Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/31—Chimeric antigen receptors [CAR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/4203—Receptors for growth factors
  • A61K40/4204—Epidermal growth factor receptors [EGFR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/4203—Receptors for growth factors
  • A61K40/4209—Hepatocyte growth factor receptor [HGFR] or c-met]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/35—Valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
• • 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
• • 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
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • 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
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

• the present application belongs to the field of biomedical technology. More specifically, the present application relates to antibodies capable of specifically binding c-Met or antigen-binding fragments thereof, as well as immunoconjugates, pharmaceutical compositions and multispecific molecules containing the same. Further, the present application also relates to the use of the antibody or antigen-binding fragment thereof that specifically binds c-Met and the multispecific molecule.
• c-Met protein is a receptor tyrosine kinase that undergoes post-translational modification from a 170kDa leader protein into a 50kDa ⁇ subunit and a 145kDa ⁇ subunit. A transmembrane dimer is formed after being linked by disulfide bonds.
• the main known ligand of c-Met is hepatocyte growth factor (HGF). After being stimulated by HGF, the tyrosine positions Y1234 and Y1235 of the intracellular end of c-Met are self-phosphorylated, and then the phosphorylation of Y1349 and Y1356 is transmitted, causing the intracellular end of c-Met to bind to the framework factors.
• HGF hepatocyte growth factor
• Signal activation pathways downstream of c-Met include PI3K/Akt, Rac1/Cdc42 and Erk/MAPK, etc., which can significantly affect cell proliferation, migration, invasion, tube tissue formation and other related characteristics.
• Cb1 ubiquitin ligase initiates ubiquitination of this protein and subsequently enters the degradation process. Negatively regulates the c-Met pathway.
• c-Met can promote cancer growth and is highly expressed in a variety of tumor tissues. And this makes it a target for anti-cancer drug development.
• RTK small molecule inhibitors the drug development of macromolecule-targeted antibodies has gradually advanced and gone into clinical research.
• anti-c-Met monoclonal antibody MetMab anti-c-Met monoclonal antibody MetMab.
• ADC molecules such as TR1801, which use the tubulin toxin tesirine to have an effect on mouse tumor PDX models with low, medium and high abundance of MET expression.
• This application obtained an antibody or an antigen-binding fragment thereof that can specifically bind to c-Met, thereby preparing a bispecific antibody that can specifically bind to c-Met and EGFR. Furthermore, an afucosylated bispecific antibody was prepared and thereby completed the present invention.
• the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding c-Met,
• the antibody or antigen-binding fragment thereof comprises:
• VH Heavy chain variable region containing the following three complementarity determining regions (CDRs):
• VH CDR1 which consists of the sequence shown in SEQ ID NO: 27 or 33, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions) , missing or added) sequence,
• VH CDR2 which consists of the sequence shown in SEQ ID NO: 28 or 34, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions) , missing or added) sequences, and
• VH CDR3 which consists of the sequence shown in any one of SEQ ID NO: 29 or 35, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) amino acid substitution, deletion or addition) sequence;
• VL light chain variable region containing the following three complementarity determining regions (CDRs):
• VL CDR1 which consists of the following sequence: SEQ ID NO: 30 or 36, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions) , missing or added) sequence,
• VL CDR2 which consists of the following sequence: SEQ ID NO: 31 or 37, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions) , missing or added) sequences, and
• VL CDR3 which consists of the following sequence: SEQ ID NO: 32 or 38, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions) , missing or added) sequences.
• substitutions of any one of (i)-(vi) are conservative substitutions.
• the CDRs described in any of (i)-(vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.
• the CDRs described in any of (i)-(vi) are defined according to the IMGT numbering system.
• the aforementioned antibody or antigen-binding fragment thereof includes: the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:27, VH CDR1 as shown in SEQ ID NO:28 VH CDR2, VH CDR3 as shown in SEQ ID NO:29; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO:30, VL CDR2 as shown in SEQ ID NO:31, VL as shown in SEQ ID NO:32 CDR3.
• the aforementioned antibody or antigen-binding fragment thereof includes: the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:33, VH CDR1 as shown in SEQ ID NO:34 VH CDR2, VH CDR3 as shown in SEQ ID NO:35; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO:36, VL CDR2 as shown in SEQ ID NO:37, VL CDR3 as shown in SEQ ID NO:38.
• the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.
• VH Heavy chain variable region comprising an amino acid sequence selected from the following:
• VL light chain variable region
• substitutions described in (ii) or (v) are conservative substitutions.
• the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 9 and a VL having the sequence set forth in SEQ ID NO: 11.
• the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 13 and a VL having the sequence set forth in SEQ ID NO: 15.
• the antibody or antigen-binding fragment thereof as described above further comprising a constant region derived from a human immunoglobulin.
• the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgGl, IgG2, IgG3, or IgG4).
• a human immunoglobulin eg, IgGl, IgG2, IgG3, or IgG4.
• the heavy chain constant region has the sequence set forth in SEQ ID NO: 19, 20, 39, or 40.
• the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
• a human immunoglobulin eg, kappa or lambda
• the light chain constant region has the sequence set forth in SEQ ID NO: 21, 22, or 41.
• the antibody or antigen-binding fragment thereof possesses ADCC activity.
• the antibody or antigen-binding fragment thereof comprises a mutated or chemically modified Fc region.
• the antibody or antigen-binding fragment thereof comprises an Fc region having a LALA mutation and/or a knob-into-hole modification.
• the antibody or antigen-binding fragment thereof comprises:
• the Fc region has the amino acid sequence set forth in SEQ ID NO: 17 or 18.
• the antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.
• the application provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above.
• the application provides a vector comprising a nucleic acid molecule as described above.
• the vector is a cloning vector or an expression vector.
• the present application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.
• the host cell is a mammalian cell.
• the host cell has low or no fucosylation activity, such as is selected from mammalian cells lacking expression of a gene encoding a fucosyltransferase (eg, CHO cells).
• the present application provides a method for preparing an antibody or an antigen-binding fragment thereof as described above, which includes culturing a host cell as described above under conditions that allow expression of the antibody or an antigen-binding fragment thereof. , and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.
• the host cell has low or no fucosylation activity, such as is selected from mammalian cells lacking expression of a gene encoding a fucosyltransferase (eg, CHO cells).
• the application provides multispecific molecules comprising an antibody or an antigen-binding fragment thereof as described above.
• the multispecific molecule specifically binds c-Met and additionally specifically binds one or more other targets.
• the multispecific molecule is a bispecific molecule.
• the bispecific molecule further comprises a molecule (eg, a second antibody) with a second binding specificity for a second target.
• a molecule eg, a second antibody
• the second target is epidermal growth factor receptor (EGFR) and the second antibody is an antibody against EGFR or an antigen-binding fragment thereof.
• EGFR epidermal growth factor receptor
• the bispecific molecule has been glycoengineered to have a lower number of fucose than the same bispecific molecule that has not been glycoengineered.
• the anti-EGFR antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.
• the antibody or antigen-binding fragment thereof as described above is hypofucosylated or afucosylated.
• the second antibody includes: the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:46, VH CDR2 as shown in SEQ ID NO:47, SEQ ID NO: VH CDR3 shown in 48; and, the following 3 light chain CDRs: VL CDR1 shown in SEQ ID NO:49, VL CDR2 shown in SEQ ID NO:50, VL shown in SEQ ID NO:51 CDR3.
• the second antibody comprises:
• VH having the sequence shown in SEQ ID NO:1 and VL having the sequence shown in SEQ ID NO:3; or
• VH having the sequence shown in SEQ ID NO:52 and VL having the sequence shown in SEQ ID NO:53.
• the second antibody comprises:
• the bispecific molecule comprises:
• a first antibody comprising VH CDR1-3 as shown in SEQ ID NO:27-29 and VL CDR1-3 as shown in SEQ ID NO:30-32; and, comprising as SEQ ID NO:46- Secondary antibodies to VH CDR1-3 set forth in SEQ ID NO: 48 and VL CDR1-3 set forth in SEQ ID NO: 49-51; or
• a first antibody comprising VH CDR1-3 as set forth in SEQ ID NO:33-35 and VL CDR1-3 as set forth in SEQ ID NO:36-38; and, comprising as set forth in SEQ ID NO:46- Secondary antibodies to VH CDR1-3 shown in 48 and VL CDR1-3 shown in SEQ ID NO: 49-51.
• the multispecific molecule comprises:
• a first antibody comprising a VH of a sequence shown in SEQ ID NO:9 and a VL of a sequence shown in SEQ ID NO:11; and, a VH and a VL comprising a sequence shown in SEQ ID NO:1
• the secondary antibody of VL with the sequence shown in SEQ ID NO:3;
• a first antibody comprising a VH of a sequence shown in SEQ ID NO:9 and a VL of a sequence shown in SEQ ID NO:11; and, a VH and a VL comprising a sequence shown in SEQ ID NO:52
• VH containing the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:15 A first antibody to a VL; and a second antibody to a VL comprising a VH of the sequence set forth in SEQ ID NO: 1 and a VL of the sequence set forth in SEQ ID NO: 3; or
• a first antibody comprising a VH of a sequence shown in SEQ ID NO: 13 and a VL of a sequence shown in SEQ ID NO: 15; and, a VH and a VL comprising a sequence shown in SEQ ID NO: 52
• a secondary antibody to VL having the sequence shown in SEQ ID NO: 53.
• the multispecific molecule comprises:
• a first antibody comprising a heavy chain of the sequence shown in SEQ ID NO:10 and a light chain of the sequence shown in SEQ ID NO:12; and, a first antibody comprising a sequence shown in SEQ ID NO:25 A secondary antibody of a heavy chain and a light chain of the sequence shown in SEQ ID NO:26;
• a first antibody comprising a heavy chain of a sequence shown in SEQ ID NO:10 and a light chain of a sequence shown in SEQ ID NO:12; and, a first antibody comprising a sequence shown in SEQ ID NO:2 A secondary antibody of a heavy chain and a light chain of the sequence shown in SEQ ID NO:4;
• a first antibody comprising a heavy chain of a sequence shown in SEQ ID NO:14 and a light chain of a sequence shown in SEQ ID NO:16; and, a first antibody comprising a sequence shown in SEQ ID NO:25 A secondary antibody of a heavy chain and a light chain of the sequence shown in SEQ ID NO:26;
• a first antibody comprising a heavy chain of the sequence shown in SEQ ID NO:14 and a light chain of the sequence shown in SEQ ID NO:16; and, a first antibody comprising a sequence shown in SEQ ID NO:2 A secondary antibody of a heavy chain and a light chain of the sequence shown in SEQ ID NO:4; or
• a first antibody comprising a heavy chain of a sequence shown in SEQ ID NO:23 and a light chain of a sequence shown in SEQ ID NO:24; and, a first antibody comprising a sequence shown in SEQ ID NO:25 A secondary antibody of a heavy chain and a light chain of the sequence shown in SEQ ID NO:26.
• a method for preparing the multispecific antibody as described above includes obtaining the antibody or antigen-binding fragment thereof as described above by the method as described above, and combining it with an anti-EGFR antibody. or an antigen-binding fragment thereof; optionally, with a reducing agent (eg, DTT).
• a reducing agent eg, DTT
• an immunoconjugate which comprises an antibody or an antigen-binding fragment thereof as described above or a multispecific molecule as described above, and is linked to said antibody or an antigen-binding fragment thereof or a multispecific molecule.
• Molecular therapeutic agents comprising an antibody or an antigen-binding fragment thereof as described above or a multispecific molecule as described above, and is linked to said antibody or an antigen-binding fragment thereof or a multispecific molecule.
• the therapeutic agent is selected from cytotoxic agents.
• the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
• the immunoconjugate is an antibody-drug conjugate (ADC).
• ADC antibody-drug conjugate
• a pharmaceutical composition which includes an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, and a pharmaceutically acceptable carrier and/or excipient.
• compositions further comprise additional pharmaceutically active agents.
• the pharmaceutical composition further comprises an EGFR inhibitor.
• the EGFR inhibitor is selected from erlotinib, gefitinib, osimertinib, or any combination thereof.
• the EGFR inhibitor and the antibody or antigen-binding fragment thereof, or the multispecific molecule are separately included as active components in different formulations and are administered simultaneously or at different times.
• the EGFR inhibitor is Osimertinib.
• the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
• a drug with anti-tumor activity such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
• the EGFR inhibitor is selected from erlotinib, gefitinib, osimertinib, or any combination thereof; in certain embodiments, the The EGFR inhibitor is Osimertinib.
• the drug is used to:
• the tumor expresses c-Met
• the tumor involves tumor cells of c-Met; preferably, the c-Met is expressed on the surface of the tumor cells;
• the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck Carcinoma, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma and other hematological malignancies , such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma,
• CHL Ho
• the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections;
• the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.
• kits in another aspect, contains an antibody or an antigen-binding fragment thereof as described above.
• the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
• a detectable label such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
• the kit further includes a second antibody that specifically recognizes an anti-EGFR antibody or an antigen-binding fragment thereof.
• the second antibody further includes a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
• a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
• the anti-EGFR antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.
• the antibody or antigen-binding fragment thereof as described above is hypofucosylated or afucosylated.
• a chimeric antigen receptor comprising the antigen-binding domain of an antibody or antigen-binding fragment thereof as described above.
• the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof as described above.
• the chimeric antigen receptor is expressed by immune effector cells (eg, T cells).
• immune effector cells eg, T cells
• a method for inhibiting the growth of c-Met-expressing tumor cells and/or killing the tumor cells includes combining the tumor cells with an effective amount of the aforementioned antibody or its antigen. fragment, or as before The multispecific molecule described above, or the immunoconjugate as described above, or the pharmaceutical composition as described above, or the chimeric antigen receptor contact as described above.
• the tumor expresses c-Met.
• the tumor involves c-Met tumor cells.
• the c-Met is expressed on the surface of the tumor cells.
• the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
• CHL
• the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections.
• the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.
• the antibody or antigen-binding fragment thereof is detectably labeled.
• the c-Met is mammalian (eg, human, monkey) c-Met.
• the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
• CHL
• the application provides a method for preventing and/or treating tumors in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody as described above or its Antigen-binding fragments, or bispecific or multispecific molecules as described above, or immunoconjugates as described above, or pharmaceutical compositions as described above, or chimeric antigen receptors as described above body, or host cell as described above.
• the tumor expresses c-Met.
• the tumor involves c-Met-expressing tumor cells.
• the c-Met is expressed on the surface of the tumor cells.
• the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
• CHL
• the subject is a mammal, such as a human.
• the method further includes administering an additional drug with anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune examinations Point inhibitors or oncolytic viruses.
• an additional drug with anti-tumor activity such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune examinations Point inhibitors or oncolytic viruses.
• the methods further include administering additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.
• additional anti-tumor therapy such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.
• the present application provides a method for detecting whether a tumor can be treated by anti-tumor therapy targeting c-Met, which includes the following steps:
• the antibody or antigen-binding fragment thereof is detectably labeled.
• the c-Met is mammalian (eg, human, monkey) c-Met.
• the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and -negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma
• CHL
• the present application provides a method for detecting the presence or amount of c-Met in a sample, which includes the following steps:
• the antibody or antigen-binding fragment thereof is detectably labeled.
• the c-Met is mammalian (eg, human, monkey) c-Met.
• antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
• Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
• Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
• the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
• Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
• the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
• Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
• the light chain constant region consists of one domain, CL.
• the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
• VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
• CDRs complementarity determining regions
• FRs framework regions
• Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
• the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196 :901-917; Chothia et al. (1989) Nature 342:878-883 definition.
• CDR complementarity determining region
• the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
• CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
• the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to known methods in the art. Various numbering systems are identified. In certain embodiments, the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof, are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
• framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
• antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
• the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
• the terms “monoclonal antibody”, “monoclonal antibody” and “mAb” have the same meaning and are used interchangeably and refer to one from a group of highly homologous antibody molecules.
• An antibody or a fragment of an antibody that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
• Monoclonal antibodies are highly specific for a single epitope on the antigen.
• Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
• the modifier "monoclonal” merely indicates that the antibody is characterized as being obtained from a highly homologous population of antibodies and is not construed as requiring any specific method to prepare the antibody.
• Monoclonal antibodies of the invention can be prepared by a variety of techniques, such as hybridoma technology (see, eg, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, eg, U.S. Patent Application 4,816,567), or phage Antibody library technology (see, eg, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).
• antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
• an antigen-binding moiety which is also called an "antigen-binding moiety.”
• Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody and such polypeptides, which contain sufficient to confer specificity to the polypeptide At least a portion of an antibody with antigen-binding capabilities.
• Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
• full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains” Antibodies composed of “chain”.
• full-length heavy chain refers to such a polypeptide chain, which consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, and the hinge region in the direction from the N end to the C end. (HR), heavy chain constant region CH2 domain, heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
• a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
• a "full-length light chain” is a polypeptide chain consisting of a light chain in the N-terminal to C-terminal direction.
• the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
• the full-length antibody of the present invention contains two antigen combinations formed by VH and VL pairs respectively. site, these two antigen-binding sites specifically recognize/bind the same antigen.
• the term “Fd” means an antibody fragment consisting of VH and CH1 domains
• the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
• the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
• the term “F(ab') 2 fragment” means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment;
• the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).
• Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
• Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
• Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
• LALA mutation refers to the mutation of amino acid 234 from L to A and amino acid 235 from L to A of the native Fc fragment.
• scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore, eds. Springer-Verlag, New York, pp. 269-315 (1994)).
• Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
• Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
• a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
• Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
• scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
• scFv can form (scFv)2, which refers to two or more individual scFvs joining in parallel to form an antibody.
• single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
• Single domain antibodies are also called nanobodies.
• Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
• Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
• antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
• chimeric antibody refers to an antibody in which part of the light chain and/or heavy chain is derived from an antibody (which may be derived from a specific species or belonging to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains the binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
• the term "chimeric antibody” may include antibodies in which the heavy and light chain variable regions of the antibody are derived from the first antibody, and the heavy and light chain constant regions of the antibody are derived from the first antibody.
• the targeting area is derived from the secondary antibody.
• the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
• the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison).
• the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence.
• Determination of percent identity between two sequences can also be accomplished using mathematical algorithms.
• One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877.
• Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
• variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide).
• polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc.
• Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
• a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
• the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
• the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction.
• KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
• the specific binding properties between two molecules can be determined using methods known in the art.
• One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
• "Association rate constant” (ka or kon) and The “dissociation rate constant” (kdis or koff) can both be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187).
• the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
• KD, kon and kdis values can be measured by any valid method.
• dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
• bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
• a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
• labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium est
• the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
• the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
• the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
• Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
• Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
• retroviruses including lentiviruses
• adenoviruses such as herpes simplex virus
• poxviruses poxviruses
• baculoviruses papillomaviruses
• papillomaviruses papillomaviruses
• Polyomavacuolating viruses such as SV40.
• a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
• the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or animal cells such as human cells.
• conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
• conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
• These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
• basic side chains e.g., lysine, arginine, and histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
• polypeptide and “protein” have the same meaning and are used interchangeably.
• amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
• the term "pharmaceutical composition” is a product containing one or more active ingredients (eg, antibodies, small molecule drugs) in an optionally specified amount, as well as any product containing one or more active ingredients (e.g., antibodies, small molecule drugs) in an optionally specified amount. Any product resulting, directly or indirectly, from the combination of one or more active ingredients. Different active ingredients in the pharmaceutical composition can be administered independently in the form of separate preparations, including administration at the same time or at different time points to achieve joint synergy. In this disclosure, “pharmaceutical composition” and “formulation” are not mutually exclusive.
• the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants Agents, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
• pH adjusting agents include, but are not limited to, phosphate buffer.
• Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
• Ionic strength enhancers include, but are not limited to, sodium chloride.
• Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
• Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
• Agents that delay absorption include, but are not limited to, monostearate and gelatin.
• Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
• Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
• Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
• the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
• such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
• WFI water for injection
• BWFI bacteriostatic water for injection
• sodium chloride solution e.g. 0.9% (w/v) NaCl
• dextrose solutions eg 5% glucose
• surfactant containing solutions eg 0.01% polysorbate 20
• pH buffer solutions eg phosphate buffer solution
• Ringer's solution any combination thereof.
• prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
• treatment refers to a method performed to obtain a beneficial or desired clinical result.
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
• treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
• the term "subject” refers to a mammal, such as a human, a cynomolgus monkey, a mouse.
• the subject eg, human, cynomolgus monkey, mouse
• the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect.
• a disease-preventing effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease
• a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already suffer from the disease. amount of symptoms. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
• single-arm antibody refers to an antigen-binding fragment that includes a Fab segment and an Fc segment, typically the Fab segment includes a heavy chain (e.g., VH and CH1) and a light chain (e.g., VL and CL), The Fc segment contains the constant regions (eg, CH2 and CH3).
• the Fab segment and Fc segment may or may not be connected through a linker.
• the single-arm antibody of the present invention can be prepared or synthesized by a variety of methods. For example, the sequence encoding the Fab heavy chain and the sequence encoding the Fc are constructed into the same vector, and the sequence encoding the Fab light chain is constructed into another vector. , transform the two vectors into host cells respectively to obtain single-arm antibodies.
• bispecific antibody refers to a conjugate formed by a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analog through a coupling arm
• Conjugation methods include, but are not limited to, chemical reactions, gene fusion, and enzymatic methods.
• Bispecific antibodies can be linked or produced by various methods, see for example the method of Songsivilai et al. (Clin. Exp. Immunol., 79:315-321 (1990)), and the method of Kostelny et al. (J. Immunol., 148:1547-1553(1992)).
• the terms “fucosylation” or “fucosylation” have the same meaning and refer to the presence of fucose residues in the oligosaccharide attached to the peptide backbone of the antibody. Accordingly, the terms “afucosylation” or “defucosylation” or “defucosylation” have the same meaning and refer to the removal of fucose present in the oligosaccharide attached to the peptide backbone of the antibody. Residues.
• This application has obtained an antibody or an antigen-binding fragment thereof that can specifically bind to c-Met. Furthermore, a bispecific antibody that can specifically bind to c-Met and EGFR, as well as a defucosylated bispecific antibody, have been prepared. Antibody. Compared with the standard antibody, the bispecific antibody or defucosylated bispecific antibody of the present application can: block HGF-dependent TKI resistance; block HGF-induced tumor cell proliferation and migration; induce ADCC Effect; inhibits tumor growth in the body, and is better than control antibodies and commercially available antibodies Rybrevant; combined with EGFR inhibitors can demonstrate synergistic tumor killing effects. Therefore, the bispecific antibodies of the present application have significant clinical value.
• Figure 1 shows the structural schematic diagram of the single-arm antibody (Figure 1A) and the anti-EGFRxc-Met bispecific antibody ( Figure 1B) used for anti-c-Met screening in this application.
• Figure 2 shows the inhibitory effect of anti-c-Met antibodies (136 single-arm antibody, 187 single-arm antibody) and control antibodies of the present application on the growth of HCC827 cells.
• Figure 3 shows the results of blocking EGF-EGFR binding by the anti-EGFRxc-MET bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187) and control antibodies of the present application.
• Figure 4 shows the inhibitory effect of the anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187) and control antibodies of the present application on the growth of HCC827 cells.
• Figure 5 shows the inhibitory effect of the anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187) and control antibodies of the present application on H596 cell proliferation.
• Figure 6 shows the blocking effect of the anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187) and control antibodies of the present application on the migration of HepG2 cells induced by HGF.
• Figure 7 shows the quality identification results of the stable cell line products, wherein Figure 7A shows the purity of the bispecific antibodies obtained by affinity purification; Figure 7B shows the proportion of correctly paired products of the purified bispecific antibodies.
• Figure 8 shows defucosylated bispecific antibody (EGFRxc-Met-136 Afu) (EGFRxc-Met-187 Afu) and control antibodies against A375 (Figure 8A), H292 ( Figure 8B), HCC827 (Figure 8C) cell binding activity.
• Figure 9 shows the application's anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187), defucosylated bispecific antibodies (EGFRxc-Met-136 Afu) and control antibodies induced ADCC effect of A375 (Figure 9A), H1975 (Figure 9B), HCC827 ( Figure 9C).
• Figure 10 shows the induction of PBMC by anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136, EGFRxc-Met-187), defucosylated bispecific antibodies (EGFRxc-Met-136 Afu) and control antibodies of the present application. The killing effect on A375.
• Figure 11 shows that the combination of defucosylated anti-EGFRxc-Met bispecific antibody (EGFRxc-Met-136 Afu) of the present application and an EGFR inhibitor induces the killing effect of PBMC on HCC827 cells.
• EGFRxc-Met-136 Afu defucosylated anti-EGFRxc-Met bispecific antibody
• Figure 12 shows the effect of defucosylated anti-EGFRxc-Met bispecific antibodies (EGFRxc-Met-136 Afu, EGFRxc-Met-187 Afu) and control antibodies of the present application in inhibiting the growth of H292 human lung cancer tumor cells.
• Figure 13 shows the effect of different doses of defucosylated anti-EGFRxc-Met bispecific antibody (EGFRxc-Met-136 Afu) of the present application on inhibiting the growth of H1975 human lung cancer tumor cells.
• Figure 14 shows the effect of defucosylated anti-EGFRxc-Met bispecific antibody (EGFRxc-Met-136 Afu) of the present application on inhibiting the growth of H292 human lung cancer tumor cells at different doses.
• Figure 15 shows the effect of a single dose of defucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) and the control commercial antibody Rybrevant in inhibiting the growth of H292 human lung cancer tumor cells.
• Figure 16 shows the effect of combined use of defucosylated anti-EGFRxc-Met bispecific antibody (EGFRxc-Met-136 Afu) and a small molecule inhibitor (osimertinib) to inhibit the growth of H1975-HGF human lung cancer tumor cells.
• EGFRxc-Met-136 Afu defucosylated anti-EGFRxc-Met bispecific antibody
• osimertinib small molecule inhibitor
• the sequences of anti-c-MET antibodies 136 and 187 used in this application were obtained from mice immunized with cellular mesenchymal epithelial transition factor (c-Met) antigen (purchased from AcroBiosystems), and total RNA was extracted and reverse transcribed. , and obtained through PCR amplification to construct a yeast display library for screening. And the sequences of antibodies 136 and 187 were obtained through sequencing. The CDR sequences of the antibodies were determined by the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).
• an anti-c-MET fully human antibody library from five immunized mice was first constructed, with a diversity of 3 ⁇ 10 ⁇ 7, and human c-MET was labeled according to the product instructions of the biotin labeling kit (purchased from Thermo).
• MET protein Yeast that can specifically bind to c-MET are enriched through MACS, and after multiple rounds of flow sorting, yeast cells that specifically bind to biotin-labeled c-MET protein with higher affinity are finally obtained.
• the sequences of the heavy and light chains of the antibody are retrieved using the finally sorted yeast as a template and constructed into an expression vector to prepare the antibody.
• the anti-cMet and EGFR double antibodies were constructed according to the method described in the patent (patent application number: 201611016435.0).
• the nucleotide sequences encoding the variable regions of anti-EGFR and anti-cMet antibodies were synthesized and connected to the nucleotide sequences that can spontaneously form heterogeneous On the constant region of the source dimer (its sequence comes from patent application number: PCT/CN2017/111310). Among them, three different anti-cMet antibodies were selected.
• the first anti-cMet antibody is the antibody disclosed in the patent number: WO2011/110642A2 (its sequence is specifically shown in Table 1), as a control antibody; the second and third The anti-cMet antibodies are antibodies 136 and 187 obtained through the above screening, and their sequences are shown in Table 1.
• the nucleotide sequence encoding the heavy chain variable region of the anti-EGFR antibody (the amino acid sequence is shown in SEQ NO: 1, disclosed in the patent number: WO02/100348A2) is conventionally synthesized and encodes a heavy chain that can spontaneously form heterodimers.
• the nucleotide sequence encoding the heavy chain variable region (SEQ NO: 9) of the anti-cMet antibody and the nucleotide sequence encoding the heavy chain constant region sequence 2 (SEQ NO: 40) that can spontaneously form heterodimers were routinely synthesized. After the sequences are connected, EcoRI and XhoI restriction enzyme sites are added to both ends and cloned into the vector pCDNA3.1 to construct plasmid cMet136-HC-pCDNA3.1 (136-2).
• the amino acid sequence of Fc is as shown in SEQ NO: 10 (shown); after conventional synthesis, the nucleotide sequence encoding the light chain variable region of the anti-cMet antibody (SEQ NO: 11) is connected to the nucleotide sequence encoding the light chain constant region sequence (SEQ NO: 41), and then added at both ends. EcoRI and XhoI restriction enzyme sites were cloned into vector pCDNA3.1 to construct plasmid cMet136-LC-pCDNA3.1 (the amino acid sequence of 136LC is shown in SEQ NO: 12).
• the nucleotide sequence encoding the anti-cMet antibody heavy chain variable region (SEQ NO: 13) is conventionally synthesized, and encoding can After the nucleotide sequence of the heavy chain constant region sequence 2 (SEQ NO: 40) that spontaneously forms a heterodimer is connected, EcoRI and XhoI restriction enzyme sites are added to both ends, and cloned into the vector pCDNA3.1.
• plasmid cMet187-HC-pCDNA3.1 (the amino acid sequence of 187-2Fc is shown in SEQ NO: 14); the nucleotide sequence encoding the anti-cMet antibody light chain variable region (SEQ NO: 15) is conventionally synthesized, and After the nucleotide sequence encoding the light chain constant region sequence (SEQ NO: 41) was connected, EcoRI and XhoI restriction enzyme sites were added to both ends, and cloned into vector pCDNA3.1 to construct plasmid cMet187-LC-pCDNA3. 1 (The amino acid sequence of 187LC is shown in SEQ NO: 16).
• the homologous recombination products were transformed into Top10 competent cells, spread on ampicillin-resistant plates, cultured at 37°C overnight, and single clones were picked for sequencing.
• the target protein was purified using a Protein A affinity chromatography column (MabSelect TM PrismA, GE Healthcare). Use 5 to 10 column volumes of equilibrium buffer (20mM Tris-HCl, 150mM NaCl, pH7.4) to equilibrate the purification column until the conductivity and pH value of the effluent remain unchanged before loading. After loading, continue to flush the column with equilibrium buffer until the UV value of the effluent no longer decreases. Samples were eluted using elution buffer (20mM glycine-HCl, pH 2.7) and the flow-through was collected. Neutralize the eluent with alkaline buffer (1M Tris-HCl, pH 8.0).
• the antibodies obtained in normal host cells were named Amivantamab analogs (control antibodies) and EGFRxMET-136. and EGFRxMET-187.
• the antibodies obtained from the defucosylation gene knockout cell line were named EGFRxMET-187 Afu and Amivantamab analog Afu.
• the nucleotide sequence encoding the heavy chain variable region of the anti-EGFR antibody as described above (SEQ ID NO: 1) and three The nucleotide sequence of the heavy chain variable region of an anti-cMet antibody (SEQ ID NO: 5, 9, 13) and the segment encoding human IgG1-CH1-Fc (LALA mutation, knob mutation) (SEQ ID NO: 17)
• the nucleotide sequences were connected to obtain the nucleotide sequence of the heavy chain of the anti-EGFR antibody (SEQ ID NO: 42) and the heavy chains of the three anti-cMet antibodies (SEQ ID NO: 43, 44, 45), using homologous recombination.
• the enzyme (purchased from Vazyme) was constructed into the EcoRI/Not I double digestion linearized pCDNA3.1 vector.
• the nucleotide sequences encoding the light chains of the four antibodies mentioned above (SEQ ID NO: 4, 8, 12, 16) were constructed into the EcoRI/Xhol I double-digested linearized pCDNA3.1 vector, and the process was according to the commercial product manual.
• the homologous recombination products were transformed into Top10 competent cells, spread on ampicillin-resistant plates, cultured at 37°C overnight, single clones were picked for sequencing, and plasmids were extracted.
• the nucleotide sequence encoding Fc-LALA-hole (SEQ ID NO: 18) was constructed into the EcoR I/Xhol I double-digestion linearized pCDNA3.1 vector.
• the extracted three plasmids of heavy chain (Fc-LALA-knob), light chain and Fc-LALA-hole were co-transfected into Expi-CHO cells to form a single Fab.
• the antibody structure ( Figure 1A) and the transfection method were in accordance with the product instructions.
• the supernatant was collected and the target protein was purified using protein A magnetic beads (purchased from GenScript). Resuspend the magnetic beads in an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads), add it to the sample to be purified, and incubate at room temperature for 1 hour, shaking gently during the period.
• binding buffer PBS+0.1% Tween 20, pH 7.4
• the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer.
• Add elution buffer (0.1M sodium citrate, pH 3.2) at 3-5 times the volume of magnetic beads and shake at room temperature for 5-10 minutes. Place it back on the magnetic stand. Collect the elution buffer and transfer it to the neutralizer.
• Buffer (1M Tris, pH 8.54) was mixed in a collection tube to obtain 4 target proteins, which were named anti-EGFR single-arm antibody, anti-MET single-arm antibody, 136 single-arm antibody and 187 single-arm antibody.
• c-MET antibodies 136 and 187 block the HGF-c-MET signaling pathway
• HCC827 cells are human non-small cell lung cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences. They highly express epidermal growth factor receptor EGFR (exon 19 loss) and c-Met receptor. Treatment with the tyrosine kinase inhibitor TKI small molecule Gefitinib (gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor) will induce apoptosis in HCC827 cells. If HGF is added simultaneously under such conditions, the c-Met pathway will be activated to induce HCC827 resistance to Gefitinib and inhibit apoptosis.
• TKI small molecule Gefitinib gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor
• Anti-c-MET antibodies 136 and 187 can restore the inhibitory effect of Gefitinib on the proliferation of HCC827 cells by blocking the HGF-c-MET signaling pathway.
• H292 cells are human non-small cell lung cancer cells purchased from (Procell) and express EGFR and c-Met receptor. Expand and culture H292 cells to a suitable density, digest and detach the cells from the cell culture flask, resuspend to 1 ⁇ 10 6 cells/ml, add 100 ⁇ L per well to a 96-well flow plate, centrifuge and set aside. Dilute the antibody to be detected with PBS, starting with 3-fold dilution at 200 nM. Add 100 ⁇ L/well of the diluted sample above to the above-mentioned 96-well flow plate with cells, and incubate at 25°C for 60 minutes.
• HCC827 cells are human non-small cell lung cancer cells purchased from (Cell Bank of the Chinese Academy of Sciences), which highly express epidermal growth factor receptor EGFR (exon 19 loss) and c-Met receptor.
• TKI small molecule Gefitinib glycofitinib, an epidermal growth factor receptor tyrosine kinase inhibitor
• HGF HCC827 resistance to Gefitinib and inhibit apoptosis
• This experiment tested the anti-EGFRxc-Met bispecific antibody to block HGF-induced cell proliferation. Adjust the density of the expanded cultured H596 cells to 3 ⁇ 10 4 cells/ml, add 100 ⁇ l/well to a 96-well cell culture plate, and culture overnight for later use. Use 1640 culture medium to dilute the antibody to be detected to 1200nM, and then dilute it 3 times in a gradient; dilute HGF to 200ng/mL, add the above diluted antibody 50 ⁇ l/well, HGF 50 ⁇ l/well, add the above cells with cells according to the experimental needs In 96 plates, add 1640 culture medium to a total volume of 200 ⁇ l/well, and culture for 5 days at 37°C and 5% carbon dioxide. After 5 days, 100 ⁇ l of culture medium was removed, and then Cell titer glo (purchased from Promega) was added at 100 ⁇ l/well, and chemiluminescent signals were collected using a microplate reader.
• HepG2 cells are human liver cancer cells purchased from (ATCC) and express EGFR and c-Met receptor. Placing HGF in the lower culture medium will induce HepG2 cells in the upper chamber to migrate to the lower chamber through the filter membrane.
• the specific experimental method is as follows: expand and culture HepG2 cells to a suitable density, digest and detach the cells from the cell culture flask, resuspend to 1 ⁇ 10 6 cells/ml, and add 100 ⁇ L per well to a 24-well cell migration plate (purchased from Corning ) for use in the small room above. Dilute the bispecific antibody to 200 nM with MEM, add 100 ⁇ L per well into the chamber above the 24-well cell migration plate, and incubate at 25°C for 60 min.
• Dilute h.HGF (purchased from R&D) with MEM to 50ng/mL, add 500 ⁇ L/well into the chamber below the above cell migration plate, and culture at 37°C and 5% carbon dioxide for 3 days. Use a cotton swab to gently wipe away the non-migrated cells above the membrane of the upper chamber. The cells that migrated to the lower layer of the membrane were lysed with Cell titer glo (purchased from Promega), and the chemiluminescence signal was collected with a microplate reader.
• This example uses CH1/CL preference mutation (Patent No.: WO2021/067404A2) and Knob in hole technology to construct the EGFRxMET-136 Afu molecule, and construct the heavy chain variable regions of the anti-cMet antibody and the anti-EGFR antibody into the CH1 mutation respectively.
• a 1+1 asymmetric anti-EGFRxcMet bispecific antibody cell line was constructed.
• the vector pCHO2.0-GS- containing the nucleotide sequence encoding the anti-c-Met antibody heavy chain (SEQ NO: 23) and the nucleotide sequence encoding the light chain (SEQ NO: 24) was used to electroporate transfection.
• Puro-H1-L1 and the vector pCHO2.0-GS-Puro-H2- containing the nucleotide sequence encoding the anti-EGFR antibody heavy chain (SEQ NO: 25) and the nucleotide sequence encoding the light chain (SEQ NO: 26) L2 was co-transfected into the host cell CHOS-ADP Fut8 KO (defucosylation gene knockout cell line), and the cells were pressure screened using Puromycin and MSX screening pressure to obtain a high-yield minipool, and then through a round of limiting dilution and single clone identification, A high-yield and stable clonal cell line was obtained. By comparing the growth status of the clones, protein expression levels, and physical and chemical properties of the expressed proteins, the cloned cell line 6F5 was finally determined to be a recombinant engineered cell line.
• Use Dynamis AGT Medium as the basic medium, inoculate at a density of (1.0 ⁇ 0.2) ⁇ 10 6 cells/ml, and culture until the 3rd, 5th, 7th, 9th, and 11th day with an initial culture of 5.0 ⁇ 0.5% (w/w).
• the weight of 7a was fed, and 0.5 ⁇ 0.05% (w/w) of the initial culture weight of 7b was fed.
• the dissolved oxygen was set to 40%, the initial culture temperature was 36.5°C, and the temperature was lowered to 33.0°C on the 4th day.
• the culture was terminated on the 14th day or when the cell viability rate was lower than 80%.
• cell density and viability were detected using Vicell (Beckman Company).
• Cedex (Roche Company) was used to detect antibody production every day. The results are shown in Table 4.
• the bispecific antibody The expression level reaches 2.432g/L.
• the one-step purification method is the same as the single-arm antibody purification method in Example 1.
• Use HPLC to detect protein purity The HPLC method is as follows, mobile phase: 150mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.5ml/min, detection time: 30min, TSKgel G3000SWXL chromatographic column.
• the SEC results show in Figure 7A that the purity of the bispecific antibody obtained by one-step affinity purification was 98.18%.
• High-performance liquid chromatography mass spectrometry was used to detect the pairing of heavy and light chains of the protein.
• the instruments used were liquid chromatography system Vanquish UHPLC (Thermo), mass spectrometer Q Exactive (Thermo) and chromatographic column Waters ACQUITY UPLC BEH C4, 2.1mm ⁇ 100mm. Take 50 ⁇ g of sample, add ultrapure water to dilute to 25 ⁇ l, centrifuge and take 20 ⁇ l of sample into the injection bottle, inject 5 ⁇ l of sample, and use LC-MS to analyze the complete molecular weight.
• the chromatographic conditions are: column temperature: 80°C; UV detection wavelength: 280nm; flow rate: 0.3mL/min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid).
• the mass spectrometry parameters are: ESI ion source: ion transfer tube temperature 320°C, voltage 3.8kV, gas flow rate 36L/min; mode: positive ion Full MS; resolution: 17500; scanning range: 600-4000m/z. The results are shown in Figure 7B, and the proportion of correctly paired products for the purified bispecific antibodies was >98%.
• the defucosylated bispecific antibody Final EGFRxMET-136 (named EGFRxMET-136 Afu) was prepared. This antibody molecule was used as the final molecule for subsequent in vivo and in vitro activity detection experiments.
• Example 7 Binding activity of anti-EGFRxc-Met bispecific antibody to EGFR and c-Met positive tumor cells
• A375 cells are human malignant melanoma cells purchased from (Addexbio) and have low expression of EGFR and c-Met receptor.
• H292 cells are human lung adenocarcinoma cells purchased from Procell and express EGFR and c-Met receptors at high levels.
• HCC827 cells are human non-small cell lung cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences and highly express EGFR and c-Met receptors. Adjust the expanded cultured cells to a suitable cell density and add them to a 96-well flow plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 1 hour.
• the EC50 value of antibody binding strength is detailed in Table 5 below.
• A375 cells are human malignant melanoma cells purchased from (Addexbio) and express EGFR and c-Met receptor.
• H1975 cells are human lung adenocarcinoma cells purchased from (Addexbio) and have high expression of EGFR and c-Met receptor.
• HCC827 cells are human non-small cell lung cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences and highly express EGFR and c-Met receptors.
• the expanded cultured A375/H1975/HCC827 cells were resuspended in 1640 medium, and the cell density was 1.2x10 6 cells/ml; CD16a-NFAT-Luc Jurkat reporter cells (self-built by Pumis, the CD16a and NFAT-Luc gene sequences were Constructed on the pCDNA3.1 vector, then transfected into Jurkat cells, and screened for antibiotic resistance to obtain CD16a-NFAT-Luc Jurkat reporter cells). Resuspend in 1640 culture medium, and adjust the cell density to 6x10 6 cells/ml cells/ ml; dilute the antibody to 300nM with 1640 culture medium, and then dilute it 4 times in a 4-fold gradient.
• luciferase fluorescein
• sequence source Uniprot P08659, constructed on the pLVX-neu vector by Universal Biosynthesis
• plasmid was transfected into A375 cells to construct a luciferase stably transduced cell line. (A375-luc).
• cDNA encoding luciferase (sequence source Uniprot, P08659) and human HGF (hepatocyte growth factor) (sequence source UniProtKB P14210) was constructed in pLVX by universal biosynthesis.
• -neu vector the plasmid was transfected into HCC827 cells to construct a cell line stably expressing luciferase and HGF (HCC827-HGF luc). Since overexpression of HGF will cause HCC827 cells to become resistant to first-generation EGFR small molecule inhibitors, we designed a combined drug experiment with antibodies and first-generation small molecules.
• the expanded cultured luciferase stably transfected cell line (HCC827-HGF Luc) was digested and centrifuged for counting. Resuspend the cells in working medium (10% FBS+RPMI 1640), adjust the cell density to 3x10 4 /ml, and add 100 ⁇ L per well to the culture plate; recover PBMC cells from liquid nitrogen, centrifuge and resuspend the cells in working medium. Add to culture flask overnight to remove monocytes. After overnight, adjust the concentration of suspended immune cells to 1.8x10 5 /ml for later use.
• the EGFRxMET-136 Afu molecule is used in combination with small molecule inhibitors targeting EGFR (Gefitinib left, Erlotinib right), and PBMC are added to simulate the killing of tumor cells by immune cells.
• PBMC small molecule inhibitors targeting EGFR
• the results are shown in Figure 11. Without adding PBMC, the EGFRxMET-136 Afu molecule blocks the HGF-MET signaling pathway, relieves the tolerance of HGF-dependent tumor cells to small molecules, and promotes the resistance of small molecules to tumors. Kill. More importantly, after adding PBMC, compared with using EGFRxMET-136 Afu or small molecule inhibitors alone, the combination showed a synergistic effect and could kill tumor cells more effectively.
• CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model and determine the anti-tumor effect of the anti-EGFRxc-Met bispecific antibody.
• Sufficient H292 cells were cultured and expanded in vitro, collected after trypsin digestion, washed three times with PBS and counted, and then inoculated into 8-week-old female CB-17 SCID mice (purchased) at 3 ⁇ 10 6 cells/mouse. from Beijing Vitong Lever) subcutaneously on the right side of the abdomen. Observe the tumor formation of tumor cells under the skin of mice every day.
• mice tumor volume and mouse body weight were measured 2-3 times a week.
• the mouse body weight and tumor volume were measured for the last time 29 days after inoculation of tumor cells, and the mice were euthanized.
• CB-17 SCID mice were subcutaneously inoculated with H1975 human lung cancer tumor cells to establish a tumor-bearing model.
• H1975 cells were cultured and expanded in vitro, collected after trypsin digestion, washed three times with PBS, counted, and inoculated at 3 ⁇ 10 6 cells/mouse.
• Female 8-week-old CB-17 SCID mice (purchased from Beijing Vitong Lever) were subcutaneously injected into the right abdomen. Observe the tumor formation of tumor cells under the skin of mice every day.
• mice tumor volume and mouse body weight were measured 2-3 times a week.
• the body weight and tumor volume of the mice were measured for the last time 24 days after inoculation of tumor cells, and the mice were euthanized.
• the bispecific antibody groups at three different doses (2 mg/kg, 8 mg/kg, and 32 mg/kg) all showed significant inhibitory effects on tumor growth.
• the TGI of the 2 mg/kg treatment group 97.4%
• the TGI of the 8 mg/kg treatment group 99.3%
• the TGI of the 32 mg/kg treatment group 99.4%. It can be seen that the bispecific antibody of the present application almost completely inhibits tumor growth, and the tumor inhibition rate has reached more than 99%.
• CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model and compare the anti-tumor effects of different doses of anti-EGFRxc-Met bispecific antibodies.
• the mouse model establishment process is the same as experimental model one. Group the patients according to the group dosing schedule in Table 8 and inject corresponding doses of antibodies.
• mice tumor volume and mouse body weight were measured 2-3 times a week.
• the body weight and tumor volume of the mice were measured for the last time 30 days after inoculation of tumor cells, and the mice were euthanized.
• CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model, and compared the anti-EGFRxc-Met bispecific antibody with the commercially available EGFRxc-Met bispecific antibody Rybrevant (purchased from Johnson & Johnson, batch number: LHS3G02) (The anti-tumor effects of this antibody, also known as JNJ-6372, have been published at various oncology conferences.
• the mouse model establishment process is the same as experimental model one. Group the patients according to the group dosing schedule in Table 9 and inject corresponding doses of antibodies.
• mice tumor volume and mouse body weight were measured 2-3 times a week.
• the mouse body weight and tumor volume were measured for the last time 27 days after inoculation of tumor cells, and the mice were euthanized.
• HGF hepatocyte growth factor
• CB-17 SCID mice were subcutaneously inoculated with H1975-HGF human lung cancer tumor cells to establish a tumor-bearing model and determine the anti-tumor effect of the anti-EGFRxc-Met bispecific antibody.
• the model construction process was the same as experimental model 2.
• mice tumor volume and mouse body weight were measured 2-3 times a week.
• the mouse body weight and tumor volume were measured for the last time 28 days after inoculation of tumor cells, and the mice were euthanized.
• the Osimertinib treatment group TGI 38.9%
• the Rybrevant treatment group TGI 44.1%
• the EGFRxMET-136 Afu treatment group TGI 72.3%
• the Rybrevant and small molecule combination group TGI 78.7%
• the EGFRxMET-136 Afu and small molecule combination group TGI 89.8%.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Immunology (AREA)
• Organic Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Cell Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• General Physics & Mathematics (AREA)
• Peptides Or Proteins (AREA)
• Pathology (AREA)
• Analytical Chemistry (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (9)

Applications Claiming Priority (4)

Publications (1)

Family

ID=88199447

Family Applications (1)

Country Status (11)

Cited By (4)

Families Citing this family (1)

Citations (8)

• 2023
  • 2023-03-31 WO PCT/CN2023/085460 patent/WO2023186092A1/zh not_active Ceased
  • 2023-03-31 EP EP23778456.6A patent/EP4506363A1/en active Pending
  • 2023-03-31 CN CN202380031276.3A patent/CN119013296A/zh active Pending
  • 2023-03-31 AU AU2023247654A patent/AU2023247654A1/en active Pending
  • 2023-03-31 KR KR1020247036568A patent/KR20240168447A/ko active Pending
  • 2023-03-31 US US18/852,602 patent/US20250223366A1/en active Pending
  • 2023-03-31 IL IL315910A patent/IL315910A/en unknown
  • 2023-03-31 JP JP2024558241A patent/JP2025514636A/ja active Pending
• 2024
  • 2024-09-30 MX MX2024012143A patent/MX2024012143A/es unknown
  • 2024-10-02 CL CL2024002974A patent/CL2024002974A1/es unknown
  • 2024-10-31 ZA ZA2024/08262A patent/ZA202408262B/en unknown

Patent Citations (9)

Non-Patent Citations (34)

Also Published As

Similar Documents

Legal Events