the tumor cells) , and hydrazone linker.
  • cytotoxic drug means a chemical molecule capable of strongly destructing normal growth in tumor cells.
  • cytotoxic drugs can kill tumor cells in high enough concentrations, but due to the lack of specificity, when killing tumor cells, it also leads to apoptosis in normal cells, leading to serious side effects.
  • the cytotoxic drug is represented as D, and non-limiting examples include tubulin inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, and DNA synthesis inhibitors, preferably topoisomerase inhibitors.
  • igand-cytotoxicity drug conjugate means that a ligand is linked to a biologically active drug through a linking unit.
  • the “ligand-cytotoxicity drug conjugate” is preferably an antibody-drug conjugate (ADC) , which means that a monoclonal antibody or antibody fragment is linked to a cytotoxic drug with biological activity through a linking unit.
  • ADC antibody-drug conjugate
  • DAR Drug to Antibody Ratio
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 4, 2 to 6, 4 to 6, 4 to 8, 6 to 8, further preferably a number of 4 to 8 or 6 to 8, and most preferably 4, 5, 6, 7 or 8.
  • the average number of drugs in each ADC molecule after the coupling reaction can be identified by conventional methods, such as UV/visible spectroscopy, mass spectrometry, ELISA test, and HPLC characteristic identification.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group comprising 1 to 20 carbon atoms, preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 10 carbon atoms, and most preferably an alkyl having 1 to 6 carbon atoms (having 1, 2, 3, 4, 5 or 6 carbon atoms) .
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 1, 2-trimethylpropyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-di
  • the alkyl group is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 1, 2-trimethylpropyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2, 3-dimethylbutyl and the like.
  • the alkyl can be substituted or unsubstituted. When substituted, the substituent group (s) can be substituted at any available connection point.
  • the substituent group (s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo.
  • haloalkyl refers to an alkyl group substituted by one or more halogen (s) , wherein the alkyl is as defined above.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (having 3, 4, 5, 6, 7 or 8 carbon atoms) .
  • monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like.
  • Polycyclic cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or bridged ring.
  • 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, an antigen-binding fragment or an antibody-drug conjugate 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.
  • 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.
  • 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.
  • the antibodies of the present invention have cell affinity activity and endocytosis activity, making them suitable for coupling with drugs to form antibody-drug conjugates for treating HER3 mediated diseases.
  • the monoclonal antibodies are purified for drug conjugation.
  • Further antibodies for conjugation may include any antibodies described herein (see Example 3) .
  • ADCs antibody drug conjugates
  • Compound D was prepared by a method disclosed in PCT Patent Application. (See WO2022161385, filed Jan 26, 2022) .
  • Deruxtecan MedChemExpress, Cat#HY-114233 are the positive control.
  • HR9106 was prepared by a method disclosed in PCT Patent Application. (See WO2020063673, filed Sept 25, 2019) .
  • Anti-HER3 antibody (5 mg/mL in PBS, pH 7.4) was treated with sufficient molar equivalence of (tris (2-carboxylethyl) phosphine (TCEP, 10 mM) at 37 °C for 1 hour.
  • Antibody B12 is the negative control
  • Patritumab and MOTA are the positive control.
  • Sufficient molar equivalence of drug linker for example, Deruxtecan or compound D, in DMSO, was added to the reduced antibody in PBS. After an hour incubation, the reaction mixture was then purified using size-exclusion chromatography (SEC) to separate ADC and free, unconjugated drug linker.
  • SEC size-exclusion chromatography
  • the drug-to-antibody ratio (DAR) of ADC was determined using a TOF LC/MS system (Agilent) and the average DAR values were summarized in Table 10.
  • the average DAR values of anti-HER3 antibodies conjugated with compound D were approximately 6.0 or 7.0.
  • the average DAR values of anti-HER3 antibodies conjugated with Deruxtecan were approximately 8.0.
  • the synthesis steps of anti-HER3 antibodies conjugated with compound D with other DAR values can refer to the above.
  • Inhibitory effect of the ADCs on tumor cells growth were examined using in vitro cell killing assay. Different tumor cells were collected in log phase growth and distributed into 96-well plates at 1000-1500 cells/well. After overnight incubation, the ADCs were added to each well. The final concentrations of the ADCs in the wells were ranging from 0.01 nM to 666.7 nM. After 4 to 7 days of incubation, the cell viability in each well was determined by Cell Titer Glo 2.0 Assay (Promega) . Curves and IC 50 values were generated in GraphPad Prism using a sigmoidal dose-response non-linear regression fit. The data from these experiments are summarized in Table 11A and 11B.
  • Figure 4A-C showed representative killing curves for all ADCs tested against the indicated cell lines. All the tested ADCs showed good in vitro toxicity on tumor cells, with ADC-02, ADC-03, ADC-04, ADC-07, ADC-10 and ADC-11 showing the most significant toxicity better than reference ADC.
  • Example 9 HER3 ADCs Showed Cytotoxicity in Tumor Cells in the Presence of NRG1, Different from Reference ADC
  • 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 different cell lines with different HER3 heterodimer status. By using flow cytometry, we discovered that the binding signal of 18E11-1, 20B5-1, 20E1-3 and 23F6-1 are only marginally inhibited by the equal molar concentration of NRG1 molecules on SKBr3 cells, whereas the binding of patritumab is significantly affected. We further examined whether the difference of binding could affect the cytotoxicity of the HER3 ADCs, and we indeed observed that ADC-07 and ADC-10 demonstrate superior cytotoxicity when NRG1 is presented on SKBr3 and HCC1569 cells.
  • Example 10 Tumor-Inhibitory Experiment of ADCs Towards HER3 Positive Cancer Cell Nude Mouse Subcutaneous Transplantation Tumor Model
  • HCC1569 cells were engrafted subcutaneously into mice (CB17/SCID) . When the tumor volume reaches approximately 120-180 mm3, the engrafted mice were randomized into four groups (5 mice per group) . The groups were vehicle control, ADC-01, ADC-03 and ADC-04. The mice were treated with ADCs (3 mg/kg) intravenously QW.
  • Mean tumor growth inhibition (TGI) was calculated utilizing the following formula:
  • TGI ( (mean (C) -mean (C0) ) - (mean (T) -mean (T0) ) ) / (mean (C) -mean (C0) ) *100%;
  • T is current group value, C is control group value, T0 and C0 represent the tumor volume at the beginning of the test.
  • mice treated with the three ADCs showed a statistically significant difference from mice in the control group, causing regression of tumors.
  • mice treated with ADC-01, ADC-03 and ADC-04 showed a statistically significant difference from mice treated with vehicle, with TGI value at Day 28 of 40.2%, 105.2%and 105.2%respectively.
  • MX-1 cells were engrafted subcutaneously into mice (CB17/SCID) .
  • the tumor volume reaches approximately 120-180 mm3
  • the engrafted mice were randomized into four groups (5 mice per group) .
  • the groups were vehicle control, ADC-01, ADC-03 and ADC-04.
  • the mice were treated with ADCs (3 mg/kg) intravenously QW.
  • Mean tumor growth inhibition (TGI) was calculated utilizing the following formula:
  • TGI ( (mean (C) -mean (C0) ) - (mean (T) -mean (T0) ) ) / (mean (C) -mean (C0) ) *100%;
  • T is current group value, C is control group value, T0 and C0 represent the tumor volume at the beginning of the test.
  • mice treated with ADC-04 showed a statistically significant difference from mice treated with vehicle, with TGI value at Day 28 of 5.0%, 48.7%and 88.4%respectively.
  • a follow-up tumor inhibitory study of ADC-01, ADC-02 and ADC-03 and ADC-04 was carried out in the same MX-1 xenograft mouse model.
  • the MX-1 engrafted mice were randomized into eight groups (5 mice per group) .
  • the groups were vehicle control, 3 mg/kg ADC-01, 6 mg/kg ADC-02, 6 mg/kg ADC-03, 6 mg/kg ADC-04, 2 mg/kg ADC-02, 2 mg/kg ADC-03 and 2 mg/kg ADC-04.
  • the mice were treated with ADCs intravenously QW.
  • mice treated with ADC-02, ADC-04 at 2 and 6 mg/kg showed a statistically significant difference from mice treated with ADC-01 at 3 mg/kg, with TGI value of 111.9%, 91.0%, 113.2%and 95.2%respectively, whereas tumors in mice treated with ADC-01 at 3 mg/kg exhibited a TGI of 58.8%.
  • ADC-02, ADC-04, ADC-06, ADC-13 and ADC-14 was carried out in the same MX-1 xenograft mouse model.
  • the MX-1 engrafted mice were randomized into six groups (5 mice per group) .
  • the groups were vehicle control, ADC-02, ADC-04, ADC-06, ADC-13 and ADC-14.
  • the mice were treated with ADCs (3 mg/kg) intravenously QW for 3 weeks.
  • mice Female Balb/C mice (6-8 weeks old) were used to evaluate the pharmacokinetics of the antibody-drug conjugates. Blank serum was collected before dosing. ADCs were administered intravenously at 3 mg/kg. After injection, at predetermined time points (1, 4, 24, 48, 72, 120, 168, 336, and 504 hours) , serum was collected from designated mice and immediately stored at -80°C until further analysis.
  • Serum concentrations of ADCs were measured by ELISA, the pharmacokinetic parameters were calculated by PK Solver 2.0. The main pharmacokinetic parameters were shown in Table 16 and 17.
  • AUCs of HER3 antibodies ADC-02 and ADC-04 in mice were similar.
  • the elimination half-lives of ADC-02 and ADC-04 are superior to that of ADC-01, which may contribute to their superior therapeutic efficacy in vivo.

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Abstract

Un anticorps présente une capacité de liaison à l'antigène humain avec une affinité élevée, et possède de meilleures propriétés d'internalisation. L'invention concerne un conjugué anticorps-médicament qui comprend l'anticorps. Les utilisations du conjugué ligand-médicament cytotoxique et des compositions pharmaceutiques comprennent la préparation de médicaments pour le traitement de cancers.
PCT/CN2023/127136 2022-10-28 2023-10-27 Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques WO2024088388A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104755500A (zh) * 2012-11-08 2015-07-01 霍夫曼-拉罗奇有限公司 结合HER3 β-发夹的HER3抗原结合蛋白
WO2015155998A1 (fr) * 2014-04-10 2015-10-15 Daiichi Sankyo Company, Limited Conjugué médicament-anticorps anti-her3
WO2019175359A1 (fr) * 2018-03-14 2019-09-19 UltraHuman Thirteen Limited Anticorps anti-erbb3 désimmunisés
CN113135995A (zh) * 2020-01-17 2021-07-20 上海生物制品研究所有限责任公司 抗her3单克隆抗体及其应用
WO2022078425A1 (fr) * 2020-10-14 2022-04-21 江苏恒瑞医药股份有限公司 Anticorps anti-her3 et conjugué anticorps anti-her3-médicament et leur utilisation médicale

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104755500A (zh) * 2012-11-08 2015-07-01 霍夫曼-拉罗奇有限公司 结合HER3 β-发夹的HER3抗原结合蛋白
WO2015155998A1 (fr) * 2014-04-10 2015-10-15 Daiichi Sankyo Company, Limited Conjugué médicament-anticorps anti-her3
WO2019175359A1 (fr) * 2018-03-14 2019-09-19 UltraHuman Thirteen Limited Anticorps anti-erbb3 désimmunisés
CN113135995A (zh) * 2020-01-17 2021-07-20 上海生物制品研究所有限责任公司 抗her3单克隆抗体及其应用
WO2022078425A1 (fr) * 2020-10-14 2022-04-21 江苏恒瑞医药股份有限公司 Anticorps anti-her3 et conjugué anticorps anti-her3-médicament et leur utilisation médicale

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