WO2025098480A1 - Anticorps bispécifique dirigé contre dr5 et ceacam5 et son utilisation - Google Patents

Anticorps bispécifique dirigé contre dr5 et ceacam5 et son utilisation Download PDF

Info

Publication number
WO2025098480A1
WO2025098480A1 PCT/CN2024/130860 CN2024130860W WO2025098480A1 WO 2025098480 A1 WO2025098480 A1 WO 2025098480A1 CN 2024130860 W CN2024130860 W CN 2024130860W WO 2025098480 A1 WO2025098480 A1 WO 2025098480A1
Authority
WO
WIPO (PCT)
Prior art keywords
ceacam5
seq
molecule comprises
recognizing
sequence shown
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/130860
Other languages
English (en)
Chinese (zh)
Inventor
何开杰
刘慧思
付凤根
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innovent Biologics Suzhou Co Ltd
Original Assignee
Innovent Biologics Suzhou Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovent Biologics Suzhou Co Ltd filed Critical Innovent Biologics Suzhou Co Ltd
Publication of WO2025098480A1 publication Critical patent/WO2025098480A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the field of antibody engineering, and in particular to a bispecific antibody that recognizes DR5 and CEACAM5, as well as therapeutic uses of the antibody and a pharmaceutical composition containing the same.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
  • DR4 DR4
  • TRAILR2 DR5
  • Many preclinical studies have found that TRAIL signaling effectively induces apoptosis in multiple tumor cell lines, but not in most normal cells. Therefore, targeting TRAIL receptors has become an important research direction for the development of cancer therapy.
  • DR5 is highly expressed in a variety of solid cancers (including colorectal tumors). At present, clinical studies have reported a variety of agonist drugs targeting DR5.
  • the first generation of agonists is anti-DR5 monoclonal antibodies or recombinant protein molecules of its ligand TRAIL. Although clinical studies have found good safety, the efficacy is poor due to its inherent weak agonistic properties. The second generation of molecules is anti-DR5 multivalent antibodies. Although clinical studies have found that the efficacy has been improved, the side effects (mainly liver toxicity that is sensitive to DR5-induced apoptosis) limit the application of DR5 targeted therapeutic drugs.
  • CEACAM5 (also known as CEA or CD66e) is a carcinoembryonic antigen (CEA) that is expressed at low levels in normal human tissues, but is expressed at high levels in colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer and bladder cancer tissues. Therefore, CEACAM5 is regarded as a tumor biomarker and is suitable as a therapeutic target for tumor-specific targeted approaches (such as immunoconjugates).
  • CEACAM5 is regarded as a tumor biomarker and is suitable as a therapeutic target for tumor-specific targeted approaches (such as immunoconjugates).
  • bispecific antibodies Although the technology for constructing bispecific antibodies has made great progress, obtaining the desired bispecific antibodies is still a time-consuming, laborious and uncertain task. How to achieve the biological function of bispecific antibodies through different formats requires consideration and optimization of multiple parameters: such as the selection of antibodies for each target in the bispecific antibody (not all antibodies are suitable for bispecific antibodies), the valency of the antibody for each target, the structural form and conformation of each antibody part, how to arrange the layout to ensure the optimal spatial distance between each antibody, etc. For example, the study in CN 110582513A showed that the arrangement order of VH and VL in scFv has a great influence on its activity.
  • the present invention provides a bispecific antibody that can specifically recognize DR5 and CEACAM5.
  • the present invention also provides the therapeutic use of the antibody and a pharmaceutical composition containing the antibody.
  • the present invention provides a bispecific antibody (also referred to as an anti-DR5 ⁇ CEACAM5 bispecific antibody, an anti-DR5/CEACAM5 bispecific antibody) that simultaneously targets DR5 and CEACAM5.
  • a bispecific antibody also referred to as an anti-DR5 ⁇ CEACAM5 bispecific antibody, an anti-DR5/CEACAM5 bispecific antibody
  • the anti-DR5 antibody is in the form of a Fab, which maintains the activity of DR5 monoclonal antibody targeted therapy
  • the anti-CEACAM5 antibody is in the form of a scFv and has been affinity optimized, so that it can better assist in improving the activity of DR5 targeted therapy.
  • the bispecific antibody of this configuration of the present invention allows the auxiliary activity of CEACAM5 to occur only in tumor tissues that express CEACAM5, but not in normal tissues that do not express CEACAM5, thereby improving the therapeutic accuracy of the anti-DR5/CEACAM5 bispecific antibody.
  • the anti-DR5/CEACAM5 bispecific antibody of the present invention has the following advantages:
  • the configuration of the anti-DR5/CEACAM5 bispecific antibody of the present invention is the best antibody form for achieving the drug efficacy of the anti-DR5/CEACAM5 bispecific antibody;
  • the anti-DR5/CEACAM5 bispecific antibody of the present invention has reduced molecular heterogeneity, high yield, high yield in a one-step purification method, and improved overall drugability;
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising two identical heavy chains with a structure of VH-CH1-Fc-scFv, and two identical light chains with a structure of VL-CL, wherein the VL of the two light chains are respectively paired with the VH of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 .
  • the present invention provides an anti-DR5/CEACAM5 bispecific An antibody comprising two identical heavy chains of structure VH-CH1-Fc-scFv, and two identical light chains of structure VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 , wherein:
  • the VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; and
  • the scFv recognizing CEACAM5 comprises a VH recognizing CEACAM5 (referred to herein as VH CEACAM5 to distinguish it from a VH recognizing DR5) and a VL recognizing CEACAM5 (referred to herein as VL CEACAM5 to distinguish it from a VL recognizing DR5), wherein VH CEACAM5 comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11, and VL CEACAM5 recognizing CEACAM5 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:39, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO: 22, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:30, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 as contained in SEQ ID NO: 22. and HCDR3, the VL recognizing the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:34, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:34, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:30, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:39, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;
  • the scFv that recognizes CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:34, and VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 19, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:30, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:38, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:40;
  • the scFv recognizing the CEACAM5 molecule comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 contained in SEQ ID NO:39, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:20.
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are paired with the VH of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 , wherein:
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:22, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:24;
  • the VH CEACAM5 that recognizes the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 34, and
  • the VL CEACAM5 that recognizes the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 36;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:22, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:24;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 30, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 32;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:26, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:28;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 34, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 36;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:26, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:28;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 30, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 32;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:26, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:28;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 34, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 36;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 19, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO: 8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 30, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 32;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:38, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:40;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH of the complete antibody recognizing DR5 comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO:8;
  • VH CEACAM5 of the scFv that recognizes CEACAM5 comprises the sequence shown in SEQ ID NO: 15, and the VL CEACAM5 that recognizes the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 16;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:41, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:43, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:45, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:47, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 49, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing CEACAM5 molecule comprises the sequence shown in SEQ ID NO:51, and the VL CEACAM5 recognizing CEACAM5 molecule comprises the sequence shown in SEQ ID NO:20;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:54;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:56;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:39
  • the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:58.
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein:
  • the VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; and
  • the VH CEACAM5 that recognizes the CEACAM5 molecule comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11, and the VL CEACAM5 that recognizes CEACAM5 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14.
  • the present invention provides a DR5/CEACAM5 bispecific antibody, comprising an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein:
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence of SEQ ID NO:8; and
  • the VH CEACAM5 of the scFv recognizing CEACAM5 comprises the sequence shown in SEQ ID NO:15
  • the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:16.
  • the present invention provides a DR5/CEACAM5 bispecific antibody, which comprises an antibody or antigen-binding fragment that recognizes a DR5 molecule and an antibody or antigen-binding fragment that recognizes a CEACAM5 molecule, wherein the antibody that recognizes a DR5 molecule is a complete antibody, and the antigen-binding fragment that recognizes a CEACAM5 molecule is selected from scFv, scFab, Fab or Fv.
  • the Fc region in an anti-DR5/CEACAM5 bispecific antibody as provided above, can be selected from the natural Fc region of any IgG, IgA, IgM class antibody known in the prior art, such as a common or germline IgG Fc region.
  • the IgG Fc region can be from human IgG1, IgG2, IgG3 or IgG4 isotypes.
  • the IgG Fc region is the Fc sequence of human IgG1.
  • the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (e.g., disulfide bridge structure), mutations that reduce effector function (e.g., L234A and L235A mutations).
  • the scFv of the anti-DR5/CEACAM5 bispecific antibody of the present invention is connected to Fc via a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence.
  • the linker is (G 3 S) n (SEQ ID NO: 62) or (G 4 S) n (SEQ ID NO: 63), wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9.
  • the linker is selected from (G 3 S) 3 (SEQ ID NO: 64), (G 4 S) 4 (SEQ ID NO: 65).
  • VH CEACAM5 and VL CEACAM5 in the scFv of the bispecific antibody of the present invention are connected by a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence.
  • the linker is (G 3 S) n or (G 4 S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9.
  • the linker is selected from (G 3 S) 3 , (G 4 S) 4 .
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, which comprises two heavy chains with the same structure of VH-CH1-Fc-scFv, and two light chains with the same structure of VL-CL, wherein the VL of the two light chains are respectively paired with the VH of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on each heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the heavy chain comprises the sequence shown in SEQ ID NO:17, and the light chain comprises the sequence of SEQ ID NO:18.
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VL of the two light chains are respectively paired with the VH of the two heavy chains to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 .
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 , wherein:
  • the VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and VL CEACAM5 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, LCDR3 as shown in SEQ ID NO:14;
  • the VH that recognizes DR5 molecules comprises HCDR1 as shown in SEQ ID NO: 1, and HCDR2 as shown in SEQ ID NO: 2.
  • VL recognizing DR5 molecule comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:30, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:32;
  • the VH that recognizes the DR5 molecule comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the VL that recognizes the DR5 molecule comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:34, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv recognizing CEACAM5 molecules comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, and VL CEACAM5 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, LCDR3 as shown in SEQ ID NO:14;
  • the VH that recognizes the DR5 molecule comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 26, and the VL that recognizes the DR5 molecule comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28;
  • the scFv recognizing the CEACAM5 molecule comprises VH CEACAM5 and VL CEACAM5 , wherein the VH CEACAM5 comprises HCDR1, HCDR2 and HCDR3 as contained in SEQ ID NO:34, and the VL CEACAM5 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:36.
  • the present invention provides an anti-DR5/CEACAM5 bispecific antibody, comprising one heavy chain with a structure of VH-CH1-Fc-scFv, one heavy chain with a structure of VH-CH1-Fc, and two light chains with a structure of VL-CL, wherein the two heavy chains have the same VH-CH1-Fc, wherein the VLs of the two light chains are paired with the VHs of the two heavy chains respectively to form two antigen recognition sites that recognize the DR5 molecule, and the scFv on the heavy chain forms an antigen recognition site that recognizes the CEACAM5 molecule, wherein the scFv is connected to the C-terminus of the Fc and has a structure of VL CEACAM5 -linker-VH CEACAM5 , wherein:
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 15, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 16;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 30, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 32;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:7, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:8;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 34, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 36;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:26, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:28;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 15, and the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO: 16;
  • the VH recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:26, and the VL recognizing the DR5 molecule comprises the sequence shown in SEQ ID NO:28;
  • the VH CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:34
  • the VL CEACAM5 recognizing the CEACAM5 molecule comprises the sequence shown in SEQ ID NO:36.
  • the Fc region in an anti-DR5/CEACAM5 bispecific antibody as provided above, can be selected from the natural Fc region of any IgG, IgA, IgM class antibody known in the prior art, such as a common or germline IgG Fc region.
  • the IgG Fc region can be from human IgG1, IgG2, IgG3 or IgG4 isotypes.
  • the IgG Fc region is the Fc sequence of human IgG1.
  • the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (e.g., disulfide bridge structure), mutations that reduce effector function (e.g., L234A and L235A mutations).
  • the scFv of the anti-DR5/CEACAM5 bispecific antibody of the present invention is connected to Fc via a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence.
  • the linker is (G 3 S) n or (G 4 S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9.
  • the linker is selected from (G 3 S) 3 , (G 4 S) 4 .
  • VH CEACAM5 and VL CEACAM5 in the scFv of the bispecific antibody of the present invention are connected by a linker, and the linker can be any universal flexible sequence known in the art or obtained in the future, usually a short flexible amino acid sequence.
  • the linker is (G 3 S) n or (G 4 S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9.
  • the linker is selected from (G 3 S) 3 , (G 4 S) 4 .
  • the present invention also provides a polynucleotide (nucleic acid) encoding the anti-DR5/CEACAM5 bispecific antibody of the present invention, and a vector comprising the polynucleotide, preferably an expression vector.
  • a polynucleotide nucleic acid
  • a vector comprising the polynucleotide, preferably an expression vector.
  • the present invention provides a host cell comprising the polynucleotide or vector of the present invention.
  • the host cell may be a prokaryotic cell or a eukaryotic cell commonly used in the art.
  • the present invention provides a method for producing the anti-DR5/CEACAM5 bispecific antibody of the present invention, comprising the steps of (i) culturing the host cell of the present invention under conditions suitable for expressing the anti-DR5/CEACAM5 bispecific antibody of the present invention, and optionally, (ii) recovering the anti-DR5/CEACAM5 bispecific antibody of the present invention.
  • the present invention provides a pharmaceutical composition comprising the anti-DR5/CEACAM5 bispecific antibody of the present invention.
  • the present invention provides a drug combination comprising the anti-DR5/CEACAM5 bispecific antibody of the present invention and other therapeutic agents, and optional pharmaceutical excipients; preferably, the other therapeutic agents are selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.
  • the present invention provides a drug kit comprising the anti-DR5/CEACAM5 bispecific antibody of the present invention and other therapeutic agents.
  • the other therapeutic agents are selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.
  • the present invention provides the use of the anti-DR5/CEACAM5 bispecific antibody, pharmaceutical composition, and drug kit of the present invention for treating cancer.
  • the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.
  • the present invention provides the use of the anti-DR5/CEACAM5 bispecific antibody, polynucleotide, vector, host cell, pharmaceutical composition or kit described in the first to third aspects and the sixth aspect in the preparation of a medicament for treating and/or diagnosing cancer.
  • the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.
  • the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the anti-DR5/CEACAM5 bispecific antibody, polynucleotide, vector, host cell, pharmaceutical composition or kit of the first to third and sixth aspects of the present invention to a patient in need thereof.
  • the cancer is, for example, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.
  • Figure 1 shows the antibody structure that was initially designed and constructed.
  • FIG2 shows the results of the activity test of the molecule having the antibody structure 1 in inducing apoptosis of tumor cells, wherein the molecule having the antibody structure 1 has a stronger activity of inducing apoptosis of tumor cells than the anti-DR5 monoclonal antibody molecule.
  • Figure 3 shows the results of the activity test of inducing tumor cell apoptosis of molecules having antibody structure 1.
  • the molecules of antibody structure 1 the molecules of anti-DR5 antibody in the Fab region and in the scFv region have stronger activity of inducing tumor cell apoptosis.
  • Figure 4 shows the results of the activity test of inducing tumor cell apoptosis by the molecule having antibody structure 2.
  • the molecule having antibody structure 1 has a stronger activity of inducing tumor cell apoptosis than the molecule having antibody structure 2.
  • Figure 5 shows the results of the activity test of inducing tumor cell apoptosis by the molecule having antibody structure 3.
  • the molecule having antibody structure 1 has a stronger activity of inducing tumor cell apoptosis than the molecule having antibody structure 3.
  • FIG6 shows the activity test results of inducing tumor cell apoptosis by molecules having antibody structure 4, molecules having antibody structure 5, and molecules having antibody structure 6.
  • the molecules having antibody structure 1 are more effective than those having The molecules having antibody structure 4, the molecules having antibody structure 5 and the molecules having antibody structure 6 have stronger activity of inducing apoptosis of tumor cells.
  • Figure 7 shows the results of the activity test of anti-DR5 monoclonal antibody and its mutants in inducing apoptosis of human colon cancer Colo205 cells.
  • KMmut1 antibody can induce apoptosis of a larger proportion of tumor cells at high concentrations.
  • FIG8 shows the results of the activity detection of KMmut1-HL1 inducing apoptosis in human colon cancer HT55 cells.
  • FIG. 9 shows the activity of KMmut1-HL1 in inducing apoptosis of various tumor cell lines in vitro.
  • FIG. 10 shows the synergistic effect of KMmut1-HL1 combined with other drugs in vitro.
  • FIG. 11 shows that KMmut1-HL1 inhibits the growth of xenograft tumor cells in mice.
  • antibody is used herein in the broadest sense to refer to a protein that contains an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single-domain antibodies, complete antibodies, and antibody fragments.
  • antibody fragment refers to a molecule that is different from an intact antibody, which comprises a portion of an intact antibody and is capable of binding to the antigen that the intact antibody binds to.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (eg scFv); single domain antibodies; bivalent or bispecific antibody fragments and camelid antibodies (heavy chain antibodies).
  • Fc region comprises at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. 91 pages (2007)).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • CDR region is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy chain are usually referred to as HCDR1, HCDR2 and HCDR3, and the CDRs of the light chain are usually referred to as LCDR1, LCDR2 and LCDR, and are numbered sequentially from the N-terminus, respectively.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • multispecific antibody refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens.
  • a multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes.
  • a multispecific antibody that has binding specificity for a first antigen and a second antigen, also referred to as a "bispecific antibody.”
  • Binding refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding "Affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be described by the binding dissociation equilibrium constant ( KD ). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
  • the KD value of an antibody of the present invention that "specifically binds" an antigen as determined by biofilm thin layer interferometry (BLI) is about 1 ⁇ 10-8 M or less, preferably about 1 ⁇ 10-9 M or less; more preferably 1 ⁇ 10-10 M or less.
  • single-chain scFv antibody or “scFv” or “single-chain scFv” refers to a single polypeptide chain comprising the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin or antibody, in which the VH region and the VL region are paired to provide an antigen binding site.
  • VH and VL are connected by an artificially synthesized connecting peptide (or "linker”).
  • scFv is suitable for use as a genetic engineering component to prepare other antigen-specific binding molecules with new properties, such as full-length antibodies, scFv-Fc, multispecific antibodies, etc.
  • connecting peptide refers to a peptide comprising one or more consecutive amino acids, such as small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.).
  • the connecting peptide generally comprises a length of 5-50 amino acids, such as 10, 15, 20, 25, 30 amino acids in length.
  • linkers can be used in embodiments of the present invention.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, without considering the number of passages.
  • Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny with the same function or biological activity that are screened or selected in the initially transformed cells.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • half effective concentration refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • treat refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.
  • prevention includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • the term "effective amount” refers to an amount or dosage of an antibody or conjugate or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • the effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of the mammal; body weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • therapeutically effective amount refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time.
  • the therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof may vary according to a variety of factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%. It can be demonstrated in animal model systems predictive of efficacy in human tumors. Compounds are evaluated for their ability to inhibit a measurable parameter (eg, cancer).
  • a measurable parameter e.g., tumor growth rate, tumor volume, etc.
  • composition refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • anti-DR5/CEACAM5 bispecific antibody refers to bispecific antibodies that can bind to the targets CEACAM5 and DR5 with sufficient affinity.
  • monoclonal antibodies targeting CEACAM5 and DR5 are suitable for constructing the bispecific antibody of the present invention, and bispecific antibody molecules of different configurations have different activities.
  • the present invention finally obtains a bispecific antibody targeting CEACAM5 and DR5 with excellent performance, and its molecular structure is shown in Figures 1A and 1C.
  • the bispecific molecule comprises a full-length monoclonal antibody targeting DR5 and a scFv antibody targeting CEACAM5.
  • the scFv molecule is fused to the heavy chain C-terminus of the above-mentioned DR5 monoclonal antibody through a linker 2.
  • the order of the scFv antibody from the N-terminus to the C-terminus is VL-linker 1-VH, wherein the linker 2 is a (G 3 S) 3 polypeptide and the linker 1 is a (G 4 S) 4 polypeptide.
  • the bispecific anti-DR5/CEACAM5 antibody of the present invention comprises an amino acid modification, such as an amino acid substitution, addition or deletion, preferably an amino acid substitution, more preferably an amino acid conservative substitution.
  • the amino acid modification described in the present invention occurs in a region outside of the CDR (e.g., in the FR).
  • the substitution is a conservative substitution.
  • a conservative substitution refers to the substitution of an amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.
  • Fc region variants may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.
  • the Fc region of the bispecific antibody described in the present application has a knob in hole structure and a disulfide bridge to promote and stabilize the structure of the bispecific antibody.
  • the bispecific anti-DR5/CEACAM5 antibody of the present invention comprises amino acid replacements at positions 234 and 235 (EU numbering) of the heavy chain.
  • the amino acid replacements are L234A and L235A (also known as "LALA mutations").
  • the present invention also provides a single-chain scFv antibody targeting CEACAM5.
  • the VH region and the VL region of the single-chain scFv antibody of the present invention are covalently linked together by a flexible linker.
  • the linker will facilitate the pairing of VH and VL, and will not interfere with the formation of a functionally effective antigen binding site for VH and VL.
  • the order of the scFv antibody from N-terminus to C-terminus is VH-VL, wherein VH is connected to VL via a linker (G4S) 4 polypeptide.
  • cysteine amino acid mutations are introduced into VH44 and VL100 (Kabat numbering convention), respectively, to form interchain disulfide bridges to stabilize the scFv structure.
  • the present invention provides nucleic acids encoding the above bispecific antibodies or antigen-binding fragments thereof targeting DR5 and CEACAM5.
  • the present invention also encompasses nucleic acids that hybridize with the above nucleic acids under stringent conditions, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions or insertions compared to the above nucleic acids, or nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above nucleic acids.
  • substitutions e.g., conservative substitutions
  • nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above nucleic acids.
  • the present invention provides a vector comprising the above-mentioned nucleic acid.
  • the vector is an expression vector. It is fully understood by those skilled in the art that the vectors commonly used in the technical field to which the present invention belongs can be applied to the present invention.
  • the present invention provides a host cell comprising the nucleic acid or the vector.
  • Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, for example, E. coli cells.
  • eukaryotic cells for example, mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, for example, E. coli cells.
  • Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
  • the present invention provides a composition comprising a bispecific antibody or antigen-binding fragment thereof targeting DR5 and CEACAM5 as described herein, preferably a pharmaceutical composition.
  • the composition further comprises a pharmaceutical excipient.
  • pharmaceutical excipient refers to a diluent, adjuvant, carrier, excipient or stabilizer, etc., which is administered together with an active substance.
  • a composition (e.g., a pharmaceutical composition) comprises an anti-DR5/CEACAM5 bispecific antibody or an antigen-binding fragment thereof of the present invention and a combination of one or more other therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators).
  • the other therapeutic agent is selected from 5-fluorouracil, oxaliplatin, exotecan, and SN-38.
  • the pharmaceutical composition of the present invention may also contain one or more other therapeutic agents, which are required for the specific indication being treated, and include any substance effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), preferably with those therapeutic agents that do not adversely affect each other's activities.
  • tumors e.g., cancer
  • infections e.g., chronic infections
  • the therapeutic agent is for the purpose of In a preferred embodiment, the other therapeutic agent is selected from 5-fluorouracil, oxaliplatin, exitecan, and SN-38.
  • the present invention provides a method for preparing an anti-DR5/CEACAM5 bispecific antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-DR5/CEACAM5 bispecific antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the anti-DR5/CEACAM5 bispecific antibody, and optionally isolating the anti-DR5/CEACAM5 bispecific antibody. In a certain embodiment, the method further comprises recovering the anti-DR5/CEACAM5 bispecific antibody from the host cell (or host cell culture medium).
  • the anti-DR5/CEACAM5 bispecific antibodies of the present invention can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art.
  • the purity of the anti-DR5/CEACAM5 bispecific antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including molecular exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.
  • the present application provides use of an anti-DR5/CEACAM5 bispecific antibody in preparing a medicament for treating cancer.
  • the therapeutically effective amount of the anti-DR5/CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same can be used to treat cancer expressing CEACAM5. In one embodiment, the therapeutically effective amount of the anti-DR5/CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same can be used to promote apoptosis of cancer cells expressing CEACAM5.
  • the cancer is colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.
  • the present invention relates to a method for treating a tumor/cancer in a subject, comprising administering to the subject an effective amount of the anti-DR5/CEACAM5 bispecific antibody disclosed herein, a nucleic acid encoding the same, or a pharmaceutical composition comprising the same.
  • the cancer is colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.
  • the subject can be a mammal, eg, a primate, preferably, a higher primate, eg, a human (eg, a human patient having or at risk of having a cancer described herein).
  • the treatment methods described herein further comprise administering to the subject or individual an anti-DR5/CEACAM5 bispecific antibody disclosed herein or a pharmaceutical composition comprising the same, in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents.
  • the prior art discloses a variety of monoclonal antibodies against DR5 or CEACAM5, no bispecific antibodies specifically targeting the two have entered clinical trials.
  • the present invention attempts to construct an anti-DR5/CEACAM5 bispecific antibody targeting the two that is suitable for clinical application.
  • the construction of a bispecific antibody that can be made into a drug needs to consider a variety of factors. Therefore, in the early stage of research and development, the inventor first constructed various forms of bispecific molecules based on the sequences of anti-DR5 or anti-CEACAM5 monoclonal antibodies known in the prior art, as shown in Figure 1.
  • the anti-DR5 antibody molecules used include conatumumab (US20190284290A1), drozitumab (US10501552B2), KMTR2 (CN100475848C), Lexatumumab (EP2016744669), Tigatuzumab (CN100506277C), LBY135 (US10501552B2), DR5mAb1 (US10501552B2), Hx01 (US20190315877) and Hx05 (US20190315877).
  • Anti-CEACAM5 antibodies include hPR1A3 (WO2012117002), labetuzumab (US20150125386A1) and tusamitamab (CN104918958A).
  • Various bispecific antibody structures as shown in FIG1 were assembled from the above-mentioned various antibody molecules. The specific assembly methods are shown in Table 1.
  • the amino acid sequences of the corresponding antibody molecules are obtained respectively, and the corresponding encoding nucleotide sequences are obtained according to conventional methods in the art, and constructed into separate expression vectors pcDNA3.1 for the heavy chain and light chain of each bispecific antibody molecule.
  • These vectors contain corresponding elements conventionally used for expression and purification in eukaryotic cells and prokaryotic cells (e.g., regulatory elements for gene expression, selection markers) for use in subsequent experiments.
  • the expression vectors encoding each antibody molecule obtained in Example 1 were used to transfect CHO cells or 293 cells to express and purify the corresponding antibody molecules.
  • ExpiCHO cells (Invitrogen) were passaged according to the required cell volume, and the cell density was adjusted to 3.5 ⁇ 10 6 cells/ml one day before transfection. The cell density was tested on the day of transfection (approximately 8-10 ⁇ 10 6 cells/ml), and the viability reached more than 95%, and the cell density was adjusted to 6x10 6 cells/ml using ExpiCHO TM Expression Medium (Gibco Catalog No.: A29100-01).
  • OptiPROTM SFM (Gibco, catalog number: 12309-019) with a final volume of 8% (v/v) as transfection buffer, add the corresponding amount (0.8 ⁇ g/mL cell) of expression vector (plasmid), mix well, filter with a 0.22 ⁇ m filter membrane for sterilization, add the reagent in ExpiFectamine TM CHO Transfection Kit (Gibco, catalog number: A29130) at 3.2 ⁇ L/mL, and incubate the complex formed by the transfection reagent and plasmid DNA at room temperature for 1 to 5 minutes, then Slowly add it to the cells, culture at 37°C, 8% CO 2 for 18 hours, then add 0.6% (v/v) Enhancer and 30% (v/v) Feed, and continue to culture for 6 days.
  • the cell culture medium was centrifuged at 4000 rpm for 50 min, and the supernatant was collected and filtered through a 0.45 ⁇ M filter membrane, and purified by affinity chromatography and gel chromatography.
  • Affinity chromatography Purify the supernatant with prepacked Hitrap Mabselect Sure (GE, 11-0034-95). Before purification, balance the column with 5 column volumes of equilibration solution (20mM Tris, 150mM NaCl, pH7.2); pass the collected supernatant through the column, and then wash the column with 10 column volumes of equilibration solution to remove non-specific binding proteins; rinse the column with 5 column volumes of elution buffer (100mM sodium citrate, pH3.5), and collect the eluate. Adjust the pH to 6.0 with 2M Tris for each 1ml of eluate, and measure the concentration.
  • Use 0.1M NaOH to remove endotoxins from the AKTApure system equipped with Superdex 200 Increase 10/300GL gel chromatography column for 2 hours, then wash the system and column with distilled water; use 2-5 column volumes of 1 ⁇ PBS to balance the column until the conductivity and pH are stable; load the protein obtained by affinity chromatography, continue to elute with 1 ⁇ PBS, remove impurities such as aggregates according to the ultraviolet absorption peak, and collect high-purity samples. Filter the sample with a 0.22 ⁇ m membrane to determine the protein concentration.
  • Expi293F cells (Thermo Fisher scientific company) were subcultured in Expi293F cell culture medium (Thermo Fisher scientific company). The cell density was checked one day before transfection, and the cell density was adjusted to 2 ⁇ 10 6 cells/ml with fresh Expi293 cell culture medium for continued culture. On the day of transfection, the cell density was adjusted to 3 ⁇ 10 6 cells/ml.
  • Opti-MEM medium (Gibco) with 1/10 of the final volume of the transfected Expi293F cells was used as the transfection buffer, 10 ⁇ g of the corresponding expression vector (plasmid) was added to each mL of the transfection buffer, and mixed, and 30 ⁇ g of polyethylenimine (PEI) (Polysciences, catalog number: 23966) was added to each mL of the transfection buffer, mixed, and incubated at room temperature for 20 minutes, and then the PEI/DNA mixture was gently poured into the Expi293F cell suspension, mixed, and placed in a shaker for culture, and the culture conditions were 8% CO 2 , 36.5° C., and 120 rpm.
  • PEI polyethylenimine
  • FEED 100 g/L Phytone Peptone + 100 g/L Difco Select Phytone
  • Cultivate continuously for 6 days collect the culture, centrifuge at 4000 rpm for 30 minutes, take the cell supernatant and filter it with a 0.22 ⁇ M filter membrane, and purify the antibody by affinity chromatography and gel chromatography as described above.
  • Example 3 In vitro efficacy of anti-DR5/CEACAM5 bispecific antibody in inducing tumor cell apoptosis
  • the various bispecific antibody molecules expressed and purified in Example 2 were used for in vitro activity evaluation experiments.
  • the tumor cells used in this example include human colon adenocarcinoma LoVo cells (ATCC), human colon adenocarcinoma LS174T cells (Cell Bank of the Chinese Academy of Sciences), human cecal adenocarcinoma NCI-H508 cells (Nanjing Kebai), human colon adenocarcinoma GP2d cells (Nanjing Kebai), and human rectal adenocarcinoma HT55 cells (Nanjing Kebai).
  • ATCC human colon adenocarcinoma LoVo cells
  • human colon adenocarcinoma LS174T cells Cell Bank of the Chinese Academy of Sciences
  • human cecal adenocarcinoma NCI-H508 cells Najing Kebai
  • human colon adenocarcinoma GP2d cells Najing Kebai
  • the relative cell viability was calculated with the control antibody Ctrl.hIgG (Merck, AG711) as 1 to reflect the activity of the antibody molecule to be tested in inducing apoptosis in vitro.
  • the bispecific antibody KM-hPR1A3 has the following heavy chain and light chain sequences:
  • the anti-DR5/CEACAM5 bispecific molecule KM-hPR1A3 has a stronger activity in inducing tumor cell apoptosis than the anti-DR5 monoclonal antibody.
  • the anti-DR5 antibody sequence and the anti-CEACAM5 antibody sequence of the KM-hPR1A3 molecule having the antibody structure 1 were optimized respectively to improve the drugability of the molecule, and finally the KMmut1-HL1 molecule sequence was optimized.
  • the sequence of the anti-DR5 antibody of the present invention is modified as shown in Table 3.
  • the modified antibody molecules are expressed and purified, and their relevant drug properties are detected.
  • the in vitro activity of the modified antibody molecules in inducing cell apoptosis is detected.
  • KMTR2 is the monoclonal antibody sequence disclosed in WO2002094880.
  • Dynamic light scattering uses laser to illuminate small particles in Brownian motion in a solution and detects the change in the intensity of the scattered light, thereby measuring the particle size distribution of the particles. Therefore, DLS can be used to detect the particle size of proteins in a solution and their stability under different temperatures or concentrations.
  • a 1 mg/ml solution of the mutant antibody molecule was centrifuged at 13000G/min for 5 minutes and then added to the sample plate, which was then centrifuged for 1 minute using a plate centrifuge to remove bubbles. The changes in the particle size of the protein molecule during the continuous temperature rise were detected.
  • the detection settings were DLS acquisition time of 5s, acquisition times of 5 times, and experimental temperature of 25-85°C. After the experiment, the changes in the sample particle size with temperature were analyzed.
  • Differential scanning calorimetry is an analytical method that measures the energy difference between a sample and a reference substance as it changes with temperature under programmed temperature conditions. As the temperature rises, the protein structure changes, and the accompanying heat changes are recorded by the differential scanning calorimeter into the DSC curve. The Tm value of the protein is obtained by analyzing the DSC curve. Specifically, the antibody mutant samples were diluted to 0.5-1 mg/ml with PBS solution, the diluted samples and PBS buffer were degassed, and then the samples were added to the left sample plate, and PBS buffer was added to the corresponding position in the right reference plate. The starting temperature was 30°C and the equilibrium time was 10min; heating was performed at a rate of 1°C/min, and the end temperature was 90°C.
  • the prior art did not modify the NTS glycosylation modification site of the KMTR2 molecule.
  • it was mutated to DTS, thereby improving the thermal stability of the antibody and reducing the heterogeneity of the bispecific antibody molecule.
  • the leucine at positions 234 and 235 of the Fc region of the KMTR2 antibody were mutated to alanine.
  • the DR5 mutant antibody KMmut1 has a higher maximum activity in inducing tumor cell apoptosis and better drugability.
  • the sequence of the anti-CEACAM5 antibody scFv part of the bispecific antibody was further modified.
  • the modified molecular sequence is shown in Table 5.
  • its variable region was constructed into a scFv in the form of VL-linker-VH from the N-terminus to the C-terminus.
  • cysteine amino acid mutations were introduced at H44 of VH and L100 of VL (Kabat numbering rules), respectively, to form an interchain disulfide bridge.
  • the complete anti-DR5/CEACAM5 bispecific antibody molecule was evaluated for in vitro activity efficacy and drugability.
  • Table 6 for molecular activity detection, please refer to Figure 8, and for molecular drugability evaluation, please refer to Table 7.
  • a KMmut1-hPR1A3-v1 molecule was constructed, in which the scFv was composed in the form of VH-linker-VL.
  • the KMmut1-HL1 molecule had the best drugability, and the protein yield was improved, and the purity after one-step affinity purification of protein A was also improved.
  • the KMmut1-hPR1A3-v1 molecule with scFv in the form of VH-linker-VL showed the worst drugability, indicating that in bispecific antibody molecules, the arrangement of VH and VL in the scFv structure has a great influence on the drugability of the antibody molecule.
  • the preferred anti-DR5/CEACAM5 bispecific antibody molecule (also called KMmut1-HL1) having the structure of Figure 1A obtained in the present application has improved yield and reduced protein molecule heterogeneity compared to the parent monoclonal antibody and the bispecific antibody molecule with unmodified sequence, and reduces the formation of multimers in the one-step purification method, thereby improving the overall drugability.
  • the anti-DR5/CEACAM5 bispecific antibody molecule (KMmut1-HL1) is composed of two identical heavy chains and two identical light chains, and the sequences of the heavy chain and light chain are as follows:
  • the CDR sequences, heavy chain variable region sequences, and light chain variable region sequences contained in the KMmut1-HL1 molecule are shown in the following table, wherein the CDR in the heavy chain variable region is determined according to the AbM coding rule; and the CDR in the light chain variable region is determined according to the Kabat coding rule:
  • the anti-DR5/CEACAM5 bispecific antibody molecule KMmut1-HL1 of the present invention can bind to human and cynomolgus monkey DR5 proteins, but not mouse DR5 protein; it can bind to human CEACAM5 protein, but not cynomolgus monkey CEACAM5 protein, and does not bind to other human CEACAM family proteins, and has good specificity.
  • the affinity (KD) of the bispecific antibody KMmut1-HL1 of the present invention for binding to DR5 and CEACAM5 was determined by biofilm thin layer interferometry (BLI). The specific method is as follows:
  • Anti-DR5/CEACAM5 bispecific antibody KMmut1-HL1 can induce apoptosis of various tumor cells
  • This example examines whether the excellent apoptotic tumor cell ability of the anti-DR5/CEACAM5 bispecific antibody of the present application is generated by anchoring the bispecific antibody molecule to the tumor cells based on CEACAM5 on the tumor cells.
  • KMmut1-HL1 of the present invention By replacing the anti-DR5 antibody sequence or the anti-CEACAM5 antibody sequence of the anti-DR5/CEACAM5 bispecific antibody molecule KMmut1-HL1 of the present invention with the GP120 antibody sequence targeting the human immunodeficiency virus (HIV) envelope glycoprotein, respectively, a KMmut1-GP120 control molecule having the same structure as the KMmut1-HL1 of the present invention, targeting DR5 but not CEACAM5, and a GP120-HL1 control molecule targeting CEACAM5 but not DR5 can be obtained.
  • HIV human immunodeficiency virus
  • the pro-apoptotic activities of the KMmut1-HL1 molecule of the present invention, the KMmut1-GP120 control molecule, the GP120-HL1 control molecule, the anti-human epidermal growth factor (EGFR) antibody Cetuximab and the topoisomerase I inhibitor irinotecan (Irinotecan, MCE, HY-16562) on various tumor cells were compared.
  • the tumor cells human colon cancer GP2d tumor cells (Nanjing Kebai), human colon cancer HT55 cells (Nanjing Kebai), human colorectal adenocarcinoma NCI-H508 cells (Nanjing Kebai), human gastric cancer MKN45 cells (Nanjing Kebai) and human pancreatic adenocarcinoma BxPC-3 cells (Cell Bank of the Chinese Academy of Sciences) were digested into single-cell suspensions with trypsin (Gibco, 25200-072), and then plated into 96-well white-bottom plates (Shanghai Wohong, WHB-96-03) at a density of 1000 cells/well, and cultured in a 37°C 5% CO2 incubator overnight;
  • the molecules to be tested were diluted in a gradient in a 96-well plate (Corning, 3799), added to the 96-well white-bottom plate with tumor cells, and cultured in a 37°C 5% CO 2 incubator for 72 hours;
  • the relative cell viability was calculated with the control antibody Ctrl.hIgG (Merck, AG711) as 1 to reflect the activity of the antibody molecule to be tested in inducing apoptosis in vitro.
  • KMmut1-HL1 can induce apoptosis of various tumor cells in vitro, and its activity is better than that of the control molecules GP120-HL1, KMmut1-GP120, Cetuximab and irinotecan ( Figure 9), indicating that CEACAM5 expressed on tumor cells further promotes the stronger apoptosis-inducing activity of DR5 antibody.
  • Example 8 Synergistic effect of anti-DR5/CEACAM5 bispecific antibody KMmut1-HL1 in combination with other drugs
  • This example tests whether the KMmut1-HL1 molecule combined with other drugs can synergistically induce tumor cell apoptosis.
  • the specific method is as follows:
  • tumor cells human gastric cancer MKN45 cells, human colon cancer HT55 cells and human pancreatic adenocarcinoma BxPC-3 cells
  • trypsin trypsin
  • the cells were plated into a 96-well white bottom plate (Shanghai Wohong, WHB-96-03) at a density of 1000 cells/well and cultured in a 37°C 5% CO2 incubator overnight;
  • KMmut1-HL1 was diluted from 100 nM to 9 concentration gradients by a 4-fold dilution method.
  • Other drugs 5-fluorouracil (5-FU, MCE, HY-90006) was diluted from 100 nM
  • oxaliplatin (Oxaliplatin, MCE, HY-17371) was diluted from 100 ⁇ M
  • exatecan Exatecan, MCE, HY-13631 was diluted from 1 ⁇ M
  • SN-38 MCE, HY-13704 was diluted from 100 nM to 5 concentration gradients by a 10-fold dilution method.
  • KMmut1-HL1 and other drugs were mixed in a chessboard cross pattern to obtain a mixture system of the two drugs at various concentrations.
  • the mixed system was added to a 96-well white-bottom plate with tumor cells, and cultured in a 37°C 5% CO2 incubator for 96 hours;
  • the relative cell viability was calculated by taking the control antibody Ctrl.hIgG (Merck, AG711) as 1, and the synergistic effect score was calculated using SynergyFinder (DOI: 10.1093/nar/gkac382).
  • Anti-DR5/CEACAM5 bispecific antibody KMmut1-HL1 can inhibit the growth of xenograft tumor cells in mice
  • This example evaluates the in vivo efficacy of the anti-DR5/CEACAM5 bispecific antibody KMmut1-HL1.
  • the specific method is as follows:
  • mice were grouped so that the average tumor size in each group was similar, and the mice were intraperitoneally injected with the drug to be tested, KMmut1-HL1 molecule; Cetuximab + irinotecan + 5-fluorouracil (5-FU, MCE, HY-90006); KMmut1-GP120 + GP120-HL1 control molecule (see Figure 11 for specific dosages);
  • Body weight was measured using an electronic balance. Mice with tumor volume exceeding 2000 mm 3 or body weight loss exceeding 20% were euthanized.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps bispécifique se liant de manière spécifique à la CEACAM5 humaine et la DR5 humaine. L'invention concerne également une molécule d'acide nucléique codant pour l'anticorps, un vecteur d'expression pour exprimer l'anticorps, une cellule hôte et son procédé de préparation. L'invention concerne en outre une méthode de traitement faisant appel à l'anticorps bispécifique de la présente invention.
PCT/CN2024/130860 2023-11-10 2024-11-08 Anticorps bispécifique dirigé contre dr5 et ceacam5 et son utilisation Pending WO2025098480A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202311498202.9 2023-11-10
CN202311498202 2023-11-10

Publications (1)

Publication Number Publication Date
WO2025098480A1 true WO2025098480A1 (fr) 2025-05-15

Family

ID=95645987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/130860 Pending WO2025098480A1 (fr) 2023-11-10 2024-11-08 Anticorps bispécifique dirigé contre dr5 et ceacam5 et son utilisation

Country Status (3)

Country Link
CN (1) CN119978136A (fr)
TW (1) TW202528350A (fr)
WO (1) WO2025098480A1 (fr)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1599754A (zh) * 2001-05-18 2005-03-23 麒麟麦酒株式会社 抗trail－r抗体
CN101247825A (zh) * 2005-02-02 2008-08-20 健泰科生物技术公司 Dr5抗体及其用途
CN102574921A (zh) * 2009-09-29 2012-07-11 罗切格利卡特公司 双特异性死亡受体激动型抗体
CN103403029A (zh) * 2011-03-02 2013-11-20 罗切格利卡特公司 抗cea抗体
US20150125386A1 (en) * 2013-11-05 2015-05-07 Immunomedics, Inc. Humanized anti-ceacam5 antibody and uses thereof
CN104918958A (zh) * 2012-11-20 2015-09-16 赛诺菲 抗ceacam5抗体及其用途
CN107250161A (zh) * 2015-01-26 2017-10-13 宏观基因有限公司 包含dr5‑结合结构域的多价分子
US20190284290A1 (en) * 2012-03-28 2019-09-19 Amgen Inc. Dr5 receptor agonist combinations
WO2021098822A1 (fr) * 2019-11-21 2021-05-27 江苏恒瑞医药股份有限公司 Anticorps bispécifiques

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1599754A (zh) * 2001-05-18 2005-03-23 麒麟麦酒株式会社 抗trail－r抗体
CN101247825A (zh) * 2005-02-02 2008-08-20 健泰科生物技术公司 Dr5抗体及其用途
CN102574921A (zh) * 2009-09-29 2012-07-11 罗切格利卡特公司 双特异性死亡受体激动型抗体
CN103403029A (zh) * 2011-03-02 2013-11-20 罗切格利卡特公司 抗cea抗体
US20190284290A1 (en) * 2012-03-28 2019-09-19 Amgen Inc. Dr5 receptor agonist combinations
CN104918958A (zh) * 2012-11-20 2015-09-16 赛诺菲 抗ceacam5抗体及其用途
US20150125386A1 (en) * 2013-11-05 2015-05-07 Immunomedics, Inc. Humanized anti-ceacam5 antibody and uses thereof
CN107250161A (zh) * 2015-01-26 2017-10-13 宏观基因有限公司 包含dr5‑结合结构域的多价分子
WO2021098822A1 (fr) * 2019-11-21 2021-05-27 江苏恒瑞医药股份有限公司 Anticorps bispécifiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRUNKER P. ET AL.: "G7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis", MOLECULAR CANCER THERAPEUTICS, vol. 15, no. 5, 1 April 2016 (2016-04-01), pages 946 - 957, XP055373631, DOI: 10.1158/1535-7163.MCT-15-0647 *
HUANG, S.Y. ET AL.: "Bispecific Antibodies Targeting Dual Tumor-associated Antigens in Cancer Therapy", JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, vol. 146, 28 September 2020 (2020-09-28), pages 3111 - 3122, XP037300178, DOI: 10.1007/s00432-020-03404-6 *
SUURS, F.V. ET AL.: "A Reviewof Bispecific Antibodies and Antibody Constructs in Oncology and Clinical Challenges", PHARMACOLOGY & THERAPEUTICS, vol. 201, 24 April 2019 (2019-04-24), pages 103 - 119, XP085764019, DOI: 10.1016/j.pharmthera.2019.04.006 *

Also Published As

Publication number Publication date
CN119978136A (zh) 2025-05-13
TW202528350A (zh) 2025-07-16

Similar Documents

Publication Publication Date Title
US11370837B2 (en) Anti-TIGIT antibody and use thereof
WO2020200210A1 (fr) Anticorps anti-pd-l1/vegf bifonctionnel et son utilisation
TWI793395B (zh) 結合ｐｄ-ｌ１和ｏｘ４０的雙特異性抗體
AU2021353368B2 (en) Anti-cd3 antibody and uses thereof
CN112794911B (zh) 人源化抗叶酸受体1抗体及其应用
AU2021352532B2 (en) Anti-claudin18.2 and cd3 bispecific antibody and use thereof
US11965037B2 (en) Anti-HER3 humanized monoclonal antibody
JP2024506626A (ja) 抗ｃｄ１１２ｒ抗体及びその用途
KR20240141178A (ko) 이중특이적 항원-결합 분자 및 이의 용도
WO2023041065A1 (fr) Anticorps ciblant ceacam5/6 humain, procédé de préparation et application
EP4628512A1 (fr) Protéine de fusion trispécifique et son utilisation
WO2017122666A1 (fr) Anticorps anti-myl9
CN118852442A (zh) 抗ptk7抗体及其用途
JP2026032010A (ja) 二重特異性抗体及びその応用
WO2023179392A1 (fr) Anticorps b7h3 et anticorps bifonctionnel le comprenant
WO2025098480A1 (fr) Anticorps bispécifique dirigé contre dr5 et ceacam5 et son utilisation
WO2021244553A1 (fr) Anticorps bispécifique tétravalent contre pd-1 et egfr
CN113164601B (zh) 一种分离的抗原结合蛋白及其用途
WO2024098940A1 (fr) Anticorps bispécifique et son utilisation
WO2022228514A1 (fr) Anticorps anti-facteur inhibiteur de la leucémie humaine et son utilisation
CN120775048A (zh) 抗il1rap抗体以及其用途
CN121794298A (zh) 抗ror1抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24888091

Country of ref document: EP

Kind code of ref document: A1