WO2024032761A1 - Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques - Google Patents

Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques Download PDF

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WO2024032761A1
WO2024032761A1 PCT/CN2023/112504 CN2023112504W WO2024032761A1 WO 2024032761 A1 WO2024032761 A1 WO 2024032761A1 CN 2023112504 W CN2023112504 W CN 2023112504W WO 2024032761 A1 WO2024032761 A1 WO 2024032761A1
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seq
antibody
chain variable
variable region
light chain
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PCT/CN2023/112504
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Qing Li
Xinquan LIU
Larry Lo
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Hansoh Bio Llc
Shanghai Hansoh Biomedical Co., Ltd.
Changzhou Hansoh Pharmaceutical Co., Ltd.
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Publication of WO2024032761A1 publication Critical patent/WO2024032761A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to a novel ROR1 antibody or functional fragments thereof, comprising engineered heavy chains and light chains.
  • the present invention further relates to conjugates of the improved ROR1 antibody with small molecule drugs.
  • the present invention further relates to use of the antibody and the conjugates in the manufacture of a drugs for treating cancers.
  • Receptor tyrosine kinase-like orphan receptor is a transmembrane protein of receptor tyrosine kinase (RTK) family, and there are ROR1 and ROR2, which are type-I transmembrane receptor tyrosine kinases.
  • ROR1 receptor tyrosine kinase family
  • ROR1 and ROR2 which are type-I transmembrane receptor tyrosine kinases.
  • ROR1 receptor tyrosine kinase
  • ROR1 and ROR2 type-I transmembrane receptor tyrosine kinases.
  • ROR1 has received attention as an anti-cancer antibody target, as it is discovered that ROR1 is overexpressed in chronic lymphocytic leukemia (CLL) .
  • CLL chronic lymphocytic leukemia
  • Antibodies to ROR1 have been described in the literature W02014031174 (UC961) , WO2017127664 (XBR1-402) , etc. And a humanized murine anti-ROR1 antibody, UC961, has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia. Monoclonal antibody has received more and more attentions due to its characteristics of high target specificity, low side effect and the like. However, when used alone, its therapeutic effect is limited. Therefore, most of the monoclonal antibody drugs are used in combination with chemotherapy. Till present, the major way for increasing the therapeutic effect of monoclonal antibody is antibody-drug conjugates (ADCs) .
  • ADCs antibody-drug conjugates
  • Antibody-drug conjugate belongs to a type of new anticancer drug, which is consisted of three parts: antibody, cytotoxin and the linker which links the two parts. Monoclonal antibody is coupled with cytotoxin through chemical coupling.
  • the antibody-drug coupling technology makes the small molecule drug and biological protein fuse together, which may have the advantages of both and increase the potency of drugs remarkably, reduce the side effects.
  • ADCs targeting ROR1 may require the antibodies that can trigger Internalization efficiently, small-molecule compounds with higher toxicity, conjugation methods with higher DAR, linkers for more efficient release of toxicants and so on. Examples include NBE-002 and LCB71/ABL202, both of which use highly toxic small-molecule compounds. At the same time, to avoid off-target toxicity caused by the release of small-molecule drugs, both of them use very stable linkers to ensure that the drug is released only in tumor cells. In view of the antibody selection, conjugation sites, linkers, and small molecule compounds all play key roles in ADC design, there is an urgent need for using more diverse components and combinations to develop more advanced anti-ROR1 ADCs. The present invention provides a technical solution to meet this need.
  • the anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen, so the insufficient internalization of ROR1-targeting monoclonal antibody is an urgent problem to be solved.
  • the technical problem to be solved by the present invention is to develop the advanced anti-ROR1 ADC which show very strong antitumor effects in cancers.
  • the antitumor effects may be due to higher DAR and robust endocytosis as well as potential effects of the antibody itself on inhibition of Wnt5a signaling.
  • the present invention encompasses the following aspects:
  • the present disclosure provides an antibody-drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvate thereof
  • D is a cytotoxic drug
  • L 1 and L 2 are linking units
  • y is a number of 1 to 20;
  • Ab is an anti-ROR1 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 as shown in SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 or SEQ ID NO: 06; b) HCDR2 as shown in SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; c) HCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20; d) LCDR1 as shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ
  • the heavy chain variable region of Ab in general formula (A) comprises: HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 07, and HCDR3 as shown in SEQ ID NO: 14, respectively; or HCDR1 as shown in SEQ ID NO: 02, HCDR2 as shown in SEQ ID NO: 08, and HCDR3 as shown in SEQ ID NO: 15 respectively; or HCDR1 as shown in SEQ ID NO: 03, HCDR2 as shown in SEQ ID NO: 09, and HCDR3 as shown in SEQ ID NO: 16 respectively; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 17 respectively; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 11, and HCDR3 as shown in SEQ ID NO: 18 respectively; or HCDR1 as shown in SEQ ID NO:
  • the light chain variable region of Ab in general formula (A) comprises: LCDR1 as shown in SEQ ID NO: 21, LCDR2 as shown in SEQ ID NO: 27, and LCDR3 as shown in SEQ ID NO: 33, respectively; or LCDR1 as shown in SEQ ID NO: 22, LCDR2 as shown in SEQ ID NO: 28, and LCDR3 as shown in SEQ ID NO: 34, respectively; or LCDR1 as shown in SEQ ID NO: 23, LCDR2 as shown in SEQ ID NO: 29, and LCDR3 as shown in SEQ ID NO: 35, respectively; or LCDR1 as shown in SEQ ID NO: 24, LCDR2 as shown in SEQ ID NO: 30, and LCDR3 as shown in SEQ ID NO: 36, respectively; or LCDR1 as shown in SEQ ID NO: 24, LCDR2 as shown in SEQ ID NO: 31, and LCDR3 as shown in SEQ ID NO: 37, respectively; or LCDR1 as shown in SEQ ID NO: 25, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ
  • the Ab in general formula (A) comprises: a) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 07 and SEQ ID NO: 14, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 33, respectively; or b) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 08 and SEQ ID NO: 15, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 34, respectively; or c) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 09 and SEQ ID NO: 16, respectively; and
  • the Ab in general formula (A) is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
  • the Ab in general formula (A) comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 41, 43, 45, 47, 49, 51 and 53, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
  • the Ab in general formula (A) comprises: a) the heavy chain variable region as shown in SEQ ID NO: 40, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 41, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or b) the heavy chain variable region as shown in SEQ ID NO: 42, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 43, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or c) the heavy chain variable region as shown in SEQ ID NO: 44, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 45, or having at least at least 80%,
  • the Ab in general formula (A) comprises: a) the heavy chain variable region as shown in SEQ ID NO: 40 and the light chain variable region as shown in SEQ ID NO: 41; b) the heavy chain variable region as shown in SEQ ID NO: 42 and the light chain variable region as shown in SEQ ID NO: 43; c) the heavy chain variable region as shown in SEQ ID NO: 44 and the light chain variable region as shown in SEQ ID NO: 45; d) the heavy chain variable region as shown in SEQ ID NO: 46 and the light chain variable region as shown in SEQ ID NO: 47; e) the heavy chain variable region as shown in SEQ ID NO: 48 and the light chain variable region as shown in SEQ ID NO: 49; f) the heavy chain variable region as shown in SEQ ID NO: 50 and the light chain variable region as shown in SEQ ID NO: 51; g) the heavy chain variable region as shown in SEQ ID NO: 52 and the light chain variable region as shown in SEQ ID NO: 53.
  • the Ab in general formula (A) is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant regions is selected from ⁇ and ⁇ chain constant regions of human antibody and conservative variants thereof; more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 68 and a human light chain constant region of SEQ ID NO: 69.
  • the Ab in general formula (A) comprises: a) the heavy chain as shown in SEQ ID NO: 54 and the light chain as shown in SEQ ID NO: 55; b) the heavy chain as shown in SEQ ID NO: 56 and the light chain as shown in SEQ ID NO: 57; c) the heavy chain as shown in SEQ ID NO: 58 and the light chain as shown in SEQ ID NO: 59; d) the heavy chain as shown in SEQ ID NO: 60 and the light chain as shown in SEQ ID NO: 61; e) the heavy chain as shown in SEQ ID NO: 62 and the light chain as shown in SEQ ID NO: 63; f) the heavy chain as shown in SEQ ID NO: 64 and the light chain as shown in SEQ ID NO: 65; g) the heavy chain as shown in SEQ ID NO: 66 and the light chain as shown in SEQ ID NO: 67.
  • the Ab in general formula (A) is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.
  • the cytotoxic drug is selected from the group consisting of toxin, chemotherapeutic, antibiotic, radioisotope and nucleolytic enzyme.
  • the cytotoxic drug is selected from the group consisting of tubulin inhibitors or topoisomerase inhibitors; preferably auristatin analogues or camptothecin derivatives; more preferably MMAE, MMAF or Exatecan.
  • the antibody-drug conjugate is as shown in general formula (I) :
  • L 1 and L 2 are linking units
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 4 to 8 or 2 to 6, further preferably a number of 6 to 8, and most preferably 4, 6 or 8;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (II) :
  • L 1 and L 2 are linking units
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6, 2 to 4, 3 to 6, 3 to 5, 4 to 6, 4 to 8, 6 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (I-A) :
  • L 2 is linking unit
  • R 1 is selected from hydrogen, C 1-6 haloalkyl or C 3-8 cycloalkyl
  • R 2 is selected from hydrogen, C 1-6 haloalkyl or C 3-8 cycloalkyl
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-8 cycloalkyl
  • y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6, 2 to 4, 4 to 6, 4 to 8, 6 to 8, most preferably 2, 4, 6, 8;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (I-A) :
  • R 1 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from hydrogen, C 1-3 haloalkyl or C 3-6 cycloalkyl
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl
  • the antibody-drug conjugate is as shown in general formula (II-A) :
  • L 2 is linking unit
  • y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 6, 2 to 4, 3 to 6, 3 to 5, 4 to 6, 4 to 8, 6 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the linking unit L 2 is as shown in general formula (III) :
  • s 1 and s 2 are each independently an integer selected from 0-8, Preferably, s 1 and s 2 are independently selected from 1, 2, 3, 4, 5 or 6;
  • s 1 is an integer from 1-8, s 2 is 0, Preferably, s 1 is selected from 4, 5, 6, 7 or 8, and s 2 is 0;
  • s 2 is selected from an integer from 2 to 8, s 1 is 2, Preferably, s 2 is selected from 2, 3, 4, 5 or 6, and s 1 is 2.
  • the antibody-drug conjugate is as the following structure:
  • y 1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 4 to 8 or 2 to 6, further preferably a number of 6 to 8, and most preferably 4, 6 or 8;
  • y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is as shown in general formula (I-B) or formula (II-B) :
  • y 1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 4 to 8 or 2 to 6, further preferably a number of 6 to 8, and most preferably 4, 6 or 8;
  • y 2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;
  • Ab is the anti-ROR1 antibody or antigen-binding fragment above-mentioned.
  • the antibody-drug conjugate is selected from the group consisting of the following compounds:
  • y 1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 4 to 8 or 2 to 6, further preferably a number of 6 to 8, and most preferably 4, 6 or 8;
  • y 2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the therapeutically effective amount of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to the present disclosure, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • the present disclosure also provides a method of treatment or prevention of a disease related to overexpression of ROR1, comprising a step of administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate, or the pharmaceutical composition according to the present disclosure to a subject in need of treatment or prevention of the disease.
  • the disease is a cancer with ROR1 expression
  • the cancer is chronic lymphocytic leukemia (CLL) , B-cell leukemia, lymphoma, acute myeloid leukemia (AML) , Burkitt lymphoma, mantle cell lymphoma (MCL) , acute lymphoblastic leukemia (ALL) , Diffuse large B-cell lymphoma (DLBCL) , follicular lymphoma (FL) and marginal zone lymphoma (MZL) , breast cancer, renal cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC) , neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.
  • CLL chronic lymphocy
  • the present disclosure also provides the use of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate and the pharmaceutical composition in the manufacture of a medicament for treating or preventing a disease related to human ROR1.
  • the present disclosure also provides the use of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate and the pharmaceutical composition in the manufacture of a medicament for treating or preventing a cancer with ROR1 expression; preferably the cancer is chronic lymphocytic leukemia (CLL) , B-cell leukemia, lymphoma, acute myeloid leukemia (AML) , Burkitt lymphoma, mantle cell lymphoma (MCL) , acute lymphoblastic leukemia (ALL) , Diffuse large B-cell lymphoma (DLBCL) , follicular lymphoma (FL) and marginal zone lymphoma (MZL) , breast cancer, renal cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC) , neuroblastoma, brain cancer, colon cancer,
  • CLL chronic
  • the antibody or its antigen-binding fragment may (1) specifically recognize or bind to a ROR1 which is expressed on a cell surface derived from human, or (2) specifically recognize or bind to an extracellular domain of ROR1 which is not present on a cell surface, or (3) have sufficient internalization in a human cell.
  • the antibody-drug conjugate have the high cytotoxicity activity in tumor cell, the tumor inhibition effect of the antibody-drug conjugate is evidently strong in low dose in Jeko-1 CDX model.
  • the elimination half-lives of the antibody-drug conjugate is superior to that of VLS-101, thus exhibiting a significant long-term effect.
  • Figure 1-A ⁇ C In vitro binding characterization of ROR1 hybridoma clones to HT29 (A) , HCC1187 (B) , and T47D (C) cell lines using flow cytometry analysis.
  • Figure 2-A ⁇ B In vitro binding characterization of recombinant ROR1 antibodies to ROR1+ Jeko-1 cells (A) and ROR1-T47D cells (B) was determined by flow cytometry analysis.
  • Figure 4-A ⁇ H In vitro cytotoxicity activity of ADCs against different tumor cell lines, as determined by CellTiter-Glo luminescent viability assay.
  • the present invention is based on the development of an antibody which can specifically bind to ROR1.
  • Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, an auristatin or maytansinoid.
  • antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antigen-binding fragment of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ROR1) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH I domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) , and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883) .
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below) , (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
  • One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.
  • the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (LCDR1, LCDR2, LCDR3 or L1, L2, L3) , as well as CDR1, CDR2 and CDR3 of heavy chain variable domain (HCDR1, HCDR2, HCDR3 or H1, H2, H3) .
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Exemplary conventions that can be used to identify the boundaries of CDRs including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • Note 1 some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note2: any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note 3: the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.
  • nucleic acid molecule refers to a DNA molecule and a RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA.
  • a nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
  • the preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.
  • the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog.
  • the homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
  • ligand is a macromolecular compound able to recognize and bind to the target cell-associated antigens or receptors.
  • the role of the ligand is to deliver the drug to the target cell population bound to the ligand.
  • the ligand includes, but is not limited to, proteinaceous hormones, lectins, growth factors, antibodies and other molecules capable of binding to cells.
  • the ligand is expressed as Ab.
  • a connecting bond can be formed between a hetero atom in the ligand and the connecting unit.
  • linking unit means the part which links the antibody with the drug in the antibody-drug conjugate (i.e. ADC) , which could be cleavable or uncleavable.
  • the cleavable linker i.e., breakable linker or biodegradable linker
  • the linking unit or linker of the present invention has very good stability and greatly decreases the release of the drug during the process of delivering to the target (e.g., in blood) , thereby reducing the side effect and toxicity.
  • the linking unit or linker of the present invention is selected from cleavable linker, such as the linker based on disulfide (which is selectively broken in the tumor cells at a higher thiol concentration) , peptide linker (which is cleaved by the enzyme in the tumor cells) , and hydrazone linker.
  • cytotoxic drug means a chemical molecule capable of strongly destructing normal growth in tumor cells.
  • cytotoxic drugs can kill tumor cells in high enough concentrations, but due to the lack of specificity, when killing tumor cells, it also leads to apoptosis in normal cells, leading to serious side effects.
  • the cytotoxic drug is represented as D, and non-limiting examples include tubulin inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, and DNA synthesis inhibitors, preferably tubulin inhibitors, topoisomerase inhibitors.
  • ligand-cytotoxicity drug conjugate means that a ligand is linked to a biologically active drug through a linking unit.
  • the “ligand-cytotoxicity drug conjugate” is preferably an antibody-drug conjugate (ADC) , which means that a monoclonal antibody or antibody fragment is linked to a cytotoxic drug with biological activity through a linking unit.
  • ADC antibody-drug conjugate
  • DAR Drug to Antibody Ratio
  • y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number of 2 to 4, 2 to 6, 4 to 6, 4 to 8, 6 to 8, further preferably a number of 4 to 8 or 6 to 8, and most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0.
  • the average number of drugs in each ADC molecule after the coupling reaction can be identified by conventional methods, such as UV/visible spectroscopy, mass spectrometry, ELISA test, and HPLC characteristic identification.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group comprising 1 to 20 carbon atoms, preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 10 carbon atoms, and most preferably an alkyl having 1 to 6 carbon atoms (having 1, 2, 3, 4, 5 or 6 carbon atoms) .
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 1, 2-trimethylpropyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-di
  • the alkyl group is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 1, 2-trimethylpropyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like.
  • the alkyl can be substituted or unsubstituted. When substituted, the substituent group (s) can be substituted at any available connection point.
  • the substituent group (s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo.
  • haloalkyl refers to an alkyl group substituted by one or more halogen (s) , wherein the alkyl is as defined above.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (having 3, 4, 5, 6, 7 or 8 carbon atoms) .
  • monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like.
  • Polycyclic cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or bridged ring.
  • transfectoma includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.
  • sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening.
  • sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.
  • the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
  • a relevant sequence can be synthesized artificially, especially when the fragment is short in length.
  • several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.
  • DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.
  • the host cell obtained is cultured.
  • the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the monoclonal antibody obtained can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) .
  • the binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980) ) .
  • the antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.
  • protein precipitant such as salt precipitation
  • centrifugation such as salt precipitation
  • cell lysis by osmosis cell lysis by osmosis
  • ultrasonic treatment supercentrifugation
  • molecular sieve chromatography gel
  • variants of a polypeptide such as for example, an antigen-binding fragment, a protein or an antibody is a polypeptide in which one or more amino acid residues are inserted, deleted, added and/or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide.
  • a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintaining biological activity of the antibody disclosed herein, for example, binding to ROR1 and specificity.
  • the variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%identical to the sequence of the antibody or its antigen-binding fragment disclosed herein.
  • the percent identity (%) or homology may be calculated with reference to the following description.
  • the percent homology or identity may be calculated as 100 x [ (identical position) /min (TGA, TGB) ] , and in the formula, TGA, TGB are the sum of the number of residues of sequences A and B compared and the internal gap position (Russell et al., J. Mol Biol., 244: 332-350 (1994) .
  • the antibody of the present invention also includes a conservative variant thereof, which means that, compared to the amino acid sequence of the antibody of the present invention, there are up to 10, preferably up to 8 and more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • K D (M)
  • M molar concentration
  • K D values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the K D of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
  • affinity is the strength of interaction between an antibody or its antigen-binding fragment and an antigen, and it is determined by properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment.
  • properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment.
  • the antibody or its antigen-binding fragment is called “specifically binding" to its target such as an antigen, when a dissociation constant (K D ) is ⁇ l0 6 M.
  • the antibody specifically binds to a target with "high affinity” , when K D is ⁇ l0 9 M.
  • the term "Pharmaceutical composition” is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological /pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell.
  • administering and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell.
  • Treatment when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • the present disclosure includes a medicament for treating a disease associated with ROR1, comprising an antibody, an antigen-binding fragment or an antibody-drug conjugate thereof of the present disclosure as an active ingredient.
  • the molecules of the present disclosure are very useful for those who suffer a tumor, cancer or infectious disease when in preparations and formulations suitable for therapeutic applications.
  • the present disclosure relates to a method for immunologically detecting or measuring ROR1, a reagent for immunologically detecting or measuring ROR1, a method for immunologically detecting or measuring cells expressing ROR1, and a diagnostic reagent for diagnosis of disease related to ROR1 positive cells, comprising the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human ROR1, as an active ingredient.
  • the method for detecting or determining the amount of ROR1 may be any known method.
  • it includes immunodetection or assay.
  • the immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody.
  • immunodetection or assay include a radioactive substance labeled immunological antibody method (RIA) , an enzyme immunoassay (EIA or ELISA) , a fluorescent immunoassay (FIA) , a luminescent immunoassay, a western blotting method, physicochemical methods, etc.
  • the above-mentioned diseases related to ROR1 positive cells can be diagnosed by detecting or measuring cells expressing ROR1 by using the antibodies or antibody fragments thereof of the present invention.
  • a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemically staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Bio system) can be used.
  • the room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.
  • HEK293 cells were transduced by lentivirus containing a gene sequence encoded for human ROR1 ectodomain and a puromycin resistance gene. A single clone from the transduced HEK293 cells with highest huROR1 expression was selected.
  • Anti-ROR1 antibodies were obtained by immunizing genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions with a combination of recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO) , engineered huROR1-HEK293 cell, and HT-29 cells (ATCC, catalog number HTB38) .
  • the antibody immune response was monitored by a ROR1-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for ROR1 specificity.
  • the spleen lymphocytes and myeloma cells Sp2/0 were fused to obtain hybridoma cells by electrofusion or PEG fusion.
  • PEG fusion was performed using Clonacell TM HY technology (STEMCELL technologies) , following manufacturer’s instructions.
  • the primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10:1 for PEG fusion.
  • EXAMPLE 1-4 Screening of hybridoma clones specifically binding to human ROR1 protein by ELISA
  • ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1 ⁇ g/ml of human ROR1-Fc (9490-R0-050) , human ROR2-Fc (8609-RO-050) or BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50 ⁇ l ROR1 hybridoma supernatant was added in duplicate wells and incubated for 1 hour.
  • EXAMPLE 1-5 Screening of hybridoma clones specifically binding to ROR1 expressing cancer cells by Flow cytometry
  • Hybridoma supernatants was subjected to binding tests on ROR1+ cell line HT29 (ATCC, HTB-38) , HCC1187 (ATCC, CRL-2322) , and ROR1-ROR2+ cell line T47D (ATCC, HTB-133) using flow cytometry analysis. Briefly, 50 ⁇ L of cells in cell staining buffer (2 ⁇ 10 6 cells/mL) was mixed with 50 ⁇ L undiluted hybridoma supernatants. The mixture was incubated on ice for 20 min and then washed with ice cold staining buffer twice. The cells were subsequently stained with 50 ⁇ L of PE labelled secondary antibody (1: 400 dilution, BioLegend, Cat#405307) for 20 min.
  • FIG. 1 shows examples of selected cell binding signals measured by flow cytometry.
  • the clones 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, 38H10 were identified with enhanced binding profile to HT29 cells and HCC1187 cells compared to T47D cells.
  • the process of cloning sequences from positive hybridomas are as follows.
  • the logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification.
  • Amplified cDNA library from each clone were subjected to next-generation sequencing.
  • the amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, and 38H10 were obtained.
  • several mutations were made in the FR region, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following tables.
  • the amino acid residues of the CDRs in VH/VL are numbered and annotated according to the Kabat &Wu numbering system.
  • Example 3-1 Molecular cloning of recombinant antibodies
  • VH and VL regions of selected clones were directly synthesized as DNA fragments with 5’-end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into selected vectors using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) .
  • VH region was cloned into pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) , which in-frame with constant region of hIgG1 heavy chain in the vector.
  • VL region was cloned into pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) , which in-frame with constant region of hIg kappa light chain in the vector.
  • the IgG form of antibodies were disclosed as the following heavy chain and light chain full-lengths in order of 1E9, 8B10, 15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K, T83S) : SEQ ID NO: 54 (heavy chain) and 55 (light chain) ; SEQ ID NO: 56 (heavy chain) and 57 (light chain) ; SEQ ID NO: 58 (heavy chain) and 59 (light chain) ; SEQ ID NO: 60 (heavy chain) and 61 (light chain) ; SEQ ID NO: 62 (heavy chain) and 63 (light chain) ; SEQ ID NO: 64 (heavy chain) and 65 (light chain) ; SEQ ID NO: 66 (heavy chain) and 67 (light chain) , respectively.
  • Example 3-2 Expression and purification of recombinant antibodies
  • the heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-Scells (ThermoFisher #A29127) using ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 1: 1. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 C, 8%CO 2 .
  • Example 4 Binding characterization of anti-ROR1 recombinant antibody to ROR1+ and ROR1-cell lines by flow cytometry
  • Binding of the hlgGl mAbs to the cell surface ROR1 was determined by FACS analysis using different cancer cell lines positive for ROR1 or ROR2 expression, including Jeko-1 and T-47D cell.
  • Jeko-1 cells (ROR1 positive) was maintained in RPMI-1640 medium supplemented with 20%FBS and 1%penicillin and streptomycin.
  • T-47D cells (ROR2 positive) was maintained in RPMI-1640 medium supplemented with 10%FBS and 1%penicillin and streptomycin. Both cell lines were cultured at 37°C with 5%CO 2 in humidified atmosphere.
  • hIgG1 mAbs To determine the binding of hIgG1 mAbs to cell surface ROR1 receptors, cells were first harvested and resuspended in cell staining buffer (BioLegend) at 2 ⁇ 10 6 cells/mL. Then, the cells were treated with human Fc receptor blocking reagent (BioLegend) on the ice for 10 min. The resulting cell suspension was aliquoted into 50 ⁇ L aliquots. 50 ⁇ L of hIgG1 mAbs at various concentrations were mixed with the cell aliquots, the final concentration of hIgG1 mAbs in the mixture ranged from 1.1 pM to 200 nM.
  • the anti-ROR1 antibody of the present invention specifically binds to the human ROR1 as originally expressed in cells in a concentration-dependent pattern. In addition, it was confirmed that it does not bind to a family member protein, human ROR2.
  • Example 5 Characterization of anti-ROR1 recombinant antibody cell internalization in ROR1 expressing cells
  • the inventors evaluated endocytosis of the antibodies induced by ROR1-antibody binding.
  • Jeko-1 cells were collected and suspended in ice cold staining buffer (PBS with 0.5%FBS) at 2 ⁇ 10 6 cells/mL, followed by treating with human Fc receptor blocking reagent (BioLegend, 422302) .
  • the cells were then pre-incubated with Alexa Fluor 488 labelled antibodies on ice for 1 hour. After incubation, cells were transfer to 37°Cincubator.
  • MFI t is the MFI of samples collected at time t; MFI t0 is the MFI of samples collected at time 0 minute. MFI total is the MFI of the additional sample collected and served as control for 100%cell surface binding signal.
  • Example 6-1 ELISA binding to human ROR1 subdomain proteins
  • ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) .
  • ELISA plates were coated with 1 ⁇ g/ml of human ROR1 protein (RO1-H522y) or human ROR1 subdomains -Ig-like (RO1-H5221) , Frizzled (RO1-H5222) and Kringle (RO1-H5223) overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50 ⁇ l of 100 ⁇ g/mL ROR1 recombinant antibodies was added in duplicate wells and incubated for 1 hour.
  • a total of five clones (15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K/T83S) ) were subjected to epitope binning and compared to anti-ROR1 antibodies UC961.
  • Antibody epitope binning was performed using an Octet Red384 system equipped with anti-species and Ni-NTA sensors from Pall Life Sciences (Menlo Park, CA) . The experiment was performed as an in-tandem binning assay.
  • the assay is comprised of a five-step binding cycle: 1) A buffer baseline was established for 30 seconds, 2) 50 nM ROR1 antigen (RO1-H522y) was coupled to Ni-NTA octet sensors using a standard 1x assay buffer (PBS + 0.02%Tween20, 0.1%BSA, 0.05%sodium azide) diluted from a 10x kinetic buffer stock (ForteBio) for 5 minutes, 3) 250 nM of each antibody (saturating mAb) was loaded to saturate the immobilized antigen for 10 minutes, 4) 250 nM of each antibody (competing mAb) was bound for 5 minutes, and 5) capture sensors were regenerated for 30 seconds.
  • five anti-ROR1 recombinant antibodies can be divided into 2 different epitope bins. 15G4 (N65S) , 29D4, 31G8 and 37F9 are in the same bin 1, while 38H10 (N81K/T83S) is in a different bin 2. In addition, UC-961 binding does not compete with any of the five anti-ROR1 recombinant antibodies (15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K/T83S) ) , indicating UC-961 binding epitope is in a separate epitope bin 3.
  • ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1 ⁇ g/ml of mouse ROR1 protein (RO1-M5221) , rat ROR1 protein (RO1-R5221) or control BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50 ⁇ l of 25 ⁇ g/mL ROR1 recombinant antibodies was added in duplicate wells and incubated for 1 hour.
  • the antibodies of the present invention have cell affinity activity and endocytosis activity, making them suitable for coupling with drugs to form antibody-drug conjugates for treating ROR1-mediated diseases.
  • 15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 are purified for drug conjugation.
  • Further antibodies for conjugation may include any antibodies described herein (see Example 3) .
  • compound D was prepared by a method disclosed in PCT Patent Application. (See Application PCT/CN2022/073943, filed Jan 26, 2022) .
  • Antibody drug conjugates (ADCs) for anti-ROR1 antibodies described herein using MC-val-cit (vc) -PAB-MMAE drug linker may also be generated similar to the procedure described in US 2005/0238649.
  • Anti-ROR1 antibody (5 mg/mL in PBS, pH 7.4) was treated with sufficient molar equivalence (tris (2-carboxylethyl) phosphine (TCEP, 10 mM) at 37 °C for 1 hour.
  • TCEP (2-carboxylethyl) phosphine
  • Sufficient molar equivalence of drug linker for example, MC-val-cit (vc) -PAB-MMAE or compound D, in DMSO, was added to the reduced antibody in PBS. After an hour incubation, the reaction mixture was then purified using size-exclusion chromatography (SEC) to separate ADC and free, unconjugated drug linker.
  • SEC size-exclusion chromatography
  • the drug-to-antibody ratio (DAR) of ADC was determined using a TOF LC/MS system (Agilent) and the average DAR values were summarized in Table 13.
  • the average DAR values of anti-ROR1 antibodies conjugated with MC-val-cit (vc) -PAB-MMAE ranged from 3.5 to 4.5.
  • the average DAR values of anti-ROR1 antibodies conjugated with compound D were approximately 8.0 or 6.0.
  • the ADC-01 ⁇ ADC-11 immunoconjugates which had different cytotoxic moieties and linker chemistry, were analyzed for this Example.
  • VLS-101 is the positive control drug.
  • ROR1-mediated cell toxicities by these conjugates were tested in cell culture to define the potency of various linker-cytotoxic agent combinations.
  • Six different types of cancer cell lines were tested: colon cancer cell line HT-29, gastric cancer cell line AGS, NSCLC cell line NCI-H1975, TNBC cell line HS578T, MCL cell line Jeko-1, and ALL cell line Kasumi-2.
  • ADC-03, ADC-05, ADC-12, ADC-13 immunoconjugates which had different antibodies and DAR values, were analyzed for this Example, wherein ADC-12 had different DAR value with ADC-03 and ADC-13 different DAR value with ADC-05.
  • VLS-101 is the positive control drug.
  • ROR1-mediated cell toxicities by these conjugates were tested in cell culture. Cancer cell line was tested: human testicular embryonic carcinoma cell line NTERA-2.
  • Cells were collected in log phase growth and distributed into 96-well plates at 1000-2000 cells/well. After overnight incubation, the immunoconjugates were added to each well. The final concentrations of the immunoconjugates in the wells were ranging from 0.01 nM to 666.7 nM. After 4 to 7 days of incubation, the cell viability in each well was determined by Cell Titer Glo 2.0 Assay (Promega) .
  • mice Jeko-1 cells (MCL) were engrafted subcutaneously into mice (CB17/SCID) . When the tumor volume reaches approximate 120-180 mm 3 , the engrafted mice were randomized into six groups (5 mice per group) . The groups were vehicle control, VLS-101, ADC-07, ADC-08, ADC-09, and ADC-10. The mice were treated with ADCs (5 mg/kg) intravenously Q4D. Mean tumor growth inhibition (TGI) was calculated utilizing the following formula:
  • TGI ( (mean (C) -mean (C 0 ) ) - (mean (T) -mean (T 0 ) ) ) / (mean (C) -mean (C 0 ) ) *100%;
  • T is current group value, C is control group value, T 0 and C 0 represent the tumor volume at the beginning of the test.
  • mice treated with the six ADCs showed a statistically significant difference from mice in the control group, causing partial or even complete regression of tumors.
  • a follow-up tumor inhibitory study of VLS-101 and ADC-10 was carried out in the same Jeko-1 xenograft mouse model. The Jeko-1 engrafted mice were randomized into five groups (5 mice per group) . The groups were vehicle control, 2 mg/kg VLS-101, 1 mg/kg VLS-101, 2 mg/kg ADC-10 and 1 mg/kg ADC-10. The mice were treated with ADCs intravenously Q1W. The results of the follow-up study are shown in Table 18 and Figure 6. Treatment with ADC-10 at 1 mg/kg exhibited stronger inhibition of tumor growth, with a TGI value of 36.9%, whereas tumor growth in mice treated with VLS-101 at 1 mg/kg was inhibited by 3.8%.
  • NCI-H1975 (EGFR-L858R-T790M-C797S) cells were engrafted subcutaneously into mice (NOD/SCID) .
  • the engrafted mice were randomized into seven groups (5 mice per group) .
  • the groups were vehicle control, VLS-101, ADC-07, ADC-08, ADC-09, ADC-10 and ADC-11.
  • the mice were treated with ADCs (3 mg/kg) intravenously Q1W. The results of the study are shown in Table 18 and Figure 7.
  • mice treated with ADC-07, ADC-08 and ADC-10 showed a statistically significant difference from mice treated with VLS-101, with TGI value of 95.4%, 94.5%and 96.8%respectively, whereas tumors in mice treated with VLS-101 exhibited a TGI of 78.4%.
  • mice Female Balb/C mice (6-8 weeks old) were used to evaluate the pharmacokinetics of the antibody-drug conjugates. Blank serum was collected before dosing. ADCs were administered intravenously at 3 mg/kg. After injection, at predetermined time points (1, 4, 24, 48, 72, 120, 168, 336, and 504 hours) , serum was collected from designated mice and immediately stored at -80°C until further analysis.
  • Serum concentrations of ADCs were measured by ELISA. Briefly, to measure the total antibody concentration in the serum, the assay plates were coated with ROR1-his protein (Acro Biosystems, Cat#RO1-H522y) at 1 ⁇ g/mL, and then blocked with blocking buffer. After washing the plate 5 times, 30 ⁇ L of samples were added to each well and incubated at room temperature for 1 hour. The plates were washed 5 times and then incubated with HRP conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat#109-035-008) . Finally, TMB substrates were added to each well. The reaction was stopped after 10 minutes. Absorbance at 450 nm was measured to calculate the sample concentrations.
  • ROR1-his protein Acro Biosystems, Cat#RO1-H522y
  • the anti-payload antibody was coated on the assay plates to capture the payload-conjugated antibody.
  • HRP conjugated goat anti-human IgG antibody Jackson ImmunoResearch Laboratories, Cat#109-035-097 was used to detect the captured analytes.
  • the pharmacokinetic parameters were calculated by PK Solver 2.0.
  • the main pharmacokinetic parameters were shown in Table 19 and 20.
  • the AUCs of ROR1 antibodies UC961, 29D4, 31G8, and 37F9 in mice were similar.
  • the elimination half-lives of ADC-08, ADC-09, and ADC-10 are superior to that of VLS-101, which may contribute to their superior therapeutic efficacy in vivo.

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Abstract

Sont divulgués un anticorps dirigé contre le récepteur orphelin 1 de type récepteur tyrosine kinase, un conjugué de l'anticorps dirigé contre le récepteur orphelin 1 de type récepteur tyrosine kinase et un médicament à petites molécules. Sont divulgués également des utilisations du conjugué ligand-médicament cytotoxique et des compositions pharmaceutiques les comprenant dans la préparation de médicaments pour le traitement de cancers.
PCT/CN2023/112504 2022-08-11 2023-08-11 Conjugués ligand-médicament cytotoxique et leurs utilisations pharmaceutiques WO2024032761A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127664A1 (fr) * 2016-01-20 2017-07-27 The Scripps Research Institute Compositions d'anticorps anti-ror1 et procédés associés
WO2018237335A1 (fr) * 2017-06-23 2018-12-27 VelosBio Inc. Immunoconjugués d'anticorps ror1
WO2020160527A1 (fr) * 2019-02-01 2020-08-06 VelosBio Inc. Traitement du cancer avec des immunoconjugués d'anticorps ror1
WO2021044208A1 (fr) * 2019-09-04 2021-03-11 Legochem Biosciences, Inc. Conjugué anticorps-médicament comprenant un anticorps contre le ror1 humain, et son utilisation
WO2022011075A1 (fr) * 2020-07-10 2022-01-13 VelosBio Inc. Nouveaux immunoconjugués d'anticorps ror1

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127664A1 (fr) * 2016-01-20 2017-07-27 The Scripps Research Institute Compositions d'anticorps anti-ror1 et procédés associés
WO2018237335A1 (fr) * 2017-06-23 2018-12-27 VelosBio Inc. Immunoconjugués d'anticorps ror1
WO2020160527A1 (fr) * 2019-02-01 2020-08-06 VelosBio Inc. Traitement du cancer avec des immunoconjugués d'anticorps ror1
WO2021044208A1 (fr) * 2019-09-04 2021-03-11 Legochem Biosciences, Inc. Conjugué anticorps-médicament comprenant un anticorps contre le ror1 humain, et son utilisation
WO2022011075A1 (fr) * 2020-07-10 2022-01-13 VelosBio Inc. Nouveaux immunoconjugués d'anticorps ror1

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