WO2022245186A1 - Conjugué anticorps-médicament qui se lie à ror1 et b7-h3, et utilisation de celui-ci - Google Patents

Conjugué anticorps-médicament qui se lie à ror1 et b7-h3, et utilisation de celui-ci Download PDF

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WO2022245186A1
WO2022245186A1 PCT/KR2022/007260 KR2022007260W WO2022245186A1 WO 2022245186 A1 WO2022245186 A1 WO 2022245186A1 KR 2022007260 W KR2022007260 W KR 2022007260W WO 2022245186 A1 WO2022245186 A1 WO 2022245186A1
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amino acid
conjugate
antibody
seq
acid sequence
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Korean (ko)
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박윤희
송호영
류현민
정철웅
김주희
권정아
이보라
유병민
이수연
정진원
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주식회사 레고켐바이오사이언스
에이비엘바이오 주식회사
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Publication of WO2022245186A1 publication Critical patent/WO2022245186A1/fr

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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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Definitions

  • the present invention relates to an antibody-drug conjugate that specifically binds to ROR1 and B7-H3 and uses thereof, and more particularly, to an antibody-drug comprising a bispecific antibody that specifically binds to ROR1 and B7-H3 It relates to conjugates and their uses.
  • ROR1 is expressed during embryonic and fetal development and regulates cell polarity, cell migration, and neurite outgrowth. As overexpression of ROR1 was observed in various cancer cells, it was classified as an oncofetal gene. In particular, it has been found that ROR1 is overexpressed in chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • MCL mantle cell lymphoma
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • follicular Blood cancers such as lymphoma (FL) and marginal zone lymphoma (MZL) as well as breast, kidney, ovarian, gastric, liver, lung, colorectal, pancreatic, skin, bladder, testicular, cervical, prostate, and non-small cell lung cancer ( NSCLC), neuroblastoma, brain cancer, colon cancer, epithelial squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.
  • Expression of ROR1 in such cancers is associated with poor prognosis of cancer patients and is known to affect cancer metastasis.
  • B7-H3 (CD276) is a member of the B7 family and is a transmembrane protein that includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the two extracellular regions of B7-H3 consist of a single pair (2Ig B7-H3) or two identical pairs (4Ig B7-H3) of immunoglobulin variable domains and immunoglobulin constant domains due to exon duplication.
  • the B7-H3 protein is not always expressed in T cells, natural killer cells (NK cells) and antigen presenting cells (APCs) in normal tissues, its expression can be induced.
  • B7-1 and B7-2 are mainly confined to immune cells such as antigen-presenting cells
  • the B7-H3 protein is expressed in osteoblasts, fibroblasts, fibroblast-like synovial cells and epithelial cells as well as human liver and lung.
  • bladder, testis, prostate, breast, placenta and lymphatic organs are also expressed. This broad expression pattern suggests more diverse immunological and non-immunological functions of B7-H3, particularly in peripheral tissues.
  • B7-H3 expression has been reported in non-small cell lung cancer, renal cell carcinoma, neuroblastoma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, prostate cancer, endometrial cancer, hepatocellular carcinoma, breast cancer, cervical cancer, osteosarcoma, oral cancer, bladder cancer, glioma, It is confirmed in various solid cancers such as melanoma, and it is also reported to be expressed in hematological cancers such as acute leukemia, multiple myeloma, and various types of lymphoma (Zhimeng Yea, Zhuojun Zhengb et al, Cell Physiol Biochem (2016), Elodie Picarda, Kim C Ohaegbulam and Xingxing Zang, clinical cancer research (2016), Wei Zhang, Yagging Wang, Jing Wang et al,international journal of oncology (2015)).
  • ADCs generally have a disadvantage of low in vivo stability compared to natural antibodies, but were developed to improve the low therapeutic effect, which is a disadvantage of natural antibodies, through combination with drugs.
  • Drugs having specific medicinal effects, such as cytotoxin are being developed in various combinations with target-specific antibodies, and antibody-drug conjugates capable of inducing cancer cell death by binding drugs to cancer cell-specific antibodies have been commercialized.
  • An object of the present invention is to provide an antibody-drug conjugate in which a bispecific antibody or an antigen-binding fragment thereof specifically binding to ROR1 and B7-H3 proteins is linked to a drug.
  • Another object of the present invention is to provide a composition for preventing or treating cancer comprising the antibody-drug conjugate.
  • the present invention provides a conjugate having the structure of general formula I:
  • Ab is a dual specific antibody or antigen-binding fragment thereof that specifically binds to ROR1 and B7-H3 proteins;
  • X is independently a chemical moiety comprising one or more active agents and a linker
  • the linker connects the antibody and the active agent
  • Said y is an integer from 1 to 20.
  • the present invention provides a composition for preventing or treating cancer comprising the conjugate.
  • the antibody-drug conjugate according to the present invention includes a bispecific antibody or an antigen-binding fragment thereof that simultaneously binds to ROR1 and B7-H3, and is superior to a single antibody that binds to ROR1 and B7-H3, respectively, in ROR1 or B7-H3 overexpressing cells. It exhibits cell binding ability and exhibits enhanced antibody-derived cell cytotoxicity (ADCC).
  • ADCC antibody-derived cell cytotoxicity
  • the antibody-drug conjugate according to the present invention is more stable in plasma and circulated in the body, and a linker containing a self-killing group capable of maximizing drug efficacy by easily releasing drugs and/or toxins in target cells Including technology to effectively exert a drug and/or toxin on a target cell.
  • each anti-ROR1 monoclonal antibody specifically binds to the extracellular domain ROR1 antigen.
  • BCMA-Fc is a negative control, and each anti-ROR1 monoclonal antibody specifically binds only to the ROR1 antigen and does not bind to BCMA protein or Fc used as a tag.
  • Figure 2 is a result of measuring the binding ability of an anti-ROR1 monoclonal phage antibody to the cell surface-expressed ROR1 antigen (FACS) according to an embodiment of the present invention, and JeKo-1 cell line was used as cells expressing ROR1 on the cell surface. did Each anti-ROR1 monoclonal antibody specifically binds to ROR1 expressed on the cell surface.
  • Figures 3a and 3b are the binding ability assay (ELISA) results for the human ROR1 antigen of the anti-ROR1 IgG antibody prepared according to one embodiment of the present invention. It is shown that each antibody binds to the human ROR1 antigen in a concentration-dependent manner. These results indicate that the monoclonal phage antibody retains its binding ability to ROR1 even after being changed to an IgG form.
  • ELISA binding ability assay
  • FIG. 4 is a result of an assay (ELISA) of anti-ROR1 IgG antibodies prepared in accordance with one embodiment of the present invention for binding ability to the mouse ROR1 antigen. It is shown that each antibody binds to the mouse ROR1 antigen in a concentration-dependent manner. Through this experiment, it was confirmed that the anti-ROR1 antibody of the present invention has cross-reactivity to mouse ROR1.
  • ELISA assay
  • Figure 5 is a result of measuring the binding capacity (FACS) of the cell surface expressed ROR1 antigen of the anti-ROR1 antibody prepared according to one embodiment of the present invention
  • CHO-human ROR1 cell line is human ROR1
  • CHO-human ROR2 is human ROR2
  • CHO-mouse ROR1 is a cell line artificially overexpressing mouse ROR1. It was shown that each antibody specifically binds to human ROR1 expressed on the cell surface and does not bind to human ROR2, a family protein.
  • the anti-ROR1 antibody of the present invention has cross-species reactivity to mouse ROR1 by confirming that it binds to a cell line artificially overexpressing mouse ROR1.
  • Figure 6 is a result of measuring the binding capacity (FACS) of the anti-ROR1 antibody prepared on the cell surface ROR1 antigen prepared according to one embodiment of the present invention, JeKo-1 and Mino cell lines as ROR1 expression-positive cell lines, and MCF7 as ROR1-negative cell lines. cell line was used. It was shown that each antibody specifically binds to ROR1 expressed on the cell surface and does not bind to MCF7, a cell line that does not express ROR1.
  • FACS binding capacity
  • FIG. 7 is a result of measuring the binding ability of an anti-ROR1 antibody prepared according to an embodiment of the present invention to the ROR1 antigen expressed on the cell surface (FACS).
  • the MC38 human ROR1 cell line in which human ROR1 was artificially overexpressed in MC38, a mouse colorectal cancer cell line, was used. Each antibody was shown to bind to the cell line overexpressing human ROR1 in a concentration-dependent manner.
  • FIG. 8 is a result of measuring the binding ability of an anti-ROR1 antibody prepared according to an embodiment of the present invention to a cell-expressed ROR1 antigen in various cancer cell lines (FACS).
  • FIG. 9 shows the results of ELISA analysis of the anti-B7-H3 antibody prepared according to one embodiment of the present application to the extra cellular domain (ECD) of the B7-H3 protein.
  • the anti-B7-H3 antibody of the present invention was found to bind to the extracellular domain of human B7-H3 protein in a concentration-dependent manner.
  • ECD binding ability assay (ELISA) result of other proteins belonging to the B7 family of the anti-B7-H3 antibody prepared according to one embodiment of the present application. Each antibody was found to specifically recognize only the B7-H3 protein without binding to other proteins belonging to the B7 family.
  • FIG. 11 is a result of analyzing the cross-species reactivity of the anti-B7-H3 antibody prepared according to one embodiment of the present application by ELISA. Each antibody was shown to bind to monkey (cynomolgus) B7-H3 and mouse B7-H3 in a concentration-dependent manner.
  • FIG. 12 is a result of comparing the degree of binding ability of various anti-B7-H3 antibodies prepared according to one embodiment of the present application to the mouse B7-H3 protein by ELISA. Although the degree of binding of the antibodies according to the present disclosure to mouse B7-H3 is different, all of them were shown to bind to mouse B7-H3 protein in a concentration-dependent manner.
  • FIG. 13 is a result of measuring the binding ability of an anti-B7-H3 antibody prepared according to one embodiment of the present application to the cell surface expressed B7-H3 antigen (FACS). It was shown that the anti-B7-H3 antibodies of the present application specifically bind only to MCF-7, a cell line that overexpresses B7-H3, and do not bind to Jurkat, a cell line that does not express B7-H3.
  • FIG. 14 is a result of measuring (FACS) the binding ability of an anti-B7-H3 antibody prepared according to one embodiment of the present application to the B7-H3 antigen expressed on the cell surface by antibody concentration.
  • Each antibody was shown to bind to cancer cell lines expressing B7-H3 in a concentration-dependent manner.
  • 16a is a graph showing the cell binding ability of a single antibody and a double antibody to a cell line overexpressing B7-H3 and ROR1 (CHO-huROR1-huB7H3) by FACS.
  • FIG. 16B is a graph showing the cell binding ability of a single antibody and a double antibody in a cell line overexpressing ROR1 (CHO-huROR1) measured by FACS.
  • Figure 16c is a graph showing the cell binding ability of a single antibody and a double antibody in a cell line overexpressing B7-H3 (MC38-huB7H3) by FACS.
  • 17a is a graph showing ADCC of single antibody and double antibody in a cell line overexpressing B7-H3 and ROR1 (CHO-huROR1-huB7H3).
  • Figure 17b is a graph of ADCC measurements of single antibody and double antibody in a cell line overexpressing B7-H3 and ROR1 (CHO-huROR1-huB7H3).
  • the present invention provides conjugates having the structure of general formula I:
  • Ab is a dual specific antibody or antigen-binding fragment thereof that specifically binds to ROR1 and B7-H3 proteins;
  • X is independently a chemical moiety comprising one or more active agents and a linker
  • the linker connects the antibody and the active agent
  • Said y is an integer from 1 to 20.
  • the bispecific antibody or antigen-binding fragment thereof specifically binding to the ROR1 and B7-H3 proteins has a first domain specifically binding to the ROR1 protein and a specific binding to the B7-H3 protein. and a second domain that binds antagonistically.
  • the present invention provides conjugates having the structure of general formula I:
  • Ab is a dual specific antibody or antigen-binding fragment thereof that specifically binds to ROR1 and B7-H3 proteins;
  • X is independently a chemical moiety comprising one or more active agents and a linker
  • the linker connects the antibody and the active agent
  • Said y is an integer from 1 to 20.
  • the bispecific antibody or antigen-binding fragment thereof specifically binding to the ROR1 and B7-H3 proteins comprises a first domain specifically binding to the ROR1 protein and a B7-H3 protein. and a second domain to which it binds.
  • the first domain may include a heavy chain variable region including the following heavy chain CDRs and a light chain variable region including the light chain CDRs:
  • HCDR1 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3
  • HCDR2 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7
  • a heavy chain variable region comprising HCDR3 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10;
  • LCDR1 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 13
  • LCDR2 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 16
  • a light chain variable region comprising LCDR3 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 19.
  • the first domain may include a heavy chain variable region including the following heavy chain CDRs and a light chain variable region including the light chain CDRs:
  • HCDR1 represented by the amino acid sequence of SEQ ID NO: 1, HCDR2 represented by the amino acid sequence of SEQ ID NO: 4, HCDR3 represented by the amino acid sequence of SEQ ID NO: 8; HCDR1 represented by the amino acid sequence of SEQ ID NO: 2, HCDR2 represented by the amino acid sequence of SEQ ID NO: 5, HCDR3 represented by the amino acid sequence of SEQ ID NO: 9; HCDR1 represented by the amino acid sequence of SEQ ID NO: 2, HCDR2 represented by the amino acid sequence of SEQ ID NO: 6, and HCDR3 represented by the amino acid sequence of SEQ ID NO: 9; or a heavy chain variable region comprising HCDR1 represented by the amino acid sequence of SEQ ID NO: 3, HCDR2 represented by the amino acid sequence of SEQ ID NO: 7, and HCDR3 represented by the amino acid sequence of SEQ ID NO: 10; and
  • LCDR2 represented by the amino acid sequence of SEQ ID NO: 11 LCDR2 represented by the amino acid sequence of SEQ ID NO: 14, LCDR3 represented by the amino acid sequence of SEQ ID NO: 17; LCDR2 represented by the amino acid sequence of SEQ ID NO: 12, LCDR2 represented by the amino acid sequence of SEQ ID NO: 15, LCDR3 represented by the amino acid sequence of SEQ ID NO: 18; Or a light chain variable region comprising LCDR2 represented by the amino acid sequence of SEQ ID NO: 13, LCDR2 represented by the amino acid sequence of SEQ ID NO: 16, and LCDR3 represented by the amino acid sequence of SEQ ID NO: 19.
  • the second domain may include a heavy chain variable region including the following heavy chain CDRs and a light chain variable region including light chain CDRs:
  • HCDR1 represented by the amino acid sequence of SEQ ID NO: 57
  • HCDR2 represented by the amino acid sequence of SEQ ID NO: 58
  • a heavy chain variable region comprising HCDR3 represented by the amino acid sequence of SEQ ID NO: 59
  • HCDR3 represented by the amino acid sequence of SEQ ID NO: 59
  • LCDR1 represented by the amino acid sequence of SEQ ID NO: 60
  • LCDR2 represented by the amino acid sequence of SEQ ID NO: 61
  • a light chain variable region comprising LCDR3 represented by the amino acid sequence of SEQ ID NO: 62.
  • the first domain may include the following heavy chain variable region and light chain variable region:
  • a heavy chain variable region comprising an amino acid sequence having 90% or more sequence homology with an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 23, 37, 42, 47, and 52, and
  • a light chain variable region comprising an amino acid sequence having 90% or more sequence homology with an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 26, 38, 43, 48, and 53.
  • the first domain may include the following heavy chain variable region and light chain variable region:
  • the second domain may include the following heavy chain variable region and light chain variable region:
  • a heavy chain variable region comprising an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 63 or 83, and
  • It may include a light chain variable region comprising an amino acid sequence having 90% or more sequence homology with the amino acid sequence of SEQ ID NO: 64 or 84.
  • the second domain may include the following heavy chain variable region and light chain variable region:
  • linker refers to a compound that covalently binds an active agent to a ligand.
  • Linkers described herein may be cleavable, non-cleavable, hydrophilic or hydrophobic.
  • a cleavable linker is cleavable under intracellular conditions such that cleavage of the linker releases the active agent from the antibody construct-active agent conjugate in the intracellular environment.
  • a cleavable linker is cleavable by a cleavage agent present in the intracellular environment (eg, in lysosomes or endosomes or caveolea).
  • a cleavable linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to lysosomal or endosomal proteases.
  • the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length.
  • Cleaving agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
  • Most common are peptidyl linkers cleavable by enzymes present in antigen expressing cells.
  • peptidyl linkers cleavable by the thiol-dependent protease cathepsin-B which is highly expressed in cancer tissue, can be used (eg, Phe-Leu or Gly-Phe-Leu-Gly linkers).
  • Other such linkers are described, for example, in US Patent No. 6,214,345.
  • the peptidyl linker cleavable by an intracellular protease is, for example, a Val-Cit linker, a Phe-Lys linker (eg, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker) see) or a Val-Ala linker.
  • the Val-Cit linker or the Val-Ala linker may contain a pentafluorophenyl group and may contain a succinimide group or maleimide group.
  • it may contain a PABA group and a pentafluorophenyl group, may contain a 4-aminobenzoic acid (PABA) group and a maleimide group, and may contain a PABA group and a succinimide group.
  • PABA 4-aminobenzoic acid
  • intracellularly cleaved and intracellular cleavage refer to a metabolic process or reaction inside a cell for an antibody construct-activator conjugate, whereby the active agent ( B) and the covalent attachment between the antibody construct (Ab), eg the linker, is broken resulting in free drug or other metabolites of the conjugate separated from the intracellular antibody.
  • cleavable linkers are pH-sensitive, i.e., they can be easily hydrolyzed at certain pH values.
  • the pH-sensitive linker can be hydrolyzed in acidic conditions.
  • acid-labile linkers that can be hydrolyzed in the lysosome (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconic amides (cis -aconitic amide), orthoesters, acetals, ketals, etc.) can be used (eg, US Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm.
  • linkers are relatively stable at neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes.
  • hydrolysable linkers include thioether linkers, such as thioethers attached to a therapeutic agent via an acylhydrazone linkage (see, eg, US Pat. No. 5,622,929).
  • the linker is cleavable under reducing conditions (eg, a disulfide linker).
  • a disulfide linker For example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl- 3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-thio)toluene)-, formed using SPDB and SMPT
  • SATA N-succinimidyl-5-acetylthioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPDB N-succinimidyl- 3-(2-pyridyldi
  • the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299- 1304), 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12), beta-glucuronide ( ⁇ -Glucuronide) linker (Jeffery et al ., 2006, Bioconjug Chem. 17(3):832-40), or a beta-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376-382 ) can be.
  • a malonate linker Johnson et al., 1995, Anticancer Res. 15: 1387-93
  • a maleimidobenzoyl linker Liau et al., 1995, Bioorg-Med
  • the non-cleavable linker may be a maleimidocaproyl linker.
  • the maleimidocaproyl linker may include N-maleimidomethylcyclohexane-1-carboxylate.
  • the maleimidocaproyl linker may contain a succinimide group.
  • Maleimidocaproyl linkers may contain pentafluorophenyl groups.
  • the linker can be a combination of a maleimide group and one or more polyethylene glycol molecules.
  • the linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules.
  • the linker may be a maleimide-PEG4 linker.
  • the linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules.
  • the linker may be a combination of a pentafluorophenyl group and a maleimidocaproyl linker containing one or more polyethylene glycol molecules.
  • the linker may contain a maleimide linked to a polyethylene glycol molecule, where the polyethylene glycol allows for more linker flexibility or allows longer linkers to be used.
  • the linker may be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker.
  • the linker may be a cleavable linker.
  • the linker is a protease cleavable linker, an acid-cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, It may be a maleimidobenzoyl linker, a 3'-N-amide analogue, a ⁇ -Glucuronide linker, or a ⁇ -galactoside linker.
  • the protease cleavable linker may include a thiolreactive spacer or a dipeptide, and more specifically, the protease cleavable linker is a thiolreactive maleimidocaproyl spacer, valine-citrulline dipeptides or p-amino-benzyloxycarbonyl spacers.
  • the linker may have a structure of Formula II.
  • G is a glucuronic acid moiety or , wherein R 3 is hydrogen or a carboxyl protecting group, and each R 4 is independently hydrogen or a hydroxyl protecting group;
  • R 1 and R 2 are each independently hydrogen, C 1-8 alkyl or C 3-8 cycloalkyl
  • W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO 2 NR'-, -P(O)R''NR'-, -SONR'- or -PO 2 NR'-, wherein C, S or P is directly bonded to a phenyl ring, and R' and R'' are each independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono- or di- C 1-8 alkylamino, C 3-20 heteroaryl or C 6-20 aryl;
  • Z is independently C 1-8 alkyl, halogen, cyano or nitro
  • n is an integer from 0 to 3;
  • L is one or more units selected from the group consisting of a first unit and a second unit, or a combination of these units;
  • the second unit connects W and R z , W and the first unit, or the first unit and the first unit, wherein the first unit connects the second unit and W, or the second unit and Connecting another second unit,
  • the second unit , , , , -(CH 2 CH 2 V) t -, -(CH 2 ) t (V(CH 2 ) u ) v -, or a combination thereof;
  • L 1 is a single bond or C 2-30 alkenyl
  • R 11 is H or C 1-10 alkyl
  • L 2 is C 2-30 alkenyl
  • t is an integer from 0 to 10
  • u is an integer from 0 to 12
  • v is an integer from 1 to 20
  • V is a single bond, -O-, or -S-,
  • the carbon atoms of the alkenyl may be substituted with one or more heteroatoms selected from the group consisting of N, O and S, and alkenyl may be one or more It may be further substituted with more than C 1-20 alkyl,
  • R v and R w are each independently H, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono- or di-C 1-8 alkyl amino, C 3-20 heteroaryl or C 5-20 aryl;
  • n1, n2, n3 and n4 are each independently an integer from 0 to 10;
  • Rz is -O-NH 2 , -NH 2 , N 3 , substituted or unsubstituted C 1-12 alkyl, C 1-12 alkynyl, C 1-3 alkoxy, substituted or unsubstituted 3 to 20-membered hetero aryl, 3 to 20 membered heterocyclyl, substituted or unsubstituted C 5-20 aryl, or ego,
  • R z ′ is N or CH
  • one or more hydrogen atoms are each independently OH, ⁇ O, halo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, oxy, carboxy, C 1-6 alkoxycarbonyl , C 1-6 alkylcarbonyl, formyl, C 3-8 aryl, C 5-12 aryloxy, C 5-12 arylcarbonyl or C 3-6 heteroaryl.
  • R z may bind to an Ab, an amino acid motif or a prenylated amino acid motif.
  • the structure may be maintained, eliminated or modified.
  • the linker may have a structure of the following general formula IIa:
  • G is sugar, sugar acid, or sugar derivatives
  • W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O) 2 NR'-, -P(O)R''NR'-, -S (O) NR'-, or -PO 2 NR'-,
  • R' and R′′ are each independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1- 8 alkoxy, C 1-8 alkylthio, mono- or di-C 1-8 alkylamino, C 3-20 heteroaryl, or C 6-20 aryl;
  • each Z is independently hydrogen, C 1-8 alkyl, halogen, cyano or nitro;
  • n is an integer from 0 to 3;
  • n 0 or 1
  • Rz is -O-NH 2 , -NH 2 , N 3 , substituted or unsubstituted C 1-12 alkyl, C 1-12 alkynyl, C 1-3 alkoxy, substituted or unsubstituted 3 to 20-membered hetero aryl, 3 to 20 membered heterocyclyl, substituted or unsubstituted C 5-20 aryl, or ego,
  • R z ′ is N or CH
  • one or more hydrogen atoms are each independently OH, ⁇ O, halo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, oxy, carboxy, C 1-6 alkoxycarbonyl , C 1-6 alkylcarbonyl, formyl, C 3-8 aryl, C 5-12 aryloxy, C 5-12 arylcarbonyl or C 3-6 heteroaryl;
  • R 1 and R 2 are each independently hydrogen, C 1-8 alkyl or C 3-8 cycloalkyl, or
  • R 1 and R 2 together with the carbon atoms to which they are attached form a C 3-8 cycloalkyl ring;
  • * indicates a site connected to an active agent.
  • the sugar or sugar acid is a monosaccharide.
  • G is a glucuronic acid moiety or a compound having the structure of Formula I:
  • R 3 is hydrogen or a carboxyl protecting group
  • Each R 4 is independently hydrogen or a hydroxyl protecting group.
  • said R 3 is hydrogen and each R 4 is hydrogen.
  • each of R 1 and R 2 is hydrogen.
  • each Z is independently C 1-8 alkyl, halogen, cyano or nitro.
  • the W is -C (O) -, -C (O) NR '- or -C (O) O-, more specifically, the W is -C (O) NR'-, where C ( O) is connected to the phenyl ring, and NR' is bonded to L.
  • n 0, 1, 2 or 3, more specifically 0, 1 or 2, more specifically 0.
  • G is a compound having the structure of Formula I:
  • R 3 is hydrogen or a carboxyl protecting group
  • each R 4 is independently hydrogen or a hydroxyl protecting group
  • W is -C(O)NR'-, wherein C(O) is linked to a phenyl ring, NR' is linked to L, each Z is C 1-8 alkyl, halogen, cyano or nitro; n is 0, m is 1, and R 1 and R 2 are each hydrogen.
  • At least one first unit is an alkylene having from 1 to 100 carbon atoms, wherein the carbon atoms of the alkylene are one or more selected from the group consisting of N, O and S Heteroatoms may be substituted, and alkylenes may be further substituted with one or more alkyls having 1 to 20 carbon atoms.
  • At least one first unit is a C 1-50 alkylene or 1-50 membered heteroalkylene, and may satisfy one or more of the following:
  • the first unit is a 1 to 50 membered heteroalkylene
  • the alkylene is substituted with one or more C 1-20 alkyl.
  • the at least one first unit is a nitrogen-containing 1-50 membered heteroalkylene
  • the linker includes two or more atoms of a hydrophilic amino acid, and the nitrogen may form a peptide bond with a carbonyl of the hydrophilic amino acid.
  • the first unit is C 2-50 alkylene, C 2-50 heteroalkylene, hydrophilic amino acid, -C(O)-, -C(O)NR′′-, -C(O )O-, -(CH 2 ) s -NHC(O)-(CH 2 ) t -, -(CH 2 ) u -C(O)NH-(CH 2 ) v -, -(CH 2 ) s - NHC(O)-(CH 2 ) t -C(O)-, -(CH 2 ) u -C(O)NH-(CH 2 ) v -C(O)-, -S(O) 2 NR''-,-P(O)R'''NR''-,-S(O)NR''-, or -PO 2 NR''-,
  • R′′ and R′′′′ are each independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono- or di-C 1 -8 alkylamino, C 3-20 heteroaryl or C 5-20 aryl, and s, t, u and v are each independently an integer from 0 to 10.
  • At least one first unit is a hydrophilic amino acid.
  • the hydrophilic amino acid can be arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.
  • a hydrophilic amino acid may be an amino acid comprising a side chain having residues that carry a charge at neutral pH in aqueous solution.
  • the hydrophilic amino acid is aspartate or glutamate.
  • the hydrophilic amino acid is ornithine or lysine.
  • the hydrophilic amino acid is arginine.
  • the at least one first unit is -C(O)-, -C(O)NR′′-, -C(O)O-, -S(O) 2 NR′′-, -P(O)R'''NR''-, -S(O)NR''-, or -PO 2 NR''-, and R'' and R'' are each independently hydrogen, C 1 -8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1-8 alkylthio, mono- or di-C 1-8 alkylamino, C 3-20 heteroaryl, or C 6-20 aryl. .
  • the at least one first unit is -C(O)NR′′-, -(CH 2 ) s -NHC(O)-(CH 2 ) t -, -(CH 2 ) u - C(O)NH-(CH 2 ) v -, -(CH 2 ) s -NHC(O)-(CH 2 ) t -C(O)-, or -(CH 2 ) u -C(O)NH -(CH 2 ) v -C(O)-, where R′′ is hydrogen, C 1-5 alkyl, C 3-8 cycloalkyl, C 1-5 alkoxy, C 3-20 heteroaryl, or C 6-20 aryl, and s, tu and v are each independently an integer from 0 to 10.
  • the at least one first unit is -C(O)NR'- and R' is hydrogen.
  • the at least one second unit is represented by Formula VIII or Formula IX:
  • V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO 2 - or -SO 2 NR 25 - ego;
  • X is -O-, C 1 -C 8 alkylene or -NR 21 -;
  • R 21 to R 25 are each independently hydrogen, C 1-6 alkyl, C 1-6 alkyl C 6-20 aryl or C 1-6 alkyl C 3-20 heteroaryl;
  • r is an integer from 0 to 10;
  • p is an integer from 0 to 10;
  • q is an integer from 1 to 20;
  • w is an integer from 1 to 20;
  • q may be 4 to 20, more specifically 6 to 20.
  • q may be 2 to 12, more specifically 2, 5, or 11.
  • r may be 2.
  • p may be 2.
  • V may be -O-.
  • r is 2
  • p is 2
  • q is 2, 5 or 11
  • V may be -O-.
  • X may be -O-.
  • w may be an integer of 6 to 20.
  • X is -O-, and w may be 6 to 20.
  • the at least one second unit is at least one polyethylene glycol unit, or It has the structure of where n is 1 to 12.
  • the at least one second unit comprises 1 to 12 -OCH 2 CH 2 -units, or 3 to 12 -OCH 2 CH 2 -units, or 5 to 12 -OCH 2 CH 2 -units, or 6 to 12 -OCH 2 CH 2 -units, or 3 -OCH 2 CH 2 -units.
  • the at least one second unit is -(CH 2 CH 2 X) w -;
  • X is a single bond, -O-, C 1-8 alkylene, or -NR 21 -;
  • R 21 is hydrogen, C 1-6 alkyl, C 1-6 alkyl-C 6-20 aryl, or C 1-6 alkyl-C 3-20 heteroaryl, and w is an integer from 1 to 20, specifically 1, 3, 6, or 12.
  • X is -O-, and w is an integer from 6 to 20.
  • the linker is 1,3-dipolar cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, A third formed by non-aldol type carbonyl reactions, addition to carbon-carbon multiple bond, oxidation reactions or click reactions Additional units may be included.
  • the linker may bind to the bispecific antibody or antigen-binding fragment thereof by a third unit.
  • the third unit is formed by a reaction between an alkynyl group and an azide, or between an aldehyde or ketone group and a hydrazine or alkoxyamine.
  • R z is -O-NH 2 , -NH 2 , N 3 , -OCH 3 , -OCH 2 CH 3 , -O(CH 2 ) 2 CH 3 , or can be
  • Rz is includes
  • a third unit may be formed by a reaction between alkynyl and azide, in detail, a click reaction.
  • Rz is Including, forming a third unit through the structure, the third unit includes
  • the third unit in one aspect of the present invention, the third unit,
  • L 1 is a single bond or an alkylene having 1 to 30 carbon atoms
  • R 11 is hydrogen or alkyl having 1 to 10 carbon atoms, specifically methyl
  • L 2 is an alkylene having 1 to 30 carbon atoms.
  • L 1 is a single bond, or an alkylene having 11 carbon atoms, or an alkylene having 12 carbon atoms.
  • the third unit in one aspect of the present invention, the third unit
  • V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO 2 -, or -SO 2 NR 25 -is;
  • R 21 to R 25 are independently hydrogen, C 1-6 alkyl, C 1-6 alkyl C 6-20 aryl, or C 1-6 alkyl C 3-20 heteroaryl;
  • r is an integer from 1 to 10;
  • p is an integer from 0 to 10;
  • q is an integer from 1 to 20;
  • L 1 is a single bond.
  • r may be 2 or 3. Also, p may be 1 or 2. Also, q may be 1 to 6.
  • r is 2 or 3; p is 1 or 2; q may be 1 to 6.
  • the linker further comprising the third unit
  • tilde indicates a site linked to Ab
  • * sign indicates a site linked to an active agent
  • n is an integer from 0 to 20.
  • the third unit comprises
  • Ab is It may further contain at least one isoprenyl unit having the structure of wherein n is at least 2 or more.
  • At least one isoprenyl unit is a substrate of an isoprenoid transferase or a product of an isoprenoid transferase.
  • the isoprenyl unit of Ab is covalently bonded to Ab via a thioether bond, and the thioether bond includes a sulfur atom of a cysteine of Ab.
  • Ab comprises an amino acid motif recognized by an isoprenoid transferase and the thioether linkage comprises a sulfur atom of a cysteine of the amino acid motif.
  • Ab comprises an amino acid motif recognized by an isoprenoid transferase and the thioether linkage comprises a sulfur atom of a cysteine of the amino acid motif.
  • the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y independently represents an aliphatic amino acid; X independently at each occurrence represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine;
  • the thioether linkage includes the sulfur atom of the cysteine of the amino acid motif.
  • the amino acid motif is a CYYX sequence
  • Y is independently in each case alanine, isoleucine, leucine, methionine, or valine.
  • the amino acid motif is a CVIM or CVLL sequence.
  • one or more of the 1 to 10 amino acids preceding the amino acid motif may each independently be selected from glycine, arginine, aspartic acid and serine.
  • at least one of the seven amino acids preceding the amino acid motif is glycine.
  • 3 or more of the 7 amino acids preceding the amino acid motif are each independently selected from glycine, arginine, aspartic acid and serine.
  • from 1 to 10 amino acids preceding the amino acid motif are glycine, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif are glycine.
  • Ab may comprise the amino acid sequence of GGGGGGGCVIM.
  • L comprises one or more branched linkers covalently bondable to Ab
  • each branched linker comprises a first unit or a third unit covalently connectable to the Ab by a primary linker (PL);
  • each branched linker connects the first active agent to the first unit and comprises a first branch (B1) comprising a second linker (SL) and a cleavage group (CG); and
  • each branched linker comprises a) a second branch (B2) wherein the second active agent is covalently linked to the first unit by a second linker (SL) and a cleavage group (CG); or b) a second branch wherein a polyethylene glycol moiety is covalently linked to the first unit;
  • Each of the cleavage groups may be hydrolyzed to release the active agent from the antigen specific binding moiety-active agent conjugate.
  • the main linker (PL) may include a terminal group that can be directly bonded to Ab or a partial structure of Ab.
  • the branched linker is , , or ego,
  • L 2 , L 3 , L 4 are each independently a direct bond or -C n H 2n -, wherein n is an integer from 1 to 30;
  • G 1 , G 2 , G 3 are independently directly bonded; , , or is,
  • R 30 is hydrogen or C 1-30 alkyl
  • R 40 is a direct bond or C 1-10 alkyl.
  • branched linker is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • B and B' represent active agents which may be the same or different
  • one of B and B' is an active agent and the other is a toxin;
  • n each independently represents an integer from 0 to 30;
  • f each independently represents an integer from 0 to 30;
  • L represents a terminal group capable of binding to Ab.
  • n is an integer from 1 to 20, more specifically an integer from 1 to 10, or an integer from 4 to 20.
  • L comprises an oxime and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.
  • the cleavage group can be cleaved within a target cell and can release one or more active agents.
  • it comprises at least one branched linker capable of covalently attaching to the Ab; and two or more active agents covalently linked to a branched linker.
  • one branched linker may bind to Ab.
  • branched linkers may bind to Ab, and each branched linker may bind to two or more active agents. More specifically, it may be that three branched linkers are combined with Ab. Alternatively, 4 branched linkers may be combined with Ab.
  • each branched linker may be associated with two or more of the same or different active agents.
  • each active agent may be linked to the branched linker by a cleavable bond.
  • each branched linker comprises a branched unit, and each active agent binds to the branched unit through a secondary linker, and can be bound to an Ab by the branched unit primary linker.
  • the branched unit may be a nitrogen atom.
  • the branched unit may be an amide, and the primary linker or secondary linker may include a carbonyl of the amide.
  • the branched unit may be a lysine unit.
  • the linker In another aspect of the present invention, the linker,
  • the second unit connects the fourth unit and the first unit
  • the one or more fourth units are capable of releasing one or more active agents
  • the first unit connects the second unit and the fourth unit, or the second unit to another second unit.
  • the linker may further contain a third unit.
  • the fourth unit has the structure of general formula IIb:
  • G is sugar, sugar acid, or sugar derivatives
  • W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O) 2 NR'-, -P(O)R''NR'-, -S (O) NR'-, or -PO 2 NR'-;
  • R' and R′′ are each independently hydrogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkoxy, C 1 -8 alkylthio, mono- or di-C 1-8 alkylamino, C 3-20 heteroaryl, or C 6-20 aryl, W is linked to the second unit or the first unit;
  • each Z is independently hydrogen, C 1-8 alkyl, halogen, cyano or nitro;
  • n is an integer from 1 to 3;
  • n 0 or 1
  • R 1 and R 2 are each independently hydrogen, C 1-8 alkyl or C 3-8 cycloalkyl, or
  • R 1 and R 2 together with the carbon atoms to which they are attached form a C 3-8 cycloalkyl ring.
  • the sugar or sugar acid is a monosaccharide.
  • G is a compound of structure I
  • R 3 is hydrogen or a carboxyl protecting group
  • Each R 4 is independently hydrogen or a hydroxyl protecting group.
  • R 3 is hydrogen and each R 4 is hydrogen.
  • W is -C(O)NR'-, wherein C(O) is linked to the phenyl ring and NR' is linked to the second unit.
  • Z is hydrogen
  • R 1 and R 2 are each hydrogen.
  • the second unit is -(CH 2 ) r (V(CH 2 ) p ) q -, -((CH 2 ) p V) q -, -(CH 2 ) r (V(CH 2 ) p ) q Y-, -((CH 2 ) p V) q (CH 2 ) r -, -Y((CH 2 ) p V) q - or -(CH 2 ) r (V(CH 2 ) p ) denoted by q YCH 2 -,
  • r is an integer from 0 to 10;
  • p is an integer from 1 to 10;
  • q is an integer from 1 to 20;
  • V and Y are each independently a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO 2 -, or - SO 2 NR 25 -,
  • R 21 to R 25 are each independently hydrogen, C 1-6 alkyl, C 1-6 alkyl-C 6-20 aryl or C 1-6 alkyl-C 3-20 heteroaryl.
  • r is 2.
  • p is 2.
  • q is an integer from 6 to 20.
  • q is 2, 5, or 11.
  • V and Y are each independently -O-.
  • the branched linker is
  • L 1 , L 2 , and L 3 are each independently a direct bond or -C n H 2n -,
  • n is an integer from 1 to 30,
  • G 1 , G 2 , G 3 are each independently directly bonded; , , or ego,
  • R 3 is hydrogen or C 1-30 alkyl
  • R 4 is hydrogen or -L 4 -COOR 5 , wherein L 4 is a direct bond or -C n H 2n - where n is an integer from 1 to 10, and R 5 is hydrogen or C 1-30 is an alkyl
  • the third unit comprises
  • L 1 is a direct bond or an alkylene having 1 to 30 carbon atoms
  • R 11 is hydrogen or alkyl having 1 to 10 carbon atoms, specifically methyl
  • L 2 is an alkylene having 1 to 30 carbon atoms
  • the third unit connects the second unit and Ab.
  • L 1 is an alkylene having 12 carbon atoms.
  • R 11 is methyl
  • L 2 is an alkylene having 11 carbon atoms.
  • the third unit comprises or to be.
  • the third unit is covalently bonded to Ab via a thioether bond, and the thioether bond includes a sulfur atom of a cysteine of Ab.
  • Ab comprises an amino acid motif recognized by an isoprenoid transferase and the thioether linkage comprises a sulfur atom of a cysteine of the amino acid motif.
  • the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y independently represents an aliphatic amino acid; X independently at each occurrence represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine;
  • the thioether linkage includes the sulfur atom of the cysteine of the amino acid motif.
  • the amino acid motif is a CYYX sequence
  • Y is independently in each case alanine, isoleucine, leucine, methionine, or valine.
  • the amino acid motif is a CVIM or CVLL sequence.
  • At least one of the seven amino acids preceding the amino acid motif is glycine.
  • 3 or more of the 7 amino acids preceding the amino acid motif may each be independently selected from glycine, arginine, aspartic acid and serine.
  • 1 to 10 amino acids preceding the amino acid motif are glycine, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif is glycine.
  • Ab may comprise the amino acid sequence of GGGGGGGCVIM.
  • L comprises one or more branched linkers covalently bondable to Ab
  • each branched linker comprises a fifth unit covalently connectable to the Ab by a first linker (PL);
  • each branched linker comprises a first branch (B1) wherein the first active agent is covalently linked to the fifth unit by a second linker (SL) and a cleavage group (CG); and
  • each branched linker comprises a) a second branch (B2) wherein the second active agent is covalently linked to the fifth unit by a second linker (SL) and a cleavage group (CG); or b) a second branch (B2) having a polyethylene glycol moiety covalently bound to the fifth unit;
  • Each of the above cleavage groups may be hydrolyzed to release the active agent from the antibody construct-active agent conjugate.
  • the first linker (PL) includes an end group that can be directly bonded to Ab or a partial structure of Ab.
  • the branched linker comprises a fifth unit (BR) covalently linked to the reactive moiety by a first linker (PL);
  • the fifth unit comprises a first active agent covalently linked to the first branch (B1) and covalently linked to the second linker (SL) and the cleavage group (CG);
  • the fifth unit is covalently bonded to the second branch (B2) and comprises a) a second active agent covalently bonded to the second linker (SL) and the cleavage group (CG) or b) a polyethylene glycol moiety, wherein In each cleavage group can be hydrolyzed to release the active agent.
  • the cleavage group is as shown in Formula II above.
  • the second linker (eg, connecting the active agent to the fifth unit) is a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction (hetero -Diels-Alder reaction, nucleophilic substitution reaction, non-aldol type carbonyl reaction, addition to a carbon-carbon multiple bond ), a first unit or a third unit formed by an oxidation reaction or a click reaction.
  • the first unit or the third unit may be formed by a reaction of acetylene with an azide, or a non-aldol type carbonyl reaction, such as a reaction of an aldehyde or ketone group with a hydrazine or alkoxyamine, as described above, This allows for mild coupling of the first unit with the active agent and/or cleavage group.
  • the fifth unit is configured to:
  • L 2 , L 3 , L 4 are each independently a direct bond or -C n H 2n -, wherein n is an integer from 1 to 30;
  • G 1 , G 2 , G 3 are independently directly bonded; , , or is,
  • R 30 is hydrogen or C 1-30 alkyl
  • R 40 is hydrogen, C 1-10 alkyl or -L 5 -COOR 50 , wherein L 5 is a direct bond or -C n H 2n -;
  • n is an integer from 1 to 10
  • R 50 is hydrogen or C 1-30 alkyl.
  • the branched linker comprising the fifth unit
  • B and B' represent active agents which may be the same or different
  • one of B and B' is an active agent and the other is a toxin;
  • n each independently represents an integer from 0 to 30;
  • f each independently represents an integer from 0 to 30;
  • L represents a terminal group capable of binding to Ab.
  • L is a linker comprising at least one fifth unit and a first linker (PL).
  • the first linker of the antibody-drug conjugate comprises an alkylene having 1 to 100 carbon atoms, preferably 1 to 50 carbon atoms, or:
  • Alkylene contains at least one unsaturated bond
  • alkylene includes at least one heteroarylene
  • the carbon atoms of the alkylene are replaced by one or more heteroatoms selected from nitrogen (N), oxygen (O), and sulfur (S); or
  • Alkylene is further substituted with one or more alkyls having from 1 to 20 carbon atoms.
  • the branched linker of the antibody-active agent comprises an amino acid having a side chain with a moiety that carries a charge at neutral pH in aqueous solution, preferably arginine, aspartate, glutamate, lysine or ornithine.
  • Amino acids may be located anywhere on the branched linker.
  • the oxime of the branched linker can be covalently linked to the polyethylene glycol unit of the branched linker.
  • these amino acids may be present in the second linker, optionally within each second linker.
  • the branched linker comprising the fifth unit
  • the active agents are each independently selected from chemotherapeutic agents, immunomodulatory agents and toxins.
  • the active agent may be an immune modulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof, and may be selectively used among the active agents listed below:
  • affinity ligand is a substrate, inhibitor, activator, neurotransmitter, radioisotope, or combination thereof;
  • radioactive label 32P, 35S, fluorescent dye, electron dense reagent, enzyme, biotin, streptavidin, dioxigenin, hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination thereof;
  • kinase inhibitors growth factor inhibitors (eg EGFR, PDGF or VEGF inhibitors), calcineurin inhibitors, CRAC inhibitors, PARP1 antagonists, PPAR ⁇ agonists, Kv1.3 antagonist, PP2A agonist, MYD88 inhibitor, BCL-2 inhibitor, Adenosine A2A receptor (A2ar) agonist, TLR agonist, TLR7/8 agonist, TLR4 agonist, TLR9 agonist, Kca3.1 (calcium-activated potassium channel) agonist, TGF-R1 inhibitor, TGF-R2 inhibitor, GLI1 inhibitor, TNKS antagonist, TNIK antagonist, imide, vitamin D receptor (VDR) antagonist, STING agonist, and indoleamine 2,3-dioxygenase1 (IDO1) Any one or more immunomodulatory compounds selected from the group consisting of inhibitors, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents anti-fungal agents, and anti-para
  • the active agent in one aspect of the invention, is selected from:
  • y is an integer from 1 to 10.
  • a pyrrolobenzodiazepine dimer precursor is provided as the active agent.
  • a pyrrolobenzodiazepine dimer precursor is provided as the active agent.
  • it is necessary to convert into an effective drug by an additional reaction when exposed to blood, so the possibility of side effects that may occur when the linker is unexpectedly decomposed can be prevented in advance, It has advantages over existing PBD drugs in that toxicity to normal cells is reduced and the drug is more stable.
  • X or a precursor thereof is
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the conjugate described above.
  • the present invention also provides the use of the conjugate described above for use as a pharmaceutical composition for the prevention or treatment of cancer.
  • the present invention also provides a method for preventing or treating cancer in a subject having cancer, comprising administering to the subject an effective amount of a conjugate for treating cancer.
  • one or more therapeutic co-agents may be additionally included.
  • a pharmaceutically acceptable excipient may be additionally included.
  • the therapeutic co-agent is an agent that exhibits a preventive, ameliorative, or therapeutic effect on cancer, or an agent that can reduce the expression of side effects that occur when administering a cancer therapeutic agent, or an agent that exhibits an immunity-enhancing effect It may be, but is not limited to, a therapeutically useful effect when applied in the form of a compounding agent together with an active agent, further improving the stability of the active agent, and/or reducing side effects that may occur during administration of the active agent / Or, it means that any agent that shows the effect of maximizing the therapeutic effect through the enhancement of immunity can be applied in combination.
  • the cancer is blood cancer, lung cancer, non-small cell lung cancer, gastrointestinal cancer, colorectal cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, skin cancer, bladder cancer, pancreatic cancer, neuroblastoma, epithelial squamous cell carcinoma, thyroid cancer, head and neck cancer, adrenal cancer, oral cancer, myeloma, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma; All possible types of cancer can be applied.
  • conjugates refers to a cell binding agent that is covalently bound to one or more molecules of a cytotoxic compound.
  • “cell binding agent” is a molecule having affinity for a biological target, and may be, for example, a ligand, a protein, an antibody, specifically a monoclonal antibody, a protein or antibody fragment, a peptide, an oligonucleotide, an oligosaccharide, In other words, binding agents function to direct biologically active compounds to biological targets.
  • conjugates can be designed to target cancer cells via cell surface antigens.
  • the antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type.
  • the target antigen may be expressed only on proliferating cells (eg, cancer cells).
  • Target antigens can usually be selected based on differential expression between proliferative and normal tissues.
  • antibody refers to an anti-ROR1 antibody that specifically binds to each of ROR1 and B7-H3 proteins, an anti-B7-H3 antibody, or a bispecific that binds to both ROR1 and B7-H3 proteins.
  • Antibodies bispecific antibodies.
  • the scope of the present invention includes complete antibody forms that specifically bind to ROR1 and B7-H3 proteins, respectively, as well as portions of complete antibodies, antigen-binding fragments of the antibody molecules, and combinations thereof.
  • a complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by disulfide bonds.
  • the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence for imparting specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof.
  • the term “light chain” refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen. I mean everything.
  • the whole antibody includes subtypes of IgA, IgD, IgE, IgM and IgG, and in particular, IgG includes IgG1, IgG2, IgG3 and IgG4.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types, and subclasses include gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • An antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like.
  • Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site.
  • Fab' is different from Fab in that it has a hinge-region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab')2 is produced when a cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • Double-chain Fv two-chain Fv
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • the regions are covalently linked or directly linked at the C-terminus, so that a dimer-like structure can be achieved, such as in double-chain Fv.
  • antibody fragments can be prepared using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of intact antibodies with papain, and F(ab')2 can be obtained by digestion with pepsin), or by genetic recombination technology. can be made using .
  • proteolytic enzymes for example, Fab can be obtained by restriction digestion of intact antibodies with papain, and F(ab')2 can be obtained by digestion with pepsin
  • genetic recombination technology can be made using .
  • the antibody of the present invention is monoclonal antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, scFv, Fab fragment, F(ab')2 fragment, disulfide-linked Fvs ( sdFv) and anti-idiotypic (anti-Id) antibodies, epitope-binding fragments of the antibodies, portions of the antibodies, or combinations thereof.
  • the heavy chain constant region may be selected from any one isotype of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4).
  • Light chain constant regions can be of the kappa or lambda type.
  • variable region of an antibody used in the present invention refers to the light and heavy chain portions of an antibody molecule that include the amino acid sequences of complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3) and frameworks (FR).
  • CDRs complementarity determining regions
  • FR frameworks
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • CDR complementarity determining region
  • a “framework (FR)” is a variable domain residue other than a CDR residue.
  • Each variable domain typically has four FRs, FR1, FR2, FR3 and FR4.
  • a scFv is an antibody fragment, a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody.
  • a polypeptide linker may further be included between the VH and VL domains to allow the scFv to form a desired structure for antigen binding.
  • the VH and VL domains may be linked through a linker.
  • the linker may be a peptide linker and may have a length of about 10-25 aa.
  • hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.
  • the linker may include, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker For example, it may be (G n S) m (n and m are each 1 to 10).
  • the antibody or antibody fragment of the present invention may include not only the sequences of the antibodies described herein, but also biological equivalents thereof to the extent that each of the ROR1 and B7-H3 proteins can be specifically recognized.
  • additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties.
  • modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody.
  • amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
  • the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences described in SEQ ID NOs.
  • the above substantial identity is at least 90% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to sequences exhibiting homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, 99% or more homology. Alignment methods for sequence comparison are known in the art.
  • an antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% as compared to the specified sequence described herein or entirely. , 99%, or greater homology.
  • homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.
  • a bispecific antibody refers to an antibody having the ability to bind or antagonize more than one target, wherein an antibody having the ability to bind or antagonize two different targets is combined, or an antibody having the ability to bind to one target and another target It means an antibody to which a substance having antagonistic activity against is bound.
  • a multispecific antibody refers to an antibody that has binding specificities for at least three different antigens.
  • a multi-specific antibody is a tri-specific or higher antibody, for example, a tri-specific antibody, a tetra-specific antibody, or one that targets a higher target. may contain antibodies.
  • Antibodies belonging to bispecific or multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. Since bispecific or multispecific antibodies can simultaneously inhibit or amplify two or more signals, they are likely to be more effective than the case of inhibiting/amplifying one signal. In addition, compared to the case where each signal is treated with each signal inhibitor, low-dose administration is possible, and theoretically, two or more signals can be suppressed / amplified in the same time and space. However, it is not easy to predict whether the above theoretical effects will actually occur when a single antibody is produced as a double antibody, whether there will be no unexpected side effects, and whether synergistic or adverse effects will appear compared to the combination of single antibodies before actually manufacturing it. .
  • bispecific or multispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy/light chain pairs, under conditions in which the two or more heavy chains have different specificities.
  • the hybrid scFv can be prepared in a heterodimeric form by combining the VL and VH of different scFvs, respectively, to form a diabody, and the different scFvs can be linked together
  • tendem ScFvs can be produced
  • heterodimeric miniantibodies can be prepared by expressing CH1 and CL of Fab at the ends of each scFv, and some amino acids in the CH3 domain, which is the homodimeric domain of Fc, can be substituted to form a 'knob'.
  • a heterodimeric scFv type minibody can be prepared by changing into a heterodimeric structure in the 'into hole' type and expressing these modified CH3 domains at different ends of each scFv.
  • individual Fabs for a specific antigen can be combined with each other using a disulfide bond or a mediator to produce a heterodimeric Fab, and the heavy or light chain of a specific Fab can be combined with each other. It can be prepared to have two antigen valencies by expressing scFv for different antigens at the terminal, or to have four antigen valencies in a homodimeric form by placing a hinge region between the Fab and scFv.
  • scFvs for different antigens to the light and heavy chain ends of Fab
  • a dual-targeting bibody with three binding valences to the antigen and by fusing different scFvs to the light chain ends and heavy chain ends of Fab, respectively, bind to the antigen. It can be obtained by chemically conjugating three different Fabs, a triple-targeted bibody with three binding valencies.
  • hybrid hybridomas also known as quadromas
  • quadromas hybrid hybridomas
  • a method for producing a bispecific antibody by using a method is known.
  • bispecific antibodies can be prepared in the so-called 'Hole and Knob' type, which is produced in a heterodimeric form by modifying some amino acids of the CH3 homodimeric domain of Fc for different heavy chains while sharing the light chain portion.
  • scFvs can be fused to constant domains instead of the light and heavy chain variable domains of IgG, respectively, to produce homodimeric (scFv)4-IgG.
  • a wide variety of recombinant antibody formats can be developed, e.g. bispecific or multispecific antibodies that are bivalent, trivalent or tetravalent or higher.
  • a bivalent, trivalent, or tetravalent antibody indicates that two or more binding domains, three or more binding domains, or four or more binding domains, respectively, are present in the antibody molecule.
  • the bispecific antibody according to the present invention may comprise an IgG complete antibody or a fragment thereof in the form of a single chain Fv, VH domain and/or VL domain, Fab or (Fab)2.
  • therapeutic agent is an agent that exerts cytotoxic, cytostatic and/or immunomodulatory effects on cancer cells or activated immune cells.
  • therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.
  • a “chemotherapeutic agent” herein is a chemical compound useful in the treatment of cancer.
  • subject is intended to include humans and non-human animals, particularly mammals.
  • An example of a subject includes a human subject, such as a concept that includes a normal subject or a human patient having a disorder described herein, more specifically cancer.
  • Non-human animal refers to all vertebrates, eg, non-mammals (eg, chickens, amphibians, reptiles) and mammals, eg, non-human primates, livestock and/or useful for agriculture. animals (eg sheep, dogs, cats, cows, pigs, etc.) and rodents (eg mice, rats, hamsters, guinea pigs, etc.).
  • the subject is a human patient.
  • Treatment refers to both therapeutic treatment and prophylactic or prophylactic measures.
  • Those in need of treatment include those already with the disease, and those prone to have the disease or those in which the disease is to be prevented.
  • the term when used in reference to a disease or subject in need of treatment, the term includes arresting or slowing the progression of a disease, preventing symptoms, reducing the severity of a disease and/or symptoms, or reducing the duration of a disease, compared to an untreated subject. However, it is not limited to this.
  • administration refers to providing, contacting, and/or delivering a compound or compounds by any suitable route to achieve a desired effect.
  • Administration can be oral, sublingual, parenteral (eg intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, administration via buccal, rectal, vaginal, intranasal, ophthalmic, inhalation and implantation.
  • unsubstituted or substituted refers to a parent group that may be unsubstituted or may be substituted
  • substituted refers to a parent group having one or more substituents
  • the substituent refers to a parent group. group) or a chemical moiety fused to a parent group.
  • halo refers to fluorine, chlorine, bromine, iodine, and the like.
  • alkyl is a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, completely unsaturated) hydrocarbon compound
  • saturated alkyl include methyl, ethyl, propyl, butyl , pentyl, hexyl, heptyl, etc.
  • saturated straight-chain alkyl include methyl, ethyl, n-propyl, n-butyl, n-pentyl (amyl), n-hexyl, n-heptyl, etc.
  • saturated branched-chain alkyl may include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl and the like.
  • alkoxy means -OR [wherein R is an alkyl group], and examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy; and the like.
  • aryl means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom.
  • alkenyl is an alkyl having one or more carbon-carbon double bonds
  • alkynyl is an alkyl group having at least one carbon-carbon triple bond
  • examples of unsaturated alkynyl groups include ethynyl and 2-propynyl.
  • aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound.
  • C 5-7 aryl means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein the moiety has 5 to 7 ring atoms
  • C 5-7 aryl"" 10 aryl means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein the moiety has 5 to 10 ring atoms.
  • the prefix refers to a number of ring atoms or a range of ring atoms, whether carbon atoms or heteroatoms.
  • C 5-6 aryl relates to an aryl group having 5 or 6 ring atoms.
  • the ring atoms may all be carbon atoms as in the "carboaryl group".
  • Examples of carboaryl groups include, but are not limited to, those derived from benzene, naphthalene, azulene, anthracene, phenanthrene, naphthacene and pyrene.
  • aryl groups comprising fused rings in which at least one is an aromatic ring include groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene and aceantrene, but Not limited.
  • the ring atoms may include one or more heteroatoms as in "heteroaryl group".
  • heteroaryl is an aryl containing one or more heteroatoms, examples of which include pyridine, pyrimidine, benzothiophene, furyl, dioxalanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, and pyrimidyl.
  • Nil et al. more specifically benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, Benzodioxole, benzofuran, benzotriazole, benzothiofuran, benzothiazole, C 9 having two fused rings derived from benzothiadiazole, chromene, isochromene, chromane, isochromane, benzo Two fused rings derived from dioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, pteridine C 10 , C 11 having two fuse
  • cycloalkyl is an alkyl group which is a cyclyl group, and relates to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon compound.
  • cycloalkyl groups include, but are not limited to, those derived from:
  • unsaturated monocyclic hydrocarbon compounds cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene and methylcyclohexene; and
  • Saturated heterocyclic hydrocarbon compounds norcarane, norfinan, norbornane.
  • heterocyclyl relates to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.
  • a prefix eg, C 1-12 , C 3-8 , etc.
  • C 3-6 heterocyclyl herein refers to a heterocyclyl group having 3 to 6 ring atoms.
  • monocyclic heterocyclyl groups include, but are not limited to, those derived from:
  • N 1 aziridine, azetidine, pyrrolidine, pyrroline, 2H- or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine;
  • N 2 imidazolidine, pyrazolidine, imidazoline, pyrazoline, piperazine;
  • O 1 oxirane, oxetane, oxolane, oxol, oxane, dihydropyran, pyran, oxepin;
  • O 2 dioxolane, dioxane and dioxepane
  • N 1 O 1 tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine
  • N 1 S 1 thiazoline, thiazolidine, thiomorpholine;
  • N 1 O 1 S 1 oxathiazine.
  • prodrug refers to pyrrolobenzodiazepine by the action of an enzyme or gastric acid under physiological conditions in vivo (eg, enzymatic oxidation, reduction, and/or hydrolysis, etc.). A compound that can be directly or indirectly converted into a zepine drug.
  • an acid addition salt formed by a pharmaceutically acceptable free acid may be used, and an organic acid or an inorganic acid may be used as the free acid.
  • the organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid.
  • the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.
  • a salt can be formed with an appropriate cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ and other cations such as Al 3+ .
  • suitable organic cations include, but are not limited to, ammonium ions (ie, NH 4 + ) and substituted ammonium ions (eg, NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine , phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine.
  • An example of a typical quaternary ammonium ion is N(CH 3 ) 4 + .
  • a compound When a compound is cationic or has a functional group that can be cationic (eg -NH 2 can be -NH 3 + ), it can form a salt with an appropriate anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid, and the like.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetioxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethane Disulfonic acid, ethanesulfonic acid, fumaric acid, glutethonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, Malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, palmic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succ
  • solvate refers to a molecular complex between the compound according to the present invention and solvent molecules, and examples of solvates include water, isopropanol, ethanol, methanol, dimethyl sulfoxide (dimethylsulfoxide), ethyl acetate, acetic acid, ethanolamine, or a mixed solvent thereof, and the compound according to the present invention combined, but is not limited thereto.
  • solvate is used herein in its conventional sense to refer to a complex of a solute (eg, an active compound, a salt of an active compound) and a solvent.
  • a solute eg, an active compound, a salt of an active compound
  • the solvent is water
  • the solvate may conveniently be referred to as a hydrate, such as a monohydrate, dihydrate, trihydrate, and the like.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include macromolecules that are usually slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like. Acceptable carriers can be appropriately selected and used by those skilled in the art.
  • composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in one or more compounds. mixed and prepared In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. have.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • injectable esters such as ethyl oleate
  • witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used as a base for the suppository.
  • the pharmaceutical composition is selected from the group consisting of injections, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. It can have any one formulation that is.
  • the active ingredient may be in the form of an aqueous solution that is pyrogen-free and has an acceptable pH, isotonicity and stability for parenteral administration.
  • aqueous solution that is pyrogen-free and has an acceptable pH, isotonicity and stability for parenteral administration.
  • isotonic vehicles such as, for example, aqueous sodium chloride solution, Ringer's solution, lactated Ringer's solution, and the like, and preservatives, stabilizers, buffers, antioxidants, or other additives may be included as desired.
  • Solid forms suitable for injection can also be prepared as emulsions or in the form of the polypeptide encapsulated in liposomes.
  • the phrase "effective amount” or “therapeutically effective amount” refers to the amount necessary (relative to dosage and duration and means of administration) to achieve the desired therapeutic result.
  • An effective amount is at least the smallest amount of active agent necessary to confer a therapeutic benefit to a subject and is less than a toxic amount.
  • dosages may range from about 100 ng to about 100 mg/kg per patient, more typically from about 1 ⁇ g/kg to about 10 mg/kg.
  • the active compound is a salt, ester, amide, prodrug or the like, the dosage is calculated based on the parent compound, so the actual weight used is proportionally increased.
  • the pyrrolobenzodiazepine compound according to the present invention may be formulated to contain 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of the active ingredient per dosage form, but is not limited thereto.
  • the active ingredient can be administered to obtain a peak plasma concentration of active compound of about 0.05 ⁇ M to 100 ⁇ M, 1 ⁇ M to 50 ⁇ M, or 5 ⁇ M to 30 ⁇ M. eg by intravenous injection of a 0.1 w/v% to 5 w/v% solution of the active ingredient, optionally in saline.
  • the concentration of active compound in a pharmaceutical composition can be determined by absorption, inactivation and excretion rates of the drug and other factors known to those skilled in the art.
  • the dosage may vary depending on the severity of the symptom/disease.
  • the dosage and administration regimen for a specific patient can be adjusted according to the professional judgment of the administration supervisor in comprehensive consideration of the patient's severity of symptoms/disease, necessity, age, reactivity to drugs, etc., and the concentrations suggested in the present invention
  • the ranges are exemplary only and are not intended to limit the embodiments of the claimed compositions thereto.
  • the active ingredient may be administered once, or smaller doses may be administered in several divided doses.
  • ROR1 ECD-Fc type protein with Fc linked to the C-terminus of the extracellular region (ECD) of human ROR1 was used.
  • residues corresponding to amino acids 1 to 406 of the ROR1 amino acid sequence represented by NCBI reference number NP_0050032 were specifically used as a protein containing the extracellular domain of ROR1.
  • cDNA from Origene was purchased and used (Origene, RC214967).
  • human IgG1 was added to the 3' end of the gene encoding the extracellular domain of ROR1.
  • a gene encoding the derived Fc protein was synthesized and ligated (hereinafter referred to as 'ROR1-Fc'), and the gene was introduced into pcDNA3.1 vector to obtain a vector encoding ROR1-Fc nucleic acid in a mammalian cell line.
  • HEK 293E cells were transiently transfected with the expression vector and cultured in DMEM/F-12 medium at 8% CO 2 at 37°C to express ROR1-Fc, and the medium was collected every 72 hours. Combined and purified the Fc-ROR1 ECD protein using protein A affinity chromatography.
  • E. coli 2 ⁇ 10 10 containing human-derived scFv (single chain variable fragment) library (Yang et al, 2009 Mol Cells 27:225) genes with binding diversity to various antigens was 2X YT (Amresco, J902-500G), Ka After incubation at 37°C for 2 to 3 hours in a medium containing 100 ⁇ g/ml of benicillin (Duchefa, C01090025) and 2% glucose (sigma, G7021) (OD600 0.5-0.7) helper phage was infected and cultured in 2X YT [2X YT, carbenicillin, kanamycin (Duchfa, K0126) 70 ⁇ g/ml, 1 mM IPTG (Duchefa, I1401)] medium at 30° C.
  • 2X YT Amresco, J902-500G
  • the cultured cells were centrifuged (6000 rpm, 15 minutes, 4°C), and 4% PEG8000 (Sigma, P2139) and 3% NaCl (Samchun, S2097) were added to the supernatant to dissolve well, and then incubated on ice for 1 hour. reacted After centrifugation (8000 rpm, 20 minutes, 4°C) again, PBS (Phosphate buffered saline, Gibco 10010-023) was added to the pellet to suspend it, followed by centrifugation (12000 rpm, 10 minutes, 4°C) to contain library phage. The supernatant to be put into a new tube and stored at 4 °C until use.
  • PBS Phosphate buffered saline, Gibco 10010-023
  • ROR1-Fc at a concentration of 10 ⁇ g/ml and negative control-Fc were added to PBS in an immunotube (immunotube, maxisorp 444202), and proteins were adsorbed on the surface of the test tube overnight at 4 ° C.
  • Bovine serum albumin (BSA, Bovine serum albumin) 3% solution was added to the test tube to protect the surface to which ROR1-Fc was not adsorbed.
  • 10 12 CFU antibody phage library dispersed in a 3% BSA solution was placed in an immunotest tube adsorbed with a control Fc protein and allowed to react at room temperature for 1 hour (negative selection).
  • phages that did not bind to the negative control group Fc were recovered and bound to an immunotest tube in which ROR1-Fc was constricted.
  • the non-specifically bound phages were removed by washing 5 to 30 times with a PBS-T (Phosphate buffered saline-0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered using a 100 mM triethylamine solution. After neutralizing the recovered phage with 1M Tris buffer (pH 7.4), ER2537 E. coli was infected at 37°C for 1 hour, and the infected E.
  • PBS-T Phosphate buffered saline-0.05% Tween 20
  • coli was spread on 2X YT agar medium containing carbenicillin and incubated overnight at 37°C. The next day, the cultured E. coli was suspended in 4 ml of 2X YT carbenicillin culture medium, 15% glycerol was added, some were stored at -80 ° C, and the rest were prepared as phage for the next experiment. This process was repeated for a total of 3 rounds to amplify and enrich the ROR1 antigen-specific phage pool.
  • the phage pool was plated on LB-tetracycline/carbenicillin agar medium and then cultured to obtain a single colony.
  • the single clone was then inoculated into a 96 deep well plate containing 400 ⁇ l/well of 2 X YT-tetracycline/carbenicillin medium and grown overnight. It was placed in a 96 deep well plate containing medium and incubated for 4 hours at 37°C. 1 mM IPTG was added to the culture medium and incubated overnight at 30°C. The culture solution cultured overnight was centrifuged to obtain the supernatant.
  • clones expressing a monoclonal soluble scFv that binds to the human ROR1-Fc antigen were selected using the ELISA method as follows (Steinberger Rader and Barbas III 2000 Phage display vectors In: Phage Display Laboratory Manual 1sted ColdSpringHarboratoryPress NY USA pp119- 1112). Specifically, 100 ng of recombinant human ROR1-Fc or BCMA-Fc prepared in Example 1.1.(1) was added per well to a 96-well microtiter plate (Nunc-Immuno Plates, NUNC, USA) and coated overnight at 4°C. .
  • BCMA-Fc is a protein used as a negative control and is a recombinant protein in which the extracellular domain region of human BCMA protein is linked to human Fc.
  • 200 ⁇ L of 3% BSA was added to each well and blocked at 37° C. for 2 hours.
  • the monoclonal phage supernatant was prepared by mixing 1:1 with 3% BSA, and 100 ⁇ L of this mixture was loaded into the wells and reacted at 37° C. for 2 hours. After washing 5 times with 300 ⁇ L of PBST, anti-HA HRP-binding antibody was added and reacted at 37° C. for 1 hour, followed by washing 5 times with PBST.
  • clones binding to cell lines expressing ROR1 were selected by flow cytometry. Specifically, 100 ⁇ l of the monoclonal scFv supernatant was reacted with a cancer cell line (JeKo-1) overexpressing ROR1, and then washed twice with PBS. After reacting with an anti-HA-FITC antibody (Sigma, H7411) at 4 ° C for 30 minutes, washing twice with PBS, suspending in 200 ⁇ l of PBS, and using a FACSCalibur flow cytometer (BD Bioscience) Clone binding to JeKo-1 cell line was selected (Fig. 2).
  • antibody clones (A2F2, BA6, C2E3) that bind to the recombinant human ROR1 protein and cell lines expressing ROR1 were selected, and A2F2M1, a mutant in which point mutations were introduced to prevent post-translational modification (PTM) in the A2F2 clone, was also selected.
  • PTM post-translational modification
  • Nucleic acid sequences encoding the variable region and CDR sequences are included as part of the nucleic acid sequences encoding the following full length heavy and light chains in the order of C2E3, A2F2, A2F2 M1 and BA6: SEQ ID NO: 27 (heavy chain) and 28 (light chain) ; SEQ ID NOs: 29 (heavy chain) and 30 (light chain); SEQ ID NOs: 31 (heavy chain) and 30 (light chain); SEQ ID NOs: 33 (heavy chain) and 34 (light chain).
  • Example 1.1 In order to convert the sequence of each ROR1-specific monoclonal phage antibody obtained in Example 1.1. into a full IgG form, encoding the heavy and light chain variable regions of each clone obtained in Example 1.1. Nucleic acids were synthesized (Genotech, Korea) Genes encoding the heavy chain (SEQ ID NO: 34) and light chain constant region (SEQ ID NO: 35 or 36) proteins of human IgG1 subtype were synthesized to encode the heavy and light chain variable regions. linked to nucleic acids. Nucleic acids encoding the light and heavy chains of each antibody were cloned into pcDNA3.1-based expression vectors, respectively, to obtain vectors encoding the antibody nucleic acids in the CHO-S mammalian cell line.
  • a chimeric antibody in which human IgG1 was linked to the variable region of 2A2 (US 9,316,646), an existing anti-ROR1 antibody, was used as a comparative antibody.
  • variable heavy chain (VH), variable light chain (VL), heavy chain and light chain sequences of the anti-ROR1 antibody (C2E3, A2F2, A2F2M1, BA6) according to the present invention in IgG form are shown in the table below Same as 2a to 2d.
  • a mutation such as K149C may be introduced into the light chain constant region for drug binding, and the light chain sequence introduced with such a mutation is also shown in the table below.
  • CHO-S cells After adjusting the concentration of CHO-S cells to 1.5 ⁇ 10 6 cells/ml in CD-CHO (Gibco, 10743) medium, they were cultured for 1 day at 8% CO 2 and 37°C. On the day of DNA transfection, cells grown to 2.5 ⁇ 3 ⁇ 10 6 cells/ml were prepared at a concentration of 2.1 ⁇ 10 6 cells/ml using CD-CHO medium containing 1% DMSO, and then in 8% CO 2 , Incubated for 3 hr at 37°C. After centrifugation at 3000 rpm for 15 min, the supernatant was removed and resuspended in RPMI 1640 medium containing 25% FBS.
  • each vector expressing the heavy chain and light chain of Example 1.2.(1) was diluted in Opti-MEM medium by 1 ⁇ g per ml of medium, respectively, and PEI (Polysciences, 23966, stock concentration: 1 mg/ml) was culture medium 8 ⁇ g per ml diluted. After mixing the vector and PEI mixture and allowing it to stand at room temperature for 10 min, put it into the flask containing the cells prepared as described above, incubate for 4 hr at 5% CO 2 , 37°C, 100 rpm, and After putting the volume of CD-CHO medium, 8% CO 2 , 37 °C, cultured for 4 days at 110 rpm.
  • Example 1.3.(2) After equilibration by passing the equilibration buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl) through Mab selectsure (GE healthcare, 5mL), the culture solution of Example 1.3.(2) was transferred to a column (Mab selectsure (GE healthcare, 5mL)). , 5 mL)) to allow the expressed antibody to bind to the column. Thereafter, the solution was eluted with 50 mM Na-citrate (pH 3.4) and 100 mM NaCl solution, and then neutralized with 1 M Tris-HCl (pH 9.0) to a final pH of 7.2. The buffer was exchanged with PBS (phosphate buffered saline, pH 7.4).
  • PBS phosphate buffered saline, pH 7.4
  • Example 1.2 The antigen-specific binding ability of the IgG antibody of each clone selected in Example 1.2, prepared in Example 1.2, was analyzed as follows.
  • Anti-ROR1 antibody-antigen binding affinity was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with the ROR1 protein described below at a concentration of 1 ⁇ g/ml in a PBS solution for 16 hours at 4° C., and the nonspecific binding site was 3% BSA (bovine serum albumin) for 2 hours. In this case, ROR1-Fc of Example 1.1 or recombinant human ROR1-His (Sino Biological, 13968-H08H) was used as the ROR1 protein in the case of human ROR1.
  • ROR1-His used in ELISA is a protein from sino biological (13968-H08H), and ROR1-His from Example 1.1. or recombinant mouse ROR1 protein was used (Acrobiosystems, RO1-M5221- 100 ⁇ g).
  • the anti-ROR1 antibody prepared in Example 1.3 was added to the microtiter plate at the concentration shown in FIG. 2 on a 96-well microtiter plate, and the binding ability was analyzed by ELISA as follows. Specifically, after incubation for 2 hours, the plate was washed 5 times with PBS containing 0.05% of tween 20, and then HRP-conjugated Fab polyclonal antibody reagent (Pierce, 31414) was added to 1: Diluted at a ratio of 10,000, added to the washed microtiter plate, and reacted at 37° C. for 1 hour to detect the ROR1 antibody bound to the plate.
  • TMB Tetramethylbenzidine, Sigma, T0440.
  • the enzyme reaction was stopped by 0.5 mol/L of sulfuric acid, and absorbance was measured at 450 nm and 650 nm using a microplate reader (molecular device) (450 nm - 650 nm).
  • FIGS. 3A and 3B and FIG. 4 The results of analyzing the binding specificity to ROR1 with several anti-ROR1 antibody clones are shown in FIGS. 3A and 3B and FIG. 4 . From this, it was confirmed that the anti-ROR1 antibody of the present invention binds to human ROR1 and mouse ROR1 in a concentration-dependent manner, and the A2F2M1 clone introduced with a point mutation in A2F2 to prevent post-translational modification (PTM) exhibits the same binding affinity as the parent clone. It was also confirmed that it has. In addition, when cross-reactivity to the mouse ROR1 protein was compared, the ROR1 antibody of the present invention showed superior binding ability compared to the 2A2 control antibody used as a comparison group (FIG. 4).
  • binding to an antigen expressed on the cell surface is an essential element.
  • Some antibodies bind purified antigen but not cell surface expressed antigen. In this case, even if the antibody is administered into the body, it is impossible to bind to the antigen, so the antibody cannot bind to cells expressing the antigen, and thus cannot show activity in vivo, such as a therapeutic antibody.
  • the ROR1 gene was transiently (CHO-human ROR1, CHO-human ROR2, CHO-mouse ROR1) or stably (MC38-human ROR1) transfected to artificially overexpress ROR1 protein expression (each 5 and FIG. 7) or ROR1 expressing cell lines (JeKo-1, Mino) (FIG. 6) or ROR1 non-expressing cell lines (MCF7) (FIG. 6) and anti-ROR1 antibodies using a FACSCalibur (BD Biosciences) instrument
  • MCF7 is a negative control that does not express ROR
  • CHO-human ROR2 is a negative control that expresses human ROR2.
  • JeKo-1, Mino, CHO-human ROR1, CHO-mouse ROR1, and MC38-human ROR1 are all cell lines expressing human ROR1 or mouse ROR1.
  • each cell line was dissociated, washed in PBS, the number of cells was counted, adjusted to 2 ⁇ 10 5 cells/200 ⁇ l PBS, and each ROR1 monoclonal antibody prepared in Example 1.3 was added at 10 ⁇ g/mL or 10 ⁇ g. After treatment by diluting 5 times from / mL, the reaction was performed at 4 ° C. for 1 hour.
  • FITC-labeled constant region (Fc) specific antibody Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 20 mg/ml
  • Fc constant region
  • the cells were washed in PBS and read using a FACSCalibur instrument.
  • the negative control group (2nd Ab) was treated with only the FITC-labeled constant region (Fc) specific antibody.
  • the shifted readout results in the experimental group treated with each ROR1 monoclonal antibody were compared with the shifted readout results of the control group (MFI Ratio: MFI of anti-ROR1 / MFI of 2nd Ab).
  • the anti-ROR1 antibody of the present invention is specific to the extracellular region of human ROR1 originally expressed in cells (FIG. 6) and artificially overexpressed human ROR1 in cells (FIGS. 5 and 7), and has a concentration-dependent pattern It was confirmed that . In addition, it was confirmed that it did not bind to human ROR2, a family protein, and had cross-species cross-reactivity with mouse ROR1 (FIG. 5). When cross-reactivity to mouse ROR1 expressed on the cell surface was compared, it was confirmed that the ROR1 antibody of the present invention had a better degree of binding than 2A2, an antibody used as a comparison group (FIG. 5).
  • ROR1 is expressed in a variety of cancer cells, including chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myelogenous leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), and diffuse giant B cell.
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • MCL mantle cell lymphoma
  • ALL acute lymphocytic leukemia
  • diffuse giant B cell diffuse giant B cell.
  • Blood cancers such as lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), as well as breast cancer, kidney cancer, ovarian cancer, stomach cancer, liver cancer, lung cancer, colon cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, It has been reported to be overexpressed in various solid cancers such as prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, epithelial squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.
  • NSCLC non-small cell lung cancer
  • NSCLC non-small cell lung cancer
  • neuroblastoma brain cancer
  • colon cancer epithelial squamous cell carcinoma
  • melanoma myeloma
  • myeloma cervical cancer
  • thyroid cancer head and neck cancer
  • adrenal cancer adrenal cancer
  • AGS ATCC CRL-1739 TM , huamn gastric adenocarcinoma
  • NCI-N87 ATCC CRL-5822 TM , human gastric carcinoma
  • MKN-28 KCLB 80102
  • each cell line was dissociated and washed in PBS, the cells were counted and adjusted to 2 ⁇ 10 5 cells/200 ⁇ l PBS, and the clone name C2E3 antibody among the ROR1 monoclonal antibodies prepared in Example 1.3 was added at 10 ⁇ g/ ⁇ g/ml. After treatment with mL, it was reacted at 4 °C for 1 hour.
  • FITC-labeled constant region (Fc) specific antibody Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 20 mg/ml
  • Fc constant region
  • results are shown in FIG. 8 .
  • the anti-ROR1 antibody of the present invention binds to ROR1 expressed in various cancer cell lines derived from gastric cancer, breast cancer, lung cancer, colorectal cancer, acute lymphocytic leukemia (ALL), and mantle cell lymphoma (MCL).
  • ALL acute lymphocytic leukemia
  • MCL mantle cell lymphoma
  • Antigens used in ELISA analysis, SPR analysis or T cell activity analysis of the following examples were purchased and used as follows.
  • recombinant B7-H3 protein (Sino Biological, 11188-H08H) containing amino acid sequence No. 1 to No. 461 of NP_001019907.1 and histidine-tag attached to C-terminus and C-terminus
  • a protein to which the Fc part of human IgG1 is bound (Sino Biological, 11188-H02H) was used.
  • 2X YT (Amresco, J902-500G), ampicillin (Ampicilin) is inoculated with 2 ⁇ 10 10 in a medium containing 100 ⁇ g / ml, 2% glucose (sigma, G7021) and incubated at 37 ° C for 2 to 3 hours so that the OD 600 value is 0.5 to 0.7 did After infecting the cultured E. coli with a helper phage, incubation in 2X YT [2X YT, Ampicillin 100 ⁇ g/ml, 1 mM IPTG (Duchefa, I1401)] medium at 30 ° C. for 16 hours paper packing was induced.
  • 2X YT [2X YT, Ampicillin 100 ⁇ g/ml, 1 mM IPTG (Duchefa, I1401)] medium at 30 ° C. for 16 hours paper packing was induced.
  • the cultured cells were centrifuged at 4°C and 4500 rpm for 20 minutes, and 4% PEG 8000 (Sigma, P2139) and 3% NaCl (Samchun, S2097) were added to the supernatant to dissolve them well, and then incubated on ice for 1 hour. reacted during After centrifugation at 4°C and 8000 rpm again, the supernatant was discarded and PBS (Phosphate buffered saline, Gibco 10010-023) was added to the cell pellet and suspended.
  • PBS Phosphate buffered saline, Gibco 10010-023
  • the non-specifically bound phages were removed by washing three times with a PBS-T (Phosphate buffered saline-0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered by adding 1 ml of a 100 mM triethylamine solution. Since the pH of the triethylamine solution is low, the recovered phages were neutralized with 1 M Tris buffer (pH 7.4) and then infected with ER2537 Escherichia coli grown at an OD of 600 to 0.8 to 1 for 1 hour and 30 minutes at 37°C and 120 rpm. made it The culture solution was centrifuged at 4 ° C.
  • PBS-T Phosphate buffered saline-0.05% Tween 20
  • coli was inoculated into 2X TB containing ampicillin, grown, infected with helper phage, and Examples 2.2.(1) and 2.2.(2) were repeated twice more to obtain a human B7-H3 protein-specific phage pool ( phage pool) was amplified and concentrated.
  • the phage pool was spread on LB-ampicillin agar medium and then cultured to obtain a single colony. Subsequently, the single clone was inoculated into a 96 deep well plate containing 200 ⁇ l of super broth (SB) medium per well, cultured at 37° C. for 4 hours, and then a part was transferred to another plate to make a cell stock. 1 mM IPTG was added to the remaining cell culture medium and incubated at 30° C. for 16 hours to induce scFv production. After centrifuging the culture medium at 4° C. and 6000 rpm for 20 minutes, the supernatant was discarded and only the cells were obtained. After dissolving the cells using TES solution, the cells were centrifuged again to obtain only the supernatant and used.
  • SB super broth
  • clones expressing a monoclonal soluble scFv binding to the B7-H3-His antigen were selected using the ELISA method as follows (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1sted. ColdSpringHarborLaboratoryPress. NY. USA. pp.11.9-11.12). Specifically, 100 ng per well of the recombinant human B7-H3-his protein prepared in Example 2.1.(1) was diluted in PBS in a 96-well plate (Nunc-Immuno Plates, NUNC, Rochester, NY, USA) and placed at 4°C.
  • an anti-HA HRP (Horseradish peroxidase) binding antibody (Roche, 12 013 819 001) was diluted 1:5000 in PBS containing 1% BSA to detect phages bound to human B7-H3. 100 ⁇ l per well was added and reacted at 37° C. for about 1 hour. After washing again with PBST, 100 ⁇ l of TMB (Tetramethylbenzidine, Thermo, 34028) was added to develop color. After reacting at RT for 5 to 10 minutes, 50 ml of 1N H 2 SO 4 was added to terminate the reaction. Absorbance was measured at 450 nm, and clones having a value of 1.0 or more were selected.
  • TMB Tetramethylbenzidine
  • antibody clone B5 binding to the recombinant human B7-H3 protein was selected, and the amino acid sequences and CDR sequences of the heavy chain variable and light chain variable regions of the B5 clone are shown in the following table.
  • Nucleic acid sequences encoding the B5 variable region are included in SEQ ID NOs: 85 (heavy chain) and 86 (light chain).
  • Example 2.1 In order to convert the sequence of each human B7-H3-specific monoclonal phage antibody obtained in Example 2.1 into a full IgG form, the heavy and light chain variable regions of each clone obtained in Example 2.1. Nucleic acids encoding were synthesized (Genotech, Korea). Genes (SEQ ID NOs: 68 and 69) encoding the heavy chain (SEQ ID NO: 65) and light chain constant region (SEQ ID NO: 66 or 67) proteins of the human IgG1 subtype were synthesized and linked to nucleic acids encoding the heavy and light chain variable regions.
  • variable heavy chain (VH), variable light chain (VL), heavy chain and light chain of the anti-B7-H3 antibody (B5) according to the present invention in IgG form are shown in Table 4 below.
  • a mutation such as K149C may be introduced into the light chain constant region for drug binding, and the light chain sequence introduced with such a mutation is also shown in the table below.
  • ExpiCHO-S TM (Thermo Fisher, A29127) cells developed by Thermo were used for expression of the anti-B7-H3 antibody, and antibody expression was performed in accordance with the manufacturer's ExpiCHO TM Expression System Kit (Thermo Fisher, A29133) protocol did
  • ExpiCHO-S cells were cultured at 120 rpm in a shaking incubator under 8% CO 2 , 37°C conditions.
  • ExpiCHO-S cells were diluted and prepared by adding ExpiCHO TM Expression Medium (Thermo Fisher, A2910001) at a cell concentration of 6 ⁇ 10 6 cells/ml.
  • each of the vectors expressing the heavy and light chains of Example 2.2.(1) was diluted in OptiPRO TM SFM medium (Thermo Fisher, 12309050) at 1 ⁇ g per ml of medium, and ExpiFectamine TM CHO included in the ExpiCHO Expression system 3.2 ⁇ l per ml was diluted in OptiPRO TM SFM medium.
  • the vector and ExpiFectamine TM CHO mixture were mixed and reacted at room temperature for 5 minutes, and then the mixture was put into prepared cells and cultured for 20 hours under conditions of 8% CO 2 , 37° C., and 120 rpm.
  • the cell culture medium was centrifuged at 4° C. and 6000 rpm for 30 minutes, and then the supernatant was separated and stored in a refrigerator.
  • Example 2.2.(2) After equilibration by passing the equilibration buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl) through Mab selectsure (GE healthcare, 5 ml), the culture solution of Example 2.2.(2) was transferred to a column (Mab selectsure (GE healthcare, 5 ml)). healthcare, 5 ml)) to allow the expressed antibody to bind to the column. Thereafter, the solution was eluted with 50 mM Na-citrate (pH 3.4) and 100 mM NaCl solution, and then neutralized with 1 M Tris-HCl (pH 9.0) to a final pH of 7.2. The buffer was exchanged with PBS (phosphate buffered saline, pH 7.4).
  • PBS phosphate buffered saline, pH 7.4
  • recombinant human B7-H3 protein diluted to a concentration of 1 ⁇ g/ml was added to a 96-well plate (Nunc-Immuno Plates, NUNC) per well, followed by reaction at 4° C. for 16 hours and coating.
  • the recombinant human B7-H3 protein used was the product purchased for analysis in Example 2.1.
  • HRP-conjugated anti-human IgG F(ab')2 antibody (Goat anti-Human IgG F(ab')2 Cross-Adsorbed Secondary Antibody, HRP , Pierce, 31414) was diluted 1:10,000 in PBS containing 1% bovine serum albumin (BSA), and 100 ⁇ l per well was added and reacted at 37° C. for about 1 hour.
  • BSA bovine serum albumin
  • 100 ⁇ l of TMB Tetramethylbenzidine, Sigma, T0440
  • 50 ⁇ l of 1 NH 2 SO 4 was added to terminate the reaction, and absorbance was measured at 450 nm and 650 nm using a microplate reader (molecular device).
  • Results are shown in FIG. 9 .
  • the anti-B7-H3 antibody of the present invention binds to the extracellular domain of human B7-H3 in a concentration-dependent manner.
  • B7 family proteins have 20-40% amino acid identity to each other and have structural relationships such as repeats of immunoglobulin domains. Therefore, whether it specifically binds to the B7-H3 protein rather than to other B7 family proteins was analyzed as follows.
  • B7 family constituent proteins having structural similarities for confirmation of immune-specific binding B7-1 (Sino Biological, Cat #: 10698-H08H), B7-2 (Sino Biological, Cat #: 10699-H08H), B7-DC ( Sino Biological, Cat #: 10292-H08H), B7-H1 (Sino Biological, Cat #: 10084-H08H), B7-H2 (Sino Biological, Cat #: 11559-H08H), B7-H4 (Sino Biological, Cat # : 10738-H08H), B7-H5(Sino Biological, Cat #: 13482-H08H), B7-H6(Sino Biological, Cat #: 16140-H08H), B7-H7(Sino Biological, Cat #: 16139-H02H) was purchased and used.
  • recombinant human B7 family protein diluted to a concentration of 1 ⁇ g/ml was added to a 96-well plate (Nunc-Immuno Plates, NUNC) per well, followed by reaction at 4° C. for 16 hours and coating.
  • the recombinant protein used was the product purchased for analysis in Example 2.1.1.
  • HRP-conjugated anti-human IgG F(ab')2 antibody (Goat anti-Human IgG F(ab')2 Cross-Adsorbed Secondary Antibody, HRP, Pierce, 31414) was diluted 1:10,000 in PBS containing 1% bovine serum albumin (BSA), and 100 ⁇ l per well was added and reacted at 37° C. for about 1 hour.
  • BSA bovine serum albumin
  • TMB Tetramethylbenzidine, Sigma, T0440
  • 1N H2SO4 1N H2SO4
  • Results are shown in FIG. 10 .
  • the anti-B7-H3 antibody specifically binds only to B7-H3 and not to B7 family proteins.
  • Evaluation in rodent or primate models is important to evaluate the antibody potency and immune modulator activity of the anti-B7-H3 antibody prior to human clinical progression.
  • the sequence of human B7-H3 shares more than 90% homology between monkey and mouse.
  • the cross-reactivity of the anti-B7-H3 antibody of the present application prepared in Example 2.2 to mouse or monkey B7-H3 was analyzed by ELISA assay as follows.
  • a recombinant mouse B7-H3 protein (Sino Biological, Cat #: 50973-M08H) with a histidine tag attached to the C-terminus and a recombinant Fc part of human IgG1 linked to the C-terminus Monkey B7-H3 protein (Sino Biological, Cat #: 90806-C02H) antigen was purchased and used.
  • Recombinant human B7-H3, mouse B7-H3, and monkey B7-H3 proteins were diluted to a concentration of 1 ⁇ g/ml, and 100 ⁇ l per well was added to a 96-well plate (Nunc-Immuno Plates, NUNC), followed by incubation at 4°C for 16 days. The coating was reacted for a period of time.
  • the recombinant protein used was the product purchased for analysis in Example 2.1.
  • HRP-conjugated anti-human IgG F(ab')2 antibody (goat anti-Human IgG F(ab')2 antibody) was used to detect antibodies bound to human B7-H3, monkey B7-H3, and mouse B7-H3. )2 Cross-Adsorbed Secondary Antibody, HRP, Pierce, 31414) was diluted 1:10,000 in PBS containing 1% bovine serum albumin (BSA), and 100 ⁇ l per well was added and reacted at 37 ° C for about 1 hour.
  • BSA bovine serum albumin
  • TMB Tetramethylbenzidine
  • Results are shown in Figures 11 and 12.
  • the anti-B7-H3 antibody of the present application exhibited similar binding to human and monkey B7-H3, but showed a relatively low binding to mouse B7-H3 (FIG. 11).
  • the degree of binding to mouse B7-H3 for each antibody clone was different, and it was observed that the 84D antibody used as a comparison antibody did not bind to mouse B7-H3 protein (FIG. 12).
  • the ability of the anti-B7-H3 antibody of the present application prepared in Example 2.2 to bind to human B7-H3 expressed on the cell surface was measured by FACS analysis.
  • binding to an antigen expressed on the cell surface is an essential element.
  • Some antibodies bind purified antigen but not cell surface expressed antigen. In this case, even if the antibody is administered in vivo, binding to the antigen is impossible, so the antibody cannot bind to cells expressing the antigen, and thus cannot show activity in vivo, such as a therapeutic antibody. Accordingly, whether the anti-B7-H3 antibody of the present application binds to cell surface-expressed B7-H3 was confirmed through FACS analysis.
  • cancer cell lines expressing human B7-H3, MCF-7 Human breast adenocarcinoma cell line, ATCC HTB-22 TM ), DLD1 (colorectal adenocarcinoma cell lines, ATCC CCL-221 TM ), HCC1954 (TNM stage IIA, grade 3, ductal carcinoma, ATCC CRL-2338 TM ), HCT116 (colon cancer cell, ATCC CCL-247 TM ) and Jurkat (acute T cell leukemia, ATCC TIB-152 TM ), a cancer cell line that does not express human B7-H3 used
  • Example 2.2 After dissociating each cell line and washing with PBS buffer, the number of cells is counted and adjusted to 2 ⁇ 10 5 cells per well, and 200 ⁇ l of PBS is prepared.
  • Each of the anti-B7-H3 antibody and control antibody (84D) of Example 2.2 was diluted in PBS containing 1% BSA at a concentration of 10 ⁇ g/ml or from a concentration of 10 ⁇ g/ml at a constant rate and mixed with cells prepared in advance. and reacted at 4°C for 1 hour.
  • FITC-labeled anti-human Fc FITC Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 2.0 mg/ml was diluted 1:500 to 100 ⁇ l per well FITC-labeled anti-human Fc FITC alone was treated as a negative control group, washed twice with PBS buffer, and the degree of anti-BCMA IgG binding was measured using a FACSCalibur instrument. did
  • B7-H3 is expressed in various cancer cells, including non-small cell lung cancer, renal cell carcinoma, neuroblastoma, colorectal cancer, pancreatic cancer, and gastric cancer.
  • gastric cancer Lung cancer, Prostate cancer, Endometrial cancer, Hepatocellular carcinoma, Lung cancer, Breast cancer, Cervical cancer ), osteosarcoma, oral carcinoma, bladder cancer, glioma, and melanoma. It has also been reported to be expressed in hematomas such as several types of lymphoma.
  • cancer cells A2780 human ovarian cancer, ECACC, 93112519
  • SKOV-3 human ovarian adenocarcinoma, ATCC HTB-77 TM
  • OVCAR-3 human ovarian adenocarcinoma, ATCC HTB-161 TM
  • HCT116 colon cancer cell, ThermoFIshcer Sci
  • HT29 ovalectal adenocarcinoma, ATCC HTB-38 TM
  • DLD-1 colonrectal adenocarcinoma cell lines, ATCC CCL-221 TM
  • Calu-6 Non-small-cell lung carcinoma, ATCC HTB-56 TM
  • HCC1954 NVM stage IIA, grade 3, ductal carcinoma, ATCC CRL-2338 TM
  • HCC1187 TMM stage IIA, ATCC CLC-2322 TM
  • renal cancer cell line 786-0 renal cell adenocarcinoma, ATCC
  • each B7-H3 monoclonal antibody prepared in Example 2.2 was added at 10 ⁇ g/ml. After treatment, it was reacted at 4°C for 1 hour. After the reaction, the cells were washed with PBS, and the FITC-labeled constant region (Fc) specific antibody (Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 2.0 mg/ml) was diluted 1:500. 100 ⁇ l per well and reacted at 4° C. for 1 hour.
  • Fc constant region
  • the cells were washed in PBS and read using a FACSCalibur instrument.
  • the negative control group was treated with only the FITC-labeled constant region (Fc) specific antibody.
  • Fc constant region
  • the anti-B7-H3 antibody of the present application was confirmed to bind to various cancer cell lines derived from ovarian cancer, colorectal cancer, non-small cell lung cancer, breast cancer, kidney cancer, pancreatic cancer, gastric cancer, cervical cancer, and lymphoma.
  • the anti-B7-H3 antibody of the present application showed a higher binding force at the same concentration than 84D, an antibody used as a comparative group, and thus showed a better degree of binding to cell surface-expressed B7-H3.
  • Example 2.3.(3) it was confirmed through ELISA that the anti-B7-H3 antibody of the present application binds to both human B7-H3 and mouse B7-H3 recombinant proteins.
  • mouse-derived cancer cell lines CT26 Mus mesculus colon carcinoma, ATCC CRL-2638 TM
  • B16F10 Mus musculus skin melanoma, ATCC CRL-6475 TM
  • TC-1 Mus musculus Lung tumor, ATCC CRL-2493 TM
  • Example 2.2 After dissociating each cell line and washing with PBS buffer, the number of cells is counted and adjusted to 2 ⁇ 10 5 cells per well, and 200 ⁇ l of PBS is prepared.
  • Each of the anti-B7-H3 antibody and comparative antibody (84D) of Example 2.2 was diluted to a concentration of 10 ⁇ g/ml or 10 ⁇ g/ml in PBS containing 1% BSA, and mixed with previously prepared cells at 4° C. It was reacted for 1 hour.
  • FITC-labeled anti-human Fc FITC Sigma, F9512
  • was diluted 1:500 treated with 100 ⁇ l per well, and reacted at 4° C. for 1 hour.
  • As a control only FITC-labeled anti-human Fc FITC was treated.
  • the degree of anti-B7-H3 IgG binding was measured using a FACSCalibur instrument.
  • the diabodies were constructed and the amino acid sequence was designed in the following way:
  • IgG is an anti-ROR1 antibody or an anti-B7-H3 antibody, and is composed of an IgG1 subtype.
  • the linker was composed of a sequence of (G4S)3 [(GGGGS)3] linked directly to the C-terminus of the CH3 domain of IgG1.
  • the scFv was constructed as a single chain Fv by connecting the VH and VL domains constituting the anti-ROR1 antibody or the anti-B7-H3 antibody with a sequence called a connector.
  • Connector is 20 amino acids in length consisting of (G4S)4 [(GGGGS)4], and depending on the configuration, the VH domain is located at the N-terminus in the order of VH-connector-VL, or the VL domain is located in the order of VL-connector-VH. It was configured to be located at the N-terminus (Table 6).
  • the heavy chain of the double antibody was designed with scFv linked to the C-terminus of the heavy chain of IgG, and the light chain was designed with the general structure of VL-CL, and Kabat 149 or Kabat 205 residues were used for drug conjugation. A configuration substituted with cysteine was additionally designed.
  • the scFv was substituted with cysteine for residues corresponding to Kabat No. 44 of the VH domain and Kabat No. 100 of the VL domain (each located within the framework) to form an intrachain disulfide bond.
  • a nucleotide sequence encoding the amino acid was derived through codon optimization, and dsDNA was produced through gene synthesis.
  • the prepared gene fragment was cloned into pcDNA3.4 vector using restriction enzymes or HIFI assembly technique.
  • A2F2M1 was additionally prepared by changing G in HCDR2 to A to prevent post-translational modification.
  • the name of the prepared double antibody is “anti-ROR1 antibody x anti-B7-H3 antibody” or “anti-ROR1 antibody y anti-B7-H3 antibody” using the anti-ROR1 antibody and anti-B7-H3 clone names used. Antibodies”.
  • the cloned diabodies were transiently expressed using the ExpiCHO system and the manufacturer's protocol was followed.
  • the antibody-expressed culture medium was centrifuged and filtered to remove suspended matter, and the antibody was purified using affinity chromatography using MabselectureSure resin and size exclusion chromatography using Superdex200 resin.
  • the purity of the purified antibody was analyzed by HPLC using TSKgel SuperSW3000, and the purity was more than 95%.
  • a 96-well microtiter plate (NUNC, 446612) was plated with recombinant human ROR1 protein (Sino biological, 13968-H08H) or recombinant human B7-H3 (Sino biological, 11188) at a concentration of 1 ⁇ g/ml in PBS solution for 16 hours at 4°C. -H08H), and nonspecific binding was blocked with 1% BSA (Bovine Serum Albumin) at 37°C for 2 hours.
  • BSA Bovine Serum Albumin
  • a 96-well microtiter plate (NUNC, 446612) was coated with recombinant human B7-H3 protein (Sinobiological, 11188-H02H) at a concentration of 1 ⁇ g/ml in PBS solution for 16 hours at 4°C, and non-specific binding was incubated with 1% BSA. (Bovine Serum Albumin) at 37°C for 2 hours.
  • BSA Bovine Serum Albumin
  • HRP-conjugated recombinant His-tag specific mouse monoclonal antibody reagent (Sigma, 11965085001) was diluted and incubated at 37°C for 1 hour, and the 96-well microtiter plate was washed 5 times with 1XPBST. Color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440), and the color reaction was stopped using 0.5 M sulfuric acid (Samjeon Pure Chemical, S1410), and then absorbance at 450 nm was measured.
  • Calu-3 cell line was prepared by washing twice with 1% BSA (Bovine Serum Albumin) to block non-specific binding.
  • BSA Bovine Serum Albumin
  • Anti-B7-H3 and anti-ROR1 antibodies were diluted 4 times each from 100 nM, incubated at 4°C for 1 hour, and washed twice with 1% BSA.
  • Anti-human Fc FITC (Sigma, F9512) was diluted 1:500, incubated again at 4°C for 1 hour, and washed twice with 1% BSA.
  • the washed cells were well dissolved in 1XPBS (pH7.4) 150 and used to measure the average fluorescence intensity of the cells using a FACSCalibur (BD Bioscience) instrument.
  • the anti-ROR1 monoclonal antibody clones A2F2, C2E3 and BA6 and the anti-B7-H3 monoclonal antibody B5 were variously combined as described in Example 3.
  • a double antibody was prepared according to the method, and cell binding ability of the double antibody was compared with that of the single antibody using cells expressing ROR1 and B7-H3 simultaneously.
  • the CHO-huROR1-huB7H3 cell line in which the ROR1 and B7-H3 proteins were artificially overexpressed by stably transfecting the ROR1 and B7-H3 genes, was used, and BD LSR Fortessa X- 20 (BD Biosciences) instrument. Specifically, after dissociating the overexpressing cell line, washing with PBS, and counting the number of cells, 20,000 cells per well were dispensed into a V-bottom 96-well plate (96 Well Plate-RV, Bioneer, 910D09).
  • the fold of induction (FOI) value of the graph was expressed by dividing the mean fluorescence intensity (MFI) of the antibody-treated experimental group by the MFI value of the control group treated with only the secondary antibody.
  • 16a shows the positions of the anti-ROR1 antibody and the anti-B7-H3 antibody in the double antibody (IgG The binding of bispecific antibodies with different forms and scFv forms) to ROR1/B7-H3 overexpressing cell lines is shown. It shows that all double antibodies used in the experiment can bind to cells at a higher level than single antibodies.
  • 16b and 16c show the binding ability of the bispecific antibody to the cell line overexpressing ROR1 alone or the cell line overexpressing B7-H3 alone, respectively. All of the tested diabodies exhibited excellent or equal or better binding ability to ROR1 or B7-H3 overexpressing cell lines than single antibodies. From this, it was confirmed that the reason why the bispecific antibodies of the present invention showed better binding ability is not simply because they target two types of antigens at the same time, but also because the bispecific antibodies themselves have excellent physical properties. In addition, this improved binding ability can be interpreted as showing selectivity for cells expressing two targets (antigens) at the same time compared to cells expressing a single target (antigen). It could be a feature that meets the goal of reducing toxicity and increasing potency.
  • Example 5 Antibody-derived cell cytotoxicity (ADCC) confirmation of single antibody and double antibody
  • the ability to induce signals by FcgRIIIA in effector cells according to antibody binding to target cells was evaluated.
  • the CHO-huROR1-huB7H3 cell line overexpressing both ROR1 and B7-H3 was plated in 96-well Solid White Flat Bottom Polystyrene TC-treated Microplates (Corning, 3917) at 15,000 cells per well. was aliquoted and incubated at 37° C. and 5% carbon dioxide for 20-24 hours.
  • Luminescence was measured using a luminescence plate reader (PHERAstar FS, BMG LABTECH). The FOI value of the graph was expressed by dividing the luminescence signal of the test group treated with the antibody by the signal of the control group not treated with the antibody.
  • the double antibody of the present invention has an improved ADCC signal inducing ability compared to the single antibody (FIGS. 17a and 17b). That is, in the ROR1/B7-H3 overexpressing cell line, the A2F2xB5 antibody and the BA6yB5 antibody induced ADCC signals to a greater extent than the corresponding single antibodies (B5, BA6, A2F2). In particular, it was confirmed that the IC50 of the A2F2xB5 antibody was reduced by about 6.7 times compared to the B5 antibody (Table 8).
  • the compounds 1 and 2 were prepared by the method described in Korean Patent Publication No. 10-2018-0110645.
  • the compound 3 was prepared by the method described in International Patent Publication No. WO2017-089895.
  • the structure of MMAE in this compound is:
  • the ADC was prepared through the following two steps, and the commonly used LCB14-0512 and LCB14-0606 were prepared by the method described in Korean Patent Publication No. 10-2014-0035393.
  • the structural formulas of LCB14-0512 and LCB14-0606 are as follows:
  • the antibody prenylation reaction was performed in a buffer solution ⁇ 50 mM Tris-HCl, (pH 7.4), 5 mM) containing 24 ⁇ M antibody, 200 nM FTase, and 0.100 mM or 0.250 mM isoprenoid (LCB14-0512 or 0606). MgCl 2 , 10 ⁇ M ZnCl 2 , 0.250 mM DTT ⁇ was used. After reacting for 4 hours or 16 hours at 30 °C, the prenylated antibody was desalted with a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.
  • AKTA purifier equilibrated with PBS buffer.
  • reaction mixture was prepared by mixing 100 mM sodium acetate (NaOAc) buffer (pH 5.2), 10% DMSO, 24 ⁇ M prenylated antibody and 240 ⁇ M linker-drug, and gently stirred at 30 ° C. did After 6 hours or 24 hours of reaction, excess low molecular weight compounds were removed using a G25 Sepharose column, and protein fractions were collected and concentrated.
  • NaOAc sodium acetate
  • reaction mixture was prepared by mixing PBS buffer solution (pH 7.4), 1% DMSO, 24 ⁇ M prenylation antibody and 120 ⁇ M linker-drug, and reacted at 25°C for 6 hours. After that, the reaction was terminated by adding an azide functional group. Excessive low-molecular-weight compounds were removed using a G25 Sepharose column, and protein fractions were collected and concentrated.
  • Example 6.3 The cytostatic activity of the ADC prepared in Example 6.3 (Table 9) against various cancer cell lines was measured.
  • commercially available human cancer cell lines Calu-3, Calu-6, HCC1954, NCI-N87 and MDA-MB-231
  • human ROR1 and B7H3 overexpressing CHO cell lines (CHO-ROR1-B7H3)
  • ROR1 overexpressing A CHO cell line (CHO-ROR1) and a mouse cell line (MC38-B7H3) overexpressing B7H3
  • 4,000 to 5,000 each cancer cell line per well was seeded in a 96-well plate, cultured for 24 hours, and ADC was treated at a concentration of 0.0015 to 10.0 nM (three-fold serial dilution).
  • IC 50 value (pM) showing 50% apoptosis was derived by 4PL curve fitting of the response curve between the absorbance by the amount of the substance reduced by the target activity of the cells and the ADC concentration.
  • ADC1 a B7H3 monoclonal antibody ADC used as a control, showed excellent cell proliferation inhibitory activity in cancer cells, and ADC2 and ADC3, ROR1 monoclonal antibody ADCs, which were other control materials, showed significantly lower cell proliferation inhibitory activity than ADC1.
  • ADC4, ADC5, ADC6, and ADC7 prepared with anti-ROR1xB7H3 bispecific antibodies showed weaker cytotoxicity than ADC1, a B7H3-only antibody ADC, but markedly increased than ADC2 and ADC3, a ROR1-only antibody ADC, in cancer cell lines expressing both ROR1 and B7H3 antigens.
  • the bispecific ADCs that is, ADC4, ADC5, ADC6, and ADC7, showed lower cytostatic activity than the control group ADC1 and ADC2, or the single antibody ADC combination treatment group treated with a 1:1 mixture of ADC1 and ADC3. , which is assumed to be due to the difference in DAR of the ADC.
  • the single antibody ADC for the control substance used in the experiment was all DAR2, but the double antibody ADC was DAR1.
  • ROR1 monoclonal antibody ADC In previous experiments, in the case of ROR1 monoclonal antibody ADC, it was confirmed that the expression of ROR1 monoclonal antibody ADC was low in several cancer cells compared to other tumor-specific antigens, resulting in insignificant efficacy. As the expression was confirmed and MTD (Maximum Tolerated Dose) was confirmed to be very low in animal experiments, ADCs targeting B7H3, in particular, ADCs loaded with drugs with relatively strong cytotoxicity, are effective against cancer cells. It can be seen that lowering toxicity to normal cells is a more important task than increasing anti-proliferative activity.
  • the bispecific ADC prepared according to the present invention is expected to be a combination capable of increasing cell proliferation inhibitory activity and lowering B7H3-derived cytotoxicity than ROR1 alone.
  • the antibody-drug conjugate of the present invention can exhibit excellent pharmacological effects due to plasma stability, body circulation limitation, target cell release, etc., and thus can be usefully used in the pharmaceutical industry.

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Abstract

La présente invention concerne un conjugué anticorps-médicament qui se lie à ROR1 et B7-H3, et une utilisation de celui-ci. En particulier, la présente invention concerne un conjugué anticorps-médicament comprenant un anticorps bispécifique qui se lie spécifiquement à ROR1 et B7-H3, et une utilisation du conjugué anticorps-médicament pour le traitement et/ou la prévention de maladies, plus spécifiquement, du cancer.
PCT/KR2022/007260 2021-05-21 2022-05-20 Conjugué anticorps-médicament qui se lie à ror1 et b7-h3, et utilisation de celui-ci WO2022245186A1 (fr)

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