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WO2018199593A1 - Anticorps bispécifique se liant à her3 et cd3 - Google Patents

Anticorps bispécifique se liant à her3 et cd3 Download PDF

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Publication number
WO2018199593A1
WO2018199593A1 PCT/KR2018/004743 KR2018004743W WO2018199593A1 WO 2018199593 A1 WO2018199593 A1 WO 2018199593A1 KR 2018004743 W KR2018004743 W KR 2018004743W WO 2018199593 A1 WO2018199593 A1 WO 2018199593A1
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WIPO (PCT)
Prior art keywords
seq
light chain
heavy chain
antibody
polypeptide
Prior art date
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Ceased
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PCT/KR2018/004743
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English (en)
Korean (ko)
Inventor
김기수
정준홍
임형권
류재환
권해냄
임양미
박용예
이은희
원종화
임옥재
신덕향
김문경
이윤정
이지원
남효정
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.)
Mogam Institute for Biomedical Research
GC Biopharma Corp
Original Assignee
Green Cross Corp Korea
Mogam Institute for Biomedical Research
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Application filed by Green Cross Corp Korea, Mogam Institute for Biomedical Research filed Critical Green Cross Corp Korea
Priority to KR1020197027313A priority Critical patent/KR102329635B1/ko
Publication of WO2018199593A1 publication Critical patent/WO2018199593A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/74—Inducing cell proliferation

Definitions

  • the present invention relates to a bispecific antibody that binds to HER3 and CD3 and a composition for preventing or treating cancer comprising the same.
  • Immune cells that directly remove cancer cells include NK cells and cytotoxic T lymphocytes (CTLs), and dendritic cells (antigenpresenting cells) that present antigens to these effector cells. dendritic cell, DC) or B cell.
  • CTLs cytotoxic T lymphocytes
  • dendritic cells antigenpresenting cells
  • dendritic cell DC
  • helper T cells Th cells
  • regulatory T cells Regulatory T cells, Treg cells
  • CD3 is a homodimeric or heterodimeric protein expressed on T cells with respect to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from two dimeric associations of four different chains: epsilon, zeta, delta and gamma. CD3 dimeric arrays include gamma / epsilon, delta / epsilon and zeta / zeta.
  • Antibodies against CD3 have been found to cause T cell activation in a manner similar to the engagement of TCR by peptide-loaded MHC molecules by clustering CD3 on T cells. Thus, anti-CD3 antibodies can be used for therapeutic purposes, including activation of T cells.
  • HER3, ErbB3 human epidermal growth factor receptor 3
  • HER3 is known to be involved in cancer cell growth, proliferation, anticancer drug resistance and cancer metastasis in various cancers such as breast cancer, lung cancer and osteosarcoma (Cancer Cell 2009 15: 353, Oncogene 2001 20, 1465, Nat Med Rev 2007 13 (6 ): 675, Proc Natl Acad Sci US A. 2008 Jan 15; 105 (2): 692-7., Science 2007 316, 1039).
  • the HER3 gene encodes a 1342 amino acid protein with significant structural similarities to EGFR and HER2.
  • Features such as full size, four extracellular subdomains (I-IV) with two cysteine clusters (domains II and IV), and tyrosine kinase domains show structural similarities to EGFR and HER2 (Cho and Leahy (2002)). Science, 297: 1330-1333.
  • the tyrosine kinase domain of HER3 shows 59% sequence homology with the tyrosine kinase domain of EGFR (Brennan et al. (2000) Oncogene, 19: 6093-6101).
  • Bispecific antibodies refer to antibodies that can bind to two different antibodies simultaneously. Such bispecific antibodies can maximize the therapeutic effect because immune cells such as T cells may be toxic to specific target cells (eg, specific cancer cells) and not to other normal cells in immunotherapy. Yet the side effects can be minimized, which can be useful in the treatment field where T-cell mediated killing is required.
  • the inventors have completed the present invention by developing a new bispecific antibody that specifically binds CD3 and HER3 and confirmed excellent anticancer effects.
  • An object of the present invention is to provide an antibody capable of effectively inducing HER3-specific cell death by effectively inducing immune cells expressing CD3 in cells expressing HER3.
  • Another object of the present invention is to provide a gene for producing the above antibody.
  • a first polypeptide of SEQ ID NO: 1 A second polypeptide of SEQ ID NO: 2; A third polypeptide selected from the group consisting of SEQ ID NOs: 3, 5, 31, 33, and 35; And a fourth polypeptide selected from the group consisting of SEQ ID NOs: 4, 6, 32, 34 and 36.
  • the first polypeptide is a first light chain variable region
  • the second polypeptide is a first heavy chain variable region
  • the third polypeptide is a second light chain variable region
  • the fourth poly The peptide is a second heavy chain variable region.
  • the first light chain heavy chain conjugate comprises a first polypeptide of SEQ ID NO: 1 and a second polypeptide of SEQ ID NO: 2
  • the second light chain heavy chain conjugate is SEQ ID NO: 3, 5, 31,
  • the first polypeptide is a first light chain variable region
  • the second polypeptide is a first heavy chain variable region
  • the third polypeptide is a second light chain variable region
  • the fourth poly The peptide is a second heavy chain variable region.
  • the second light chain heavy chain conjugate comprises SEQ ID NO: 9 A second light chain comprising a second light chain and SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 11, and a second heavy chain comprising SEQ ID NO: 12, a second light chain comprising SEQ ID NO: 37, and SEQ ID NO: 38 A second heavy chain, a second light chain comprising SEQ ID NO: 39 and a second heavy chain comprising SEQ ID NO: 40, or a second light chain comprising SEQ ID NO: 41 and a second heavy chain comprising SEQ ID NO: 42, Antibodies.
  • the second light chain heavy chain conjugate comprises any one selected from the group consisting of SEQ ID NO: 14, 15, 43, 44 and 45 That is, antibody.
  • An antibody-drug conjugate which is at least one selected from the group consisting of inhibitors, DNA intercalators, protein preparations, miRNAs, shRNAs, siRNAs, and radioisotopes.
  • the pharmaceutical composition of claim 14, wherein the cancer is any one selected from the group consisting of HER3-positive solid cancer and metastatic cancer.
  • the pharmaceutical composition of claim 14, wherein the cancer is any one selected from the group consisting of HER3-positive gastric cancer, breast cancer, lung cancer, esophageal cancer, thyroid, head and neck cancer, pancreatic cancer, and colon cancer.
  • the antibody of 14 above wherein the antibody is a toxin, a chemotherapeutic agent, an anticancer agent, an antibiotic, an ADP-ribosyl transferase, a nucleic acid degrading enzyme, a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase
  • a pharmaceutical composition which is combined with at least one selected from the group consisting of inhibitors, DNA intercalators, protein preparations, miRNAs, shRNAs, siRNAs, and radioisotopes.
  • the antibody according to the present invention can bind to HER3 with high specificity and high binding force while simultaneously binding to CD3 with high specificity and high binding force.
  • the antibody according to the present invention can effectively induce the death of cells expressing HER3.
  • the antibody according to the present invention can effectively inhibit the proliferation of cells expressing HER3.
  • the antibody according to the present invention can effectively induce the death of cancer cells.
  • the antibody according to the present invention can effectively inhibit the proliferation and metastasis of cancer cells.
  • Antibodies according to the invention can effectively induce the death of HER3-positive solid and metastatic cancers.
  • the antibodies according to the invention can effectively inhibit the proliferation and metastasis of HER3-positive solid and metastatic cancers.
  • the antibody according to the present invention can effectively induce the death of HER3-positive solid cancer and metastatic cancer cells resistant to anticancer agents.
  • the antibodies according to the present invention can effectively inhibit the proliferation and metastasis of HER3-positive solid cancers and metastatic cancers resistant to anticancer agents.
  • Antibodies according to the invention can exhibit a high level of cell killing efficacy specifically for HER3-positive cancer cells only in the presence of HER3-positive cancer cells.
  • Antibodies according to the invention may not cause side effects due to nonspecific cytotoxicity (eg, antibody-dependent cytotoxicity).
  • FIG. 3 shows the purification of HER3 / CD3 double target antibodies by size exclusion chromatography after expression.
  • FIG. 5 shows the relative expression levels of HER3 in cancer cell lines of SNU719, BT-20, T47D, MDA-MB-453.
  • FIG. 6 shows Y1103 / cOKT3-LALA, Y1103 / Hu38-LALA, Y1103 / A15-LALA, Y1103 / E15-LALA, prepared with an IgG form of chimeric OKT3 antibody, Hu38 antibody, A15, E15, A07 antibody and double antibody
  • Figure 7 shows the binding pattern of Y1103 / A07-LALA to the H9 cell line.
  • Fig. 7 is a schematic diagram showing the in vitro cell killing efficacy evaluation experiment of the T cell inducing antibody candidate according to Example 4.
  • Fig. 8 is a Y1103 according to Example 4 In vitro cell killing efficacy of / Hu38-LALA, Y1103 / A15-LALA, Y1103 / E15-LALA.
  • FIG. 9 shows the in vitro cell killing efficacy of Y1103 / Hu38-LALA and Y1103 / OKT3-LALA according to Example 4.
  • FIG. 9 shows the in vitro cell killing efficacy of Y1103 / Hu38-LALA and Y1103 / OKT3-LALA according to Example 4.
  • the present invention relates to a bispecific antibody that binds to HER3 and CD3, comprising: a first polypeptide of SEQ ID NO: 1; A second polypeptide of SEQ ID NO: 2; A third polypeptide selected from the group consisting of SEQ ID NOs: 3, 5, 31, 33, and 35; And a fourth polypeptide selected from the group consisting of SEQ ID NOs: 4, 6, 32, 34, and 36, and thus have excellent specific engaging effects on HER3-positive cells of immune cells,
  • the present invention relates to an antibody having excellent growth inhibitory and killing effects and that does not cause side effects due to nonspecific cytotoxicity.
  • Antibodies of the invention the first polypeptide of SEQ ID NO: 1; A second polypeptide of SEQ ID NO: 2; A third polypeptide selected from the group consisting of SEQ ID NOs: 3, 5, 31, 33, and 35; And a fourth polypeptide selected from the group consisting of SEQ ID NOs: 4, 6, 32, 34, and 36.
  • Antibodies of the invention can specifically bind to CD3 while binding specifically to HER3.
  • the antibody according to an embodiment of the present invention may include an antigen binding portion that specifically binds to HER3 and an antigen binding portion that specifically binds to CD3.
  • the first polypeptide and the second polypeptide can form an antigen binding site that specifically binds to HER3, and the third polypeptide and the fourth polypeptide form an antigen binding site that specifically binds to CD3. can do.
  • the first to fourth polypeptides of an antibody according to an embodiment of the present invention may be a light chain variable region or a heavy chain variable region of the antibody, respectively.
  • the first polypeptide may be a first light chain variable region
  • the second polypeptide may be a first heavy chain variable region
  • the third polypeptide may be a second light chain variable region
  • the fourth polypeptide may be a second heavy chain variable region.
  • the first polypeptide is a first heavy chain variable region
  • the second polypeptide is a first light chain variable region
  • the third polypeptide is a second light chain variable region
  • the fourth polypeptide is a second heavy chain variable region.
  • the first polypeptide is the first light chain variable region
  • the second polypeptide is the first heavy chain variable region
  • the third polypeptide is the second heavy chain variable region
  • the fourth polypeptide is the second light chain variable region.
  • the first polypeptide is a first heavy chain variable region
  • the second polypeptide is a first light chain variable region
  • the third polypeptide is a second heavy chain variable region
  • the fourth polypeptide is a second light chain variable region.
  • the first light chain variable region and the first heavy chain variable region may be included in separate polypeptide chains (eg, in the form of a general IgG), or may be included in one polypeptide chain. (Eg, in the form of a light chain heavy chain conjugate or a single chain antibody fragment (ScFv)).
  • the light chain heavy chain conjugate may include a light chain and a heavy chain in one polypeptide chain.
  • the light chain variable region and the heavy chain variable region may be included in one polypeptide chain.
  • the polypeptide chain including the first light chain variable region and the polypeptide chain including the first heavy chain variable region are disulfide bonds with each other. Or the like.
  • the first light chain variable region and the first heavy chain variable region are included in one polypeptide chain, the first light chain variable region and the first heavy chain variable region are directly linked, linked by a linker, or other additional polypeptide sequence. Can be connected by.
  • the second light chain variable region and the second heavy chain variable region may each be included in separate polypeptide chains (eg, in the form of a general IgG), or may be included in one polypeptide chain (eg, a light chain heavy chain conjugate). Or in the form of a single chain antibody fragment (ScFv).
  • the polypeptide chain including the second light chain variable region and the polypeptide chain including the second heavy chain variable region are disulfide bonds with each other. Or the like.
  • the second light chain variable region and the second heavy chain variable region are included in one polypeptide chain, the second light chain variable region and the second heavy chain variable region are directly linked, linked by a linker, or other additional polypeptide sequence. Can be connected by.
  • the antibody comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8; And a second light chain including SEQ ID NO: 9 and a second heavy chain including SEQ ID NO: 10.
  • the antibody comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8; And a second light chain including SEQ ID NO: 11 and a second heavy chain including SEQ ID NO: 12.
  • the antibody comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8; And a second light chain comprising SEQ ID NO: 37 and a second heavy chain comprising SEQ ID NO: 38.
  • the antibody comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8; And a second light chain comprising SEQ ID NO: 39 and a second heavy chain comprising SEQ ID NO: 40.
  • the antibody comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8; And a second light chain including SEQ ID NO: 41 and a second heavy chain including SEQ ID NO: 42.
  • the antibody according to one embodiment of the present invention may include a first light chain heavy conjugate and a second light chain heavy conjugate.
  • the first light chain heavy chain conjugate comprises a first polypeptide of SEQ ID NO: 1 and a second polypeptide of SEQ ID NO: 2, and the second light chain heavy chain conjugate is SEQ ID NO: 3, 5, 31, It may include a third polypeptide selected from the group consisting of 33, and 35 and a fourth polypeptide selected from the group consisting of SEQ ID NO: 4, 6, 32, 34 and 36.
  • Each of the first to fourth polypeptides may be a light chain variable region or a heavy chain variable region of the antibody.
  • the first polypeptide is a first light chain variable region
  • the second polypeptide is a first heavy chain variable region
  • the third polypeptide is a second light chain variable region
  • the fourth polypeptide is a second heavy chain. It may be a variable region.
  • the first light chain heavy chain conjugate comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8
  • the second light chain heavy chain conjugate comprises SEQ ID NO: 9 It may include a second light chain and a second heavy chain comprising SEQ ID NO: 10.
  • the first light chain heavy chain conjugate comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8
  • the second light chain heavy chain conjugate comprises SEQ ID NO: 11. It may include a second light chain and a second heavy chain comprising SEQ ID NO: 12.
  • the first light chain heavy chain conjugate comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8, and the second light chain heavy chain conjugate comprises SEQ ID NO: 37. It may include a second light chain and a second heavy chain comprising SEQ ID NO: 38.
  • the first light chain heavy chain conjugate comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8 and the second light chain heavy chain conjugate comprises SEQ ID NO: 39. It may include a second light chain and a second heavy chain comprising SEQ ID NO: 40.
  • the first light chain heavy chain conjugate comprises a first light chain comprising SEQ ID NO: 7 and a first heavy chain comprising SEQ ID NO: 8
  • the second light chain heavy chain conjugate comprises SEQ ID NO: 41. It may include a second light chain and a second heavy chain comprising SEQ ID NO: 42.
  • the first light chain heavy chain conjugate and the second light chain heavy chain conjugate may be bonded to each other by a disulfide bond or a knob-into-hole.
  • the first light chain heavy chain conjugate and the second light chain heavy chain conjugate may be coupled to each other by a knob-into-hole.
  • a knob When combined with a knob-into-hole, a knob may be formed in the first light chain heavy chain conjugate and a hole may be formed in the second light chain heavy chain conjugate.
  • a knob may be formed in the heavy chain (eg, the heavy chain constant region) of the first light chain heavy chain conjugate, and a hole may be formed in the heavy chain (eg, the heavy chain constant region) of the second light chain heavy chain conjugate.
  • the heterodimer of the first light chain conjugate and the second light chain conjugate is heterodimer formation efficiency can be improved.
  • the antibody according to an embodiment of the present invention may further include a light chain constant region and a heavy chain constant region in addition to the light chain variable region and the heavy chain variable region described above.
  • the antibody may include a first light chain variable region, a first light chain constant region, a first heavy chain variable region, and a first heavy chain constant region.
  • the first heavy chain constant region may be included in two or more (eg three).
  • the antibody may include a first light chain variable region, a first light chain constant region, a first heavy chain variable region, a first heavy chain constant region A, a first heavy chain constant region B, and a first heavy chain constant region C.
  • the antibody may include a second light chain variable region, a second light chain constant region, a second heavy chain variable region, and a second heavy chain constant region.
  • the second heavy chain constant region may be comprised of two or more (eg three).
  • the antibody may include a second light chain variable region, a second light chain constant region, a second heavy chain variable region, a second heavy chain constant region A, a second heavy chain constant region B, and a second heavy chain constant region C.
  • the first light chain variable region and the first heavy chain variable region may form a first antigen binding portion that specifically binds to HER3, and the second light chain variable region and the second heavy chain variable region specifically bind to CD3. It may be to form a two antigen binding portion.
  • the antibody according to the present invention can bind to HER3 with high specificity and high binding force and at the same time bind to CD3 with high specificity and high binding force, thereby expressing HER3 in immune cells (eg T cells, natural killer cells, etc.) expressing CD3.
  • immune cells eg T cells, natural killer cells, etc.
  • CD3 eg CD3
  • target eg cancer cells
  • the antibody may be one in which the LALA mutations (L243A, L245A) are present in the Fc portion.
  • Antibodies having a LALA mutation in the Fc portion according to an embodiment of the present invention do not exhibit antibody-dependent cell cytotoxicity (ADCC), thereby expressing or overexpressing HER3 (eg, cancer cells). ) Can only selectively exhibit cytotoxicity (eg apoptosis efficacy) against the cancer cells in question.
  • ADCC antibody-dependent cell cytotoxicity
  • the antibody according to the present invention can induce death, inhibit proliferation or inhibit metastasis of cells expressing or overexpressing HER3.
  • the antibodies according to the invention can induce the death of cancer cells, inhibit their proliferation or inhibit metastasis.
  • the cancer cells of the stomach may be any one of the cancer cells selected from the group consisting of HER3-positive solid cancer and metastatic cancer, such as breast cancer, lung cancer or osteosarcoma cells, for example metastatic cancer cells,
  • it may be cancer cells resistant to anticancer agents.
  • the anticancer agent of the stomach may be, for example, lapatinib.
  • the present invention provides an antibody-drug conjugate (ADC) to which the antibody and the drug are coupled according to the present invention.
  • the drug is a toxin, chemotherapy agent, anticancer agent, antibiotic, ADP-ribosyl transferase (ADP-ribosyl transferase), nuclease, microtubulin inhibitor, mitosis inhibitor, topoiso Merase inhibitors, DNA intercalators, protein preparations, miRNA, shRNA, siRNA, and at least one selected from the group consisting of radioisotopes, but is not limited thereto.
  • ADC antibody-drug conjugate
  • the drug may be maytansinoid, auristatin, dolastatin, tricortesene, CC1065 (cytotoxic compound), calicheamicin and other enedyne antibiotics, taxanes, anthracyclines, methotrexate, Adriamycin, vindesine, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, Carminomycin, aminopterin, dactinomycin, mitomycin, bleomycin, esperamicin, 5-fluorouracil, melphalan and other nitrogen mustards, stereoisomers, isomers, homologues or derivatives, cis Platinum and cis-platinum homologues, other intercalating enzymes and fragments thereof, such as nucleolytic enzymes
  • the radioisotope is 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I and At least one selected from the group consisting of 186 Re, but is not limited thereto.
  • the toxin is etoposide, teniposide, adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, cis-platinum and cis-platinum homologue, bleo At least one selected from mycins, esperamicins, 5-fluorouracil, melphalan, and other nitrogen mustards.
  • Antibodies according to the invention can be converted to chimeric antibodies, humanized antibodies and the like which have reduced immunogenicity for application to the human body.
  • the chimeric antibody may be obtained by recombining the light chain variable region of the present invention with a light chain constant region and a heavy chain constant region of a human antibody.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer or cancer metastasis, comprising the antibody of the present invention and the antibody-drug conjugate.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier In the case of oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. may be used. , Stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.
  • the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • the anticancer composition may typically include a surfactant that facilitates movement across the membrane.
  • surfactants are steroid derived or cationic lipids such as N- [1- (2,3-dioleoyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemisuccinate
  • DOTMA steroid derived or cationic lipids
  • the pharmaceutical composition of the present invention may be administered at 0.1 mg / kg (body) to 100 mg / kg (body).
  • it may be administered at 0.5 mg / kg (body) to 50 mg / kg (body), and also, for example, 1 mg / kg (body) to 10 mg / kg (body).
  • the present invention also provides a method of preventing or treating cancer or cancer metastasis by administering to a subject an effective amount of an antibody, an antibody-drug conjugate, and a pharmaceutical composition comprising the same.
  • the effective amount may vary depending on a variety of factors, including the type of cancer, the age, weight of the patient, the nature and extent of the symptoms, the type of current treatment, the number of treatments, the dosage form and route.
  • the antibodies, antibody-drug conjugates of the invention and pharmaceutical compositions comprising the same may be administered in combination (eg, simultaneously or sequentially) with other pharmacological or physiological components, such administration being single or multiple Administration.
  • an “individual” means a mammal (eg, a person suffering from or at risk of a condition or disease that can be alleviated, inhibited or treated by administering an antibody, an antibody-drug conjugate, and a pharmaceutical composition comprising the same according to the invention). Human).
  • administration means introducing a predetermined substance into an individual in any suitable manner, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, It may be, but is not limited to, intranasal administration, pulmonary administration, rectal administration.
  • the present invention also provides novel genes.
  • the gene of the present invention is any one selected from the group consisting of SEQ ID NOs: 16 to 30, and 46 to 60.
  • genes of SEQ ID NOs: 16 to 30 are nucleotide sequences corresponding to the amino acid sequences of SEQ ID NOs: 1 to 15, and the genes of SEQ ID NOs: 46 to 60 are nucleotide sequences corresponding to SEQ ID NOs: 31 to 45.
  • amino acid sequences may be included as components in an antibody, and the gene of the present invention is a gene encoding such components.
  • Expi293F cells are passaged at 2.0 ⁇ 10 6 cells / ml at 125 ⁇ 10 rpm in an 8% CO 2 shaking incubator at 37 ° C, using Expi293 media to meet the expected requirements.
  • the culture solution is centrifuged at 4000 rpm for 30 minutes, filtered through a 0.22 ⁇ m filter to remove cell debris to obtain supernatant.
  • the mobile phase was used with 20 mM sodium phosphate (pH 7.0) containing 200 mM sodium chloride and flowed at a flow rate of 1.0 ml / min. Based on the peak value of the AKTA system, a peak eluting after about 50 to 70 minutes is collected in 1 ml units. The elution fraction is identified by coomassie blue staining using 4-12% Bis-Tris PAGE. Based on the band pattern of the staining gel, fractions containing 150 kDa antibody were collected and concentrated using a 30kDa Amicon centrifugal filter unit.
  • the antibody was purified with protein A from the supernatant cultured by transfection of the HER3 target antibody expression vector and the CD3 target antibody expression vector to Expi293F, the sample was collected by size exclusion chromatography (SEC), and only 150 kDa protein samples were taken. It was. ( Figure 3)
  • the secondary antibody labeled with fluorescent dye which can specifically bind to the primary antibody, is treated with 0.2 ⁇ g of 100 ⁇ l FACS buffer, blocked for 30 minutes, and incubated at 4 ° C.
  • HER3 antigens were compared with those of SNU719, BT-20, T47D, MDA-MB-453 and 4 cancer cell lines, and the expression of HER3 was decreased in the order of BT-20 ⁇ SNU719 ⁇ T47D ⁇ MDA-MB-453. It was confirmed to be high. In addition, all four cells did not bind to the negative control antibody used as irrelevant control (FIG. 5).
  • H9 (ATCC, HTB-176 TM , T lymphoma) cell line was used to evaluate the binding of the antibody to CD3.
  • the secondary antibody labeled with fluorescent dye which can specifically bind to the primary antibody, is treated with 0.2 ⁇ g of 100 ⁇ l FACS buffer, blocked for 30 minutes, and incubated at 4 ° C.
  • Y1103 / A07-LALA it was confirmed that the degree of binding of the double antibody to CD3 was the highest in Y1103 / Hu38, and Y1103 / A15 and Y1103 / E15 were shown in the following order.
  • Y1103 / cOKT3 showed low binding force and Y1103 / A07 showed little binding force. ( Figure 6).
  • the cells were harvested with lx Trypsin-EDTA solution and then centrifuged for 5 minutes at 4 ° C. at 1200 rpm.
  • PBMC peripheral blood mononuclear cells
  • Each donor was suspended in cRPMI and adjusted to a concentration of 1 ⁇ 10 6 cells / ml.
  • Each antibody was diluted with cRPMI and then diluted 1/5 by 10 nM and treated with target cells plated wells one day before.
  • the supernatant for CBA assay was removed by 50 ⁇ l / well using a multichannel micropipette and transferred to a 96-well plate. And, 30 ⁇ l / well of CellTiter 96 ® AQueous One Solution was added using a multichannel micropipette. And, incubated for 1 to 2 hours in 37 °C CO 2 incubator. And it measured by the microplate reader (490nm).
  • T cell activation was analyzed using IL2-luc2P Jurkat cell line (Promega) for two HER3-expressing cancer cell lines.
  • Target cell line preparation MDA-MB-453 and T47D cells, HER3 expressing breast cancer cell lines, were plated with 100 ⁇ l Culture Medium (Culture Medium) at 3x10 4 cells per well in 96 well plates, respectively, at 37 ° C and 5% CO. 2 hours incubated in a humidified incubator (humidified incubator).
  • Effector cell line preparation Dissolve GloResponse TM Frozen Thaw and Use (FTU) IL-2-luc2P Jurkat Effector Cells in a 37 ° C water bath for 2 minutes and 4 ml pre-warmed Assay Medium in a 15 ml conical centrifuge tube. (RPMI medium containing 10% FBS) was added, and then 1 ml of the dissolved effector cell was added and mixed slowly.
  • FTU Dissolve GloResponse TM Frozen Thaw and Use
  • IL-2-luc2P Jurkat Effector Cells in a 37 ° C water bath for 2 minutes and 4 ml pre-warmed Assay Medium in a 15 ml conical centrifuge tube. (RPMI medium containing 10% FBS) was added, and then 1 ml of the dissolved effector cell was added and mixed slowly.
  • Antibodies were prepared by diluting 1/3 from 1 nM to make 3X doses 10 points. In addition, 25 ⁇ l each of 10 concentrations of antibodies prepared in a 96-well plate containing FTU IL2-Luc2P Jurkat cells prepared above was added to 1 ⁇ dose.

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Abstract

La présente invention concerne un anticorps bispécifique se liant à HER3 et CD3, l'anticorps comprenant un premier polypeptide de SEQ ID NO : 1, un deuxième polypeptide de SEQ ID NO : 2, un troisième polypeptide choisi dans le groupe constitué par SEQ ID NO : 3 et SEQ ID NO : 5, et un quatrième polypeptide choisi dans le groupe constitué par SEQ ID NO : 4 et SEQ ID NO : 6, et présentant d'excellents effets de mise en contact de cellules immunitaires spécifiquement avec des cellules HER3 positives, d'induction de la suppression de la croissance et d'induction de la mort des cellules HER3 positives, sans provoquer d'effets secondaires attribués à une cytotoxicité non spécifique.
PCT/KR2018/004743 2017-04-24 2018-04-24 Anticorps bispécifique se liant à her3 et cd3 Ceased WO2018199593A1 (fr)

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WO2020236797A1 (fr) * 2019-05-21 2020-11-26 Novartis Ag Domaines de cd58 variants et leurs utilisations
WO2021053587A1 (fr) 2019-09-18 2021-03-25 Klaus Strein Anticorps bispécifiques dirigés contre ceacam5 et cd3
US20220135679A1 (en) * 2019-04-08 2022-05-05 Green Cross Corporation Bispecific antibody specifically binding to gpnmb and cd3, and use thereof
WO2024129897A3 (fr) * 2022-12-13 2024-08-29 Bj Bioscience Inc. Anticorps anti-cd3 et anticorps bispécifiques
US12221481B2 (en) 2019-05-21 2025-02-11 Novartis Ag CD19 binding molecules and uses thereof

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KR102556049B1 (ko) 2021-09-02 2023-07-13 사회복지법인 삼성생명공익재단 신규 her3 억제제 및 이를 포함하는 암 예방 또는 치료용 약학 조성물

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KR20150029714A (ko) * 2012-07-13 2015-03-18 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 이중특이적 항체의 공-도입에 의한 ｃａｒ 세포의 활성 증강
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CN111315779A (zh) * 2017-10-20 2020-06-19 株式会社绿十字 抗-cd3抗体及包含其的用于癌症治疗的药物组合物
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US11498965B2 (en) 2017-10-20 2022-11-15 Green Cross Corporation Anti-CD3 antibody and pharmaceutical composition for cancer treatment comprising same
CN111315779B (zh) * 2017-10-20 2024-06-21 株式会社绿十字 抗-cd3抗体及包含其的用于癌症治疗的药物组合物
US20220135679A1 (en) * 2019-04-08 2022-05-05 Green Cross Corporation Bispecific antibody specifically binding to gpnmb and cd3, and use thereof
US12319746B2 (en) * 2019-04-08 2025-06-03 Green Cross Corporation Bispecific antibody specifically binding to GPNMB and CD3, and use thereof
WO2020236797A1 (fr) * 2019-05-21 2020-11-26 Novartis Ag Domaines de cd58 variants et leurs utilisations
US12037378B2 (en) 2019-05-21 2024-07-16 Novartis Ag Variant CD58 domains and uses thereof
US12221481B2 (en) 2019-05-21 2025-02-11 Novartis Ag CD19 binding molecules and uses thereof
WO2021053587A1 (fr) 2019-09-18 2021-03-25 Klaus Strein Anticorps bispécifiques dirigés contre ceacam5 et cd3
US12441807B2 (en) 2019-09-18 2025-10-14 Lamkap Bio Alpha AG Bispecific antibodies against CEACAM5 and CD3
WO2024129897A3 (fr) * 2022-12-13 2024-08-29 Bj Bioscience Inc. Anticorps anti-cd3 et anticorps bispécifiques

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